Maximal Clique Enumeration and Related Tools for Microarray Data Analysis

The purpose of this study was to investigate the utility of exact maximal clique enumeration in DNA microarray analysis, to analyze and improve upon existing exact maximal clique enumeration algorithms, and to develop new clique-based algorithms to assist in the analysis as indicated during the course of the study. As a first test, microarray data sets comprised of pre-classified human lung tissue samples were obtained through the Critical Assessment of Microarray Data Analysis (CAMDA) conference. A combination of exact maximal clique enumeration and approximate dominating set was used to attempt to classify the samples. In another test, maximal clique enumeration was used for a priori clustering of microarray data from Mus musculus (mouse). Cliques from this graph, though smaller than the anticipated groups of co-regulated genes, exhibited a high degree of overlap. Many genes within the overlap are either known or suspected to be involved in one or more gene regulatory networks. Experimental tests of four exact maximal clique enumeration algorithms on graphs derived from Mus musculus data normalized by either RMA or MAS 5.0 software were performed. A branch and bound Bron and Kerbosch algorithm was shown to perform the best on the widest range of inputs. A base Bron and Kerbosch algorithm was faster on very sparse graphs, but slowed considerably as edge density increased. Both the Kose and greedy algorithms were significantly slower than both Bron and Kerbosch algorithms on all inputs. Means to improve further the branch and bound Bron and Kerbosch algorithm were then considered. Two preprocessing rules and more exacting bounds were added to the algorithm both together and separately. The low degree preprocessing rule was found to improve performance most consistently, though significant improvement was only observed with the sparsest graphs, where improvement is least necessary. Finally, a first attempt at developing an algorithm that would integrate genes that were likely excluded from a clique as a result of noise into the appropriate group was made. Initial testing of the resulting paraclique algorithm revealed that the algorithm maintains the desired high level of inter-group edge density while expanding the core clique to a more acceptable size. Research in this area is ongoing

EVALUATING THE SUB-LETHAL TOXICITY OF THE ORGANOPHOSPHATE PESTICIDE, CHLORPYRIFOS, ON THE AMPHIBIAN, XENOPUS LAEVIS

Chlorpyrifos (CPF) is an organophosphate pesticide used extensively in Canada and around the world. Due to its highly conserved mechanism of action involving inhibition of acetylcholinesterase (AChE), CPF has the ability to exert toxicity on non-target species in aquatic systems. In fish species, exposure to CPF has been associated with a range of adverse effects across physiological endpoints including abnormal development, inhibition of AChE, immunomodulation, and molecular level effects such as altered expression of specific genes and global transcriptomes. However, the literature on amphibians exposed to CPF is not as extensive despite the known global declines of amphibian species and the hypothesized links between these declines and anthropogenic pesticide contamination of aquatic systems worldwide. The overall objective of this thesis was to gain a better understanding of the sub-lethal effects of CPF exposure on the model amphibian, Xenopus laevis, across levels of biological organization from molecular to whole animal. The first study (Chapter 2) examined the molecular toxicity pathways and mechanisms of toxicity after short-term exposure of early life-stage (ELS) X. laevis to CPF using whole body transcriptome analyses. The ELS transcriptomic responses were then compared to apical outcomes of chronic exposure to CPF to determine if identified dysregulated pathways could provide early indicators of these adverse outcomes. Post-hatch individuals were exposed to nominal CPF concentrations of 0.4, 2, or 10 μg L-1. A subset of individuals were sampled at 96 hours (h) for whole-body transcriptomic analysis and remaining individuals were transferred to tanks for long-term exposure through to metamorphic climax (~ 75 days). Pathway analysis revealed dysregulated pathways that were related to outcomes known to be associated with exposure to CPF such as altered serine hydrolase activity, impacted metabolic processes, and immune-related outcomes. Other dysregulated pathways with less precedence in the literature included vasculature development and sensory perception of light stimulus. Apical outcomes of chronic CPF exposure included inhibition of AChE activity, increased relative liver weight, and a decrease in percentage of individuals that reached metamorphic climax. Dysregulation of serine hydrolase associated pathways after ELS CPF exposure is in agreeance with the decrease in AChE (a serine hydrolase enzyme) activity observed in the brains of individuals at metamorphic climax. Additionally, an increase in relative liver weight after chronic CPF exposure could be related to dysregulation of ELS pathways associated with metabolic processes and immune function. In fact, several pathways related to immune function were depleted. In Chapter 3, we more closely examined the potential immunotoxicity of sub-lethal CPF exposure. Post-metamorphic individuals were exposed 1 or 10 μg L-1 CPF (nominal) for 7 days (d), then administered a phosphate buffered sodium (PBS) control injection or a lipopolysaccharide (LPS) injection to stimulate an inflammatory response. At 24 h post-injection, morphometric indices were recorded and tissues were sampled for differential leukocyte counts (flow cytometry), liver pro-inflammatory cytokine expression (qPCR), and kidney histopathology. At 1 µg L−1 CPF, there was a decrease in circulating lymphocytes, an increase in circulating granulocytes, and an increase in the granulocyte:lymphocyte (GL) ratio regardless of immune state. Liver expression of pro-inflammatory cytokines TNF-α and CSF-1 was increased in individuals exposed to 10 µg L−1 CPF, independent of immune state. Exposure to 10 μg L-1 CPF increased kidney epithelial cell height (by 18 %) and decreased lumen space in the convoluted tubules of the kidney. This study provided evidence that exposure to CPF can lead to changes in key biomarkers of immune status in amphibians in both immune-rested (PBS-injected) and immune-stimulated (LPS-injected) states. Additionally, we found that LPS was an effective mitogen in our study, capable of inducing a robust and measurable stress response in X. laevis. This response included a decrease in circulating lymphocytes, and increase in circulating monocytes, and an increase in the GL ratio. In addition, increased liver expression of pro-inflammatory cytokines TNF-α, IL-1β, and CSF-1 was induced by LPS injection. We conclude that LPS is an appropriate immunostimulatory agent in an immune challenge assay using X. laevis and that exposure to CPF does not appear to impact the response to LPS exposure. Overall, our findings show that exposure to environmentally relevant concentrations of CPF has the ability to impact amphibians at multiple levels of biological organization. A number of affected molecular pathways warrant further study in terms of the underlying mechanisms of CPF-mediated toxicity as well as the associated outcomes of CPF exposure in amphibians. This research provides novel data on the effects of CPF exposure to amphibians, which are generally overlooked and under-represented in the literature despite links between pesticide exposure and globally declining amphibian populations

Breaking the paradigm: Dr Insight empowers signature-free, enhanced drug repurposing

Motivation: Transcriptome-based computational drug repurposing has attracted considerable interest by bringing about faster and more cost-effective drug discovery. Nevertheless, key limitations of the current drug connectivity-mapping paradigm have been long overlooked, including the lack of effective means to determine optimal query gene signatures. Results: The novel approach Dr Insight implements a frame-breaking statistical model for the ‘hand-shake’ between disease and drug data. The genome-wide screening of concordantly expressed genes (CEGs) eliminates the need for subjective selection of query signatures, added to eliciting better proxy for potential disease-specific drug targets. Extensive comparisons on simulated and real cancer datasets have validated the superior performance of Dr Insight over several popular drug-repurposing methods to detect known cancer drugs and drug–target interactions. A proof-of-concept trial using the TCGA breast cancer dataset demonstrates the application of Dr Insight for a comprehensive analysis, from redirection of drug therapies, to a systematic construction of disease-specific drug-target networks

What Can General Chemistry Students Learn from External Representations of Acid- Base Titrations?

Laboratory activities are a prevalent and essential part of chemistry learning because of their potential to help students develop problem solving abilities, visualize chemistry concepts learned in lecture, and gain practical skills. However, learning in the laboratory environment is not without its challenges. For example, cookbook-style chemistry laboratories can promote superficial learning, and cognitive overload can result from the study of new concepts and the use of new procedures in this environment. Multiple pedagogies and supports have been developed to address challenges such as these. The current research focuses upon external representations that are commonly used to support learning in the general chemistry laboratory. External representations are visual tools—such as graphs, pictures, charts, and equations—that are used to illustrate, model, and communicate ideas. These representations are often used in chemistry lecture and laboratory to illustrate concepts that are beyond the senses. Although external representations are used with the intent of supporting student learning, research indicates that students have difficulty interpreting and learning from them. Therefore, it is important to examine how students’ learning from these figures, graphs, and symbols correlates with what their instructors intend for them to learn.External representations are commonly used in the chemistry laboratory environment; however, very few studies have examined what instructors intend for their students to learn from these external representations, what is possible for students to learn from these external representations, and what students actually learn from these representations. Specifically, I focus on external representations of acid-base titrations. I have chosen this subject because titrations are an important skill for practicing scientists; therefore, it is important that students master titrations during their training. Furthermore, external representations are commonly used (1) to illustrate how students should set up their laboratory equipment, (2) to demonstrate how to carry out a titration, and (3) to help students understand the chemical reactions that occur during a titration. This research utilizes variation theory to determine what instructors intend for their students to understand about acid-base titrations, what they can learn from the images we use to instruct, and what they ultimately do learn about acid-base titrations. In this dissertation, I first describe the 18 features which instructors deem as critical for their general chemistry students to understand about acid-base titrations. These include pH, chemical equations, and excess base, among others. The images the instructor participants then chose to share with the student participants included most of the critical features mentioned by the instructors, indicating that typical images used to teach students about acid-base titrations are consistent with the concepts that instructors want their students to understand about titrations. Students were interviewed about their understandings of acid-base titrations both before and after viewing the external representations selected by the instructors. Evidence suggests that students learned from the representations. In fact, the external representations used in the study facilitated learning. The external representations used in the study facilitated learning for many of the critical features; in some cases, they were essential for learning. Therefore, it is critical that external representations used to teach acid-base titrations are chosen with consideration to the critical features identified by instructors, the affordances that the representations can give for learning, and the features that students focus upon when learning about these important chemistry concepts. Based on the findings of the current study, recommendations are provided for the design, the selection, and the utilization of external representations used in teaching acid- base titrations

Quality of Life and Self-Determination: Youth with Chronic Health Conditions Make the Connection

While optimizing quality of life(QOL) is a key goal of rehabilitation care,no previous study has reported on what ‘QOL’ means to youth with chronic health conditions. In addition, no qualitative studies have explored the relationship between QOL and self-determination(SD). Objectives of this qualitative study were to examine: what the terms ‘quality of life’ and ‘self-determination’ mean to youth with chronic conditions; the factors these youth think are linked with these concepts; the relationship they see between concepts, the types of future goals youth have and how they view the connection between their SD and these goals. A descriptive methodology was used. A purposive sample of 15 youth aged 15 to 20 years was obtained. Youth had cerebral palsy, a central nervous system disorder, or autism spectrum disorder. Semi-structured interviews were conducted first, followed by a focus group. Line-by-line coding of transcripts was completed, codes were collapsed into categories, and themes identified. Participants viewed QOL as an overarching personal evaluation of their life, and use

Youth with Disabilities Talk About Spirituality:A Qualitative Descriptive Study

There is little known about what spirituality means for youth with disability or about the potential relevance of youths’ spirituality in pediatric rehabilitation. This study explored perceptions of spirituality for youth with disabilities. Using a qualitative descriptive methodology, we examined the lived experiences of eighteen youth ages 11-20 years with disabilities including cerebral palsy, central nervous system disorder or autism spectrum disorder. In individual interviews, followed by a focus group, youth identified key spiritual themes – the importance of their beliefs, personal sources of comfort and strength, finding purpose in helping others, significance of personal connections, and strengths-based perspectives on disability. This study makes a unique contribution by informing health care professionals about the relevance of youths’ spirituality in service delivery

Biomagnetic recovery of selenium: Bioaccumulating of selenium granules in magnetotactic bacteria

Using microorganisms to remove waste and/or neutralize pollutants from contaminated water is attracting much attention due to the environmentally friendly nature of this methodology. However, cell recovery remains a bottleneck and a considerable challenge for the development of this process. Magnetotactic bacteria are a unique group of organisms that can be manipulated by an external magnetic field due to the presence of biogenic magnetite crystals formed within their cells. In this study, we demonstrated the first account of accumulation and precipitation of amorphous elemental selenium nanoparticles within magnetotactic bacteria alongside and independently to magnetite crystal biomineralisation when grown in a medium containing selenium oxyanion (SeO3 (2-)). Quantitative analysis shows that magnetotactic bacteria accumulate the highest amount of target molecules (Se) per cell than any other previously reported of non-ferrous metal/metalloid. For example, 2.4 and 174 times more Se is accumulated when compared to Te uptaken into cells and Cd(2+) adsorption onto the cell surface respectively. Crucially, the bacteria with high levels of Se accumulation were successfully recovered with an external magnetic field. This biomagnetic recovery and effective accumulation of target elements demonstrate the potential for application in bioremediation of polluted water. IMPORTANCE: The development of a technique for effective environmental water remediation is urgently required across the globe. A biological remediation process of waste removal and/or neutralization of pollutant from contaminated water using microorganism has great potential, but cell recovery remains a bottleneck. Magnetotactic bacteria synthesize magnetic particles within their cells, which can be recovered by a magnetic field. Herein, we report the first example of accumulation and precipitation of amorphous elemental selenium nanoparticles within magnetotactic bacteria independent of magnetic particle synthesis. The cells were able to accumulate the highest amount of Se compared to other foreign elements. More importantly, the Se accumulating bacteria were successfully recovered with an external magnetic field. We believe magnetotactic bacteria confer unique advantages of biomagnetic cell recovery and of Se accumulation, providing a new and effective methodology for bioremediation of polluted water

Computational, Integrative, and Comparative Methods for the Elucidation of Genetic Coexpression Networks

Gene expression microarray data can be used for the assembly of genetic coexpression network graphs. Using mRNA samples obtained from recombinant inbred Mus musculus strains, it is possible to integrate allelic variation with molecular and higher-order phenotypes. The depth of quantitative genetic analysis of microarray data can be vastly enhanced utilizing this mouse resource in combination with powerful computational algorithms, platforms, and data repositories. The resulting network graphs transect many levels of biological scale. This approach is illustrated with the extraction of cliques of putatively coregulated genes and their annotation using gene ontology analysis and cis-regulatory element discovery. The causal basis for coregulation is detected through the use of quantitative trait locus mapping

The effect of modulating the quantity of enzymes in a model ethanol pathway on metabolic flux in <i>Synechocystis</i> sp. PCC 6803

Synthetic metabolism allows new metabolic capabilities to be introduced into strains for biotechnology applications. Such engineered metabolic pathways are unlikely to function optimally as initially designed and native metabolism may not efficiently support the introduced pathway without further intervention. To develop our understanding of optimal metabolic engineering strategies, a two-enzyme ethanol pathway consisting of pyruvate decarboxylase and acetaldehyde reductase was introduced into Synechocystis sp. PCC 6803. We characteriseda new set of ribosome binding site sequences in Synechocystis sp. PCC 6803 providing a range of translation strengths for different genes under test. The effect of ribosome-bindingsite sequence, operon design and modifications to native metabolism on pathway flux was analysed by HPLC. The accumulation of all introduced proteins was also quantified using selected reaction monitoring mass spectrometry. Pathway productivity was more strongly dependent on the accumulation of pyruvate decarboxylase than acetaldehyde reductase. In fact, abolishment of reductase over-expression resulted in the greatest ethanol productivity, most likely because strains harbouringsingle-gene constructs accumulated more pyruvate decarboxylase than strains carrying any of the multi-gene constructs. Overall, several lessons were learned. Firstly, the expression level of the first gene in anyoperon influenced the expression level of subsequent genes, demonstrating that translational coupling can also occur in cyanobacteria. Longer operons resulted in lower protein abundance for proximally-encoded cistrons. And, implementation of metabolic engineering strategies that have previously been shown to enhance the growth or yield of pyruvate dependent products, through co-expression with pyruvate kinase and/or fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase, indicated that other factors had greater control over growth and metabolic flux under the tested conditions