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Microsporidia are unicellular fungi that are intracellular parasites of animals, including humans. They are both complex and simple, armed with a sophisticated infection apparatus and possessing the smallest eukaryotic nuclear genomes. The microsporidian Encephalitozoon cuniculi has a genome size of 2.9 Mb, which is smaller than many bacterial genomes. Genome reduction and compaction in size, content, and form has been interpreted as an adaptation to parasitism. One of the effects of genome size reduction concerns intron evolution — E. cuniculi has retained only a few extremely short spliceosomal introns. This thesis examines the splicing of introns in the spore stage. The introns were retained in spores, suggesting life-stage specific splicing and splicing inhibition. How the short introns are recognized was also examined. Unique splicing signal motifs were predicted, and were used to find additional introns. The intron density was doubled for this species, and I also obtained data that counter current views about intron evolution in compacted genomes with low intron densities. I also predict that E. cuniculi introns are recognized in a unique way by the spliceosome.

The fossil record is the only direct source of data for studying modes (patterns) and rates of morphological change over geologic time periods. Determining modes is critical for understanding macroevolutionary processes, but just how modes can vary within a taxon, and why, have hitherto been largely understudied. To address this, I examined patterns of morphological change in the shell of the Mesozoic marine bivalve genus Buchia over its geographic and temporal range. Buchia was chosen as a test subject because it is abundant, well-preserved across a variety of facies, and is widely distributed across the Northern Hemisphere where the likelihood of multiple lineages is low. While the focus of this thesis is on evolutionary patterns, it is also necessary to address issues of taxonomy and geographical variation, making this research applicable to a wide-variety of fields. Previous to this study there was no protocol for measuring buchiid valves, nor was the genus studied in a quantitative manner. Throughout this research I used ten morphological characters to describe shell shape and size. Multivariate methods (principle component and canonical variate analyses) were employed to discriminate between species of Buchia and examine how morphological characters change through time and space within the genus. Evolutionary patterns were delineated using two well-established programs that discriminate between multiple modes of evolution. Overall, nearly 2000 specimens from eight geographical locations around the world were studied for this thesis. I found the genus Buchia was a useful tool for evolutionary studies as it can be studied quantitatively in space and time. Specically I have found that buchiid species can be delineated using morphometrics; the genus is restricted to the Northern Hemisphere; while the environment significantly affects morphology, there is no evidence of a latitudinal gradient; diversity and disparity within Buchia are not correlated; most evolutionary modes conformed to random walks or stasis; and modes and rates vary across the geographical range of the genus. Overall, I have found that the environment plays an important role in shaping both morphology and modes.

Copper-containing nitrite reductase (NiR) is a homotrimer of two cupredoxin domains and catalyzes the single electron reduction of NO2- to NO during dissimilatory denitrification. To investigate the evolution of NiR, methods of mutagenic library generation and high-throughput variant screening from E. coli colonies were developed. These methods allow for facile screening of 105 mutants for folding efficiency or substrate specificity. Initial proof of principle studies yielded several variants that oxidized the artificial substrate ο-dianisidine up to 8 times faster than wild type NiR, suggesting that this methodology has the potential to engineer NiR to acquire other reductase functions. A crystal structure was solved for a putative multicopper oxidase (MCO) and NiR homologue from Arthrobacter sp. (AMMCO) to 1.8 Å resolution. The overall folds of AMMCO and NiR are very similar (r.m.s.d. of 2.0 Å over 250 Cα atoms); Like NiR, AMMCO is a trimer with type-1 Cu sites in the N-terminal domain of each monomer; however, the active site of AMMCO contains trinuclear Cu site characteristic of MCOs instead of a the mononuclear type-2 Cu site found in NiR. Detailed structural analysis supports the theory that two-domain MCOs similar to AMMCO were intermediaries in the evolution of NiR and the more common three-domain MCOs. The physiological function of AMMCO remains uncertain, but genomic, crystallographic and functional analysis suggests that the enzyme is involved in metal regulation. Considering the extensive similarity between AMMCO and NiR, particularly at the active site, engineering a trinuclear cluster into NiR appears feasible with a modest number of alterations to the polypeptide chain. With the aid of my newly developed high-throughput screening technique and site-directed mutagenesis, the mononuclear NiR active site was remodelled into a trinuclear Cu site similar to that of MCO. A crystal structure of this variant was solved to 2.0 Å and the presence of three copper atoms at the engineered cluster was confirmed by Cu-edge anomalous diffraction data. Although the trinuclear copper cluster is present and catalyzes the reduction of oxygen, achieving rates of catalysis seen in native MCOs has proven more difficult. With the framework provided, further engineering NiR into a robust MCO is likely to provide further insights into the structural basis of oxygen reduction by trinuclear copper sites.

Much scientific and media attention has been devoted to the growing body of research into the genetic correlates of human phenomena. However, many of the resulting reports lead to a deterministic interpretation of the role of genes, and involve fundamental misunderstandings of genetics and heredity. Hence, questions arise regarding the ways in which people make sense of the behavioural genetics research they encounter in everyday life. Furthermore, essentialist accounts are often embedded within popular understanding of politically sensitive topics, such as eugenics, race, and sex, and therefore it is important to examine how people comprehend genetic influences on behaviour. In this dissertation, I review current findings regarding the effects of genetic attributions on beliefs, attitudes, and behaviours in the context of the social world. Particular attention is paid to such effects in the context of gender issues. Specifically, in three studies I examine the effects of exposure to scientific theories concerning human sexuality on attitudes towards and evaluations of men’s dubious sexual behaviors. The results indicate that among men exposure to evolutionary psychology arguments leads to more lenient evaluations and judgments of an array of dubious sexual behaviors, compared with exposure to social constructivist arguments. It also seems that men implicitly hold nativist perceptions with regards to male sexuality and promiscuity. The findings were less conclusive among women, with some indication that women are less affected by such exposure as well as less likely to naturally hold a nativist perspective in the context of human sexuality. This empirical research has direct implications for previously suggested intervention programs and adds to the incurrent resurgence of interest in the effects of genetic theories. Finally, I identify areas where further exploration is needed, suggest potential solutions for specific problems, and evaluate related individual and social implications.

It has been almost 150 years since Darwin’s “The Origin of Species” was published, yet the mechanisms that lead to the creation of new species are still a topic of fervent research in Evolutionary Biology. In the last several decades, there has been considerable interest in how genetic drift and adaptive processes internal to populations may lead to population divergence. It has been recognized that intersexual co-evolution may drive the rapid evolution of reproductive traits, leading to reproductive isolation between populations in allopatry. To explore the nature of this process, I conducted a series of interpopulation interaction experiments using a set of laboratory-evolved Drosophila melanogaster populations. These “B” (for baseline) populations share a common ancestor and had evolved in identical habitats in allopatry for 650-700 generations prior to my experiments. When different populations were combined in competitive fitness assays, I found that foreign males usually suffer depressed fitness when competing with males who share a common evolutionary history with the focal females. I selected a subset of B populations to investigate potential sources of variation that could explain this result. I examined; 1) characters involved in post-copulatory interactions between mates and 2) traits involved in mating behaviour. I found that sperm precedence patterns varied significantly depending on female population of origin and suggestive evidence that males between populations vary in ability to compete in sperm offence. Males also varied in mating behaviour between populations, however we found no evidence that divergence of these traits was correlated to that of traits involved in post-copulatory interactions, suggesting that understanding how intersexual co-evolution drives population divergence requires considering independent components of the reproductive phenotype and their contributions to overall fitness differences. / Thesis (Master, Biology) -- Queen's University, 2008-08-13 12:29:52.602

A major hurdle facing in vitro protein characterization is obtaining soluble protein from targets that tend to aggregate and form insoluble inclusion bodies. Soluble protein is essential for any biophysical data collection and new methods are needed to approach this significant problem. Directed evolution can be used to discover mutations which lead to improved solubility using an appropriate screening method. Green fluorescent protein (GFP) has been shown to be an effective solubility reporter which can be used to screen for soluble protein variants. We have chosen three diverse enzymes as targets for improving protein solubility using this technique: arachidonate 5-lipoxygenase—an enzyme which converts fatty acids into leukotrienes, PhnG—an enzyme belonging to the bacterial carbon-phosphorus lyase pathway, and RebG—a glycosyltransferase. Error-prone PCR and DNA shuffling were used to generate libraries of mutants which were subsequently cloned into a GFP-fusion screening vector. From the evolution of 5LO and RebG, much was learned about the optimization of the protocols involved in this methodology, including valuable information about how to avoid common “false-positive” results in which fluorescent colonies arise while screening but do not represent an improvement of the target. Evolution of these two targets did not result in an improvement of solubility, however truncation strategies may still prove to be effective, and more work needs to be done in this area. Evolution of PhnG successfully produced one variant, named clone B6, which showed both an improvement in expression and folding over wild type PhnG. It was also discovered that GFPuv can act as an effective solubility enhancing fusion tag for PhnG. Prior to the current studies PhnG had not been effectively expressed and purified in E. coli , however purification and refolding of resolubilized inclusion bodies of the clone B6 PhnG-GFP fusion construct was shown to yield enough soluble protein for future crystallographic studies. / Thesis (Master, Chemistry) -- Queen's University, 2009-10-09 12:26:03.353

Code clones are duplicated code fragments that are copied to re-use functionality and speed up development. However, due to the duplicate nature of code clones, inconsistent updates can lead to bugs in the software system. Existing research investigates the inconsistent updates through analysis of the updates to code clones and the bug fixes used to fix the inconsistent updates. We extend the work by investigating other factors that affect clone evolution, such as the number of developers. On two levels of analysis, the method and clone class level, we conduct an empirical study on clone evolution. We analyze the factors affecting bug fixes and co-change (i.e. update cloned methods at the same time) using our new metrics. Our metrics are related to the developers, code complexity, and stages of development. We use these metrics to find ways to improve the maintenance of cloned code. We discover that one way to improve maintenance of code clones is the decrease of code complexity. We find that increased code complexity leads to a decrease in co-change, which can lead to bugs in the software. We perform our study on 6 applications. To maximize the number of clones detected, we use two existing code clone detection tools: SimScan and Simian. SimScan was used to find clones in 5 of the applications due to its versatility in finding code clones. Simian was used to detect clones due to its reliability to find code clones regardless of language or compilation problems. To analyze and determine the significance of the metrics, we use the R Statistical Toolkit. / Thesis (Master, Computing) -- Queen's University, 2009-11-25 14:18:05.884

Constructing software bears many similarities to constructing buildings. In a building project, each floor builds on the previous structures (walls of the previous floors) with some structures being more foundational (i.e. essential) for other structures and some periods of construction being more foundational (i.e. critical) to the final structure. In a software project, each change builds on the structures created by prior changes with some changes creating foundational structure and with some time periods of changes being more foundational than others. This thesis is inspired by this similarity between constructing buildings and constructing software. The thesis makes use of the similarity to study the evolution of software projects. In particular, we develop the concept of time dependence between code changes to study software evolution through empirical studies on two large open source projects (PostgreSQL and FreeBSD) with more than 25 years of development history. We show that time dependence can be used to study how changes build on prior changes and the impact of this building process on the quality of a project. We show how a development period impacts the development of future periods in a project. We also show how a subsystem (module) of a project builds on other subsystems and we identify the subsystems that have high impact on a project's development. Using this knowledge, managers can better monitor the progress of the projects and better plan for future changes. / Thesis (Master, Computing) -- Queen's University, 2010-05-29 11:25:39.005