The osmoadaptive response of the wine yeast Saccharomyces cerevisiae K1-V1116 during icewine fermentation

The adapted metabolic response of commercial wine yeast under prolonged exposure to concentrated solutes present in Icewine juice is not fully understood. Presently, there is no information regarding the transcriptomic changes in gene expression associated with the adaptive stress response ofwine yeast during Icewine fermentation compared to table wine fermentation. To understand how and why wine yeast respond differently at the genomic level and ultimately at the metabolic level during Icewine fermentation, the focus ofthis project was to identify and compare these differences in the wine yeast Saccharomyces cerevisiae KI-Vll16 using cDNA microarray technology during the first five days of fermentation. Significant differences in yeast gene expression patterns between fermentation conditions were correlated to differences in nutrient utilization and metabolite production. Sugar consumption, nitrogen usage and metabolite levels were measured using enzyme assays and HPLC. Also, a small subset of differentially expressed genes was verified using Northern analysis. The high osmotic stress experienced by wine yeast throughout Icewine fermentation elicited changes in cell growth and metabolism correlating to several fermentation difficulties, including reduced biomass accumulation and fermentation rate. Genes associated with carbohydrate and nitrogen transport and metabolism were expressed at lower levels in Icewine juice fermenting cells compared to dilute juice fermenting cells. Osmotic stress, not nutrient availability during Icewine fermentation appears to impede sugar and nitrogen utilization. Previous studies have established that glycerol and acetic acid production are increased in yeast during Icewine fermentation. A gene encoding for a glycerollW symporter (STL1) was found to be highly expressed up to 25-fold in the i Icewine juice condition using microarray and Northern analysis. Active glycerol transport by yeast under hyperosmotic conditions to increase cytosolic glycerol concentration may contribute to reduced cell growth observed in the Icewine juice condition. Additionally, genes encoding for two acetyl CoA synthetase isoforms (ACSl and ACS2) were found to be highly expressed, 19- and II-fold respectively, in dilute juice fermenting cells relative to the Icewine juice condition. Therefore, decreased conversion of acetate to acetyl-CoA may contribute to increased acetic acid production during Icewine fermentation. These results further help to explain the response of wine yeast as they adapt to Icewine juice fermentation. ii

Environmental assessment and biomonitoring of the Twelve Mile Creek watershed, Niagara Peninsula, Ontario

In light of the heavy reliance of the people of the Niagara Peninsula on the T\\'elve Mile Creek (TMC) watershed for recreational activities and for municipal and industrial uses ( e.g., drinking water, shipping and discharge of effluents), it was deemed prudent to assess the envirol1tnental health of the system by analysing the sediments total and exchangeable metal, and TPH contents. The MOEE has set guidelines with limits for the protection and management of aquatic sediments, and the sediments from the headwaters of the TMC have total metal and TPH (subset of O&G) contents well below the lower provincial limits. Areas of environmental concern where total metal contents in sediments, either individually or collectively, exceed the guideline, are the south side of Lake Gibson, the Old WeIland Canal, a segment of TMC just south of the QEW and Martindale Pond. The total metal content of sediments does not in all instances identify areas of biological concern. Instead, it has been found that the exchangeable metal fraction of sediments is a better indicator of metal availability and thus potential accumulation in organisms. In some instances, the exchangeable metal fraction agrees with the total metal fraction defining areas of environmental concern, but it does vary from site to site reflecting the natural variability of the ambient environment. Overall, the exchangeable metal fraction of sediments appears to be a better indicator of anthropogenic pollution and ecosystem impact. A histochemical study of Anodon.ta sp., Elliptio sp. and zebra mussels (Dreissena polyn'101pha) was done in conjunction with passive biomonitoring of zebra and quagga mussels (Dreissena bugensis) from the Twelve Mile Creek watershed and Lake 51. Clair (Jeanette's Creek, Chatham, Ontario). The highest concentrations of divalent metals such as Cu, Ni, Cd, and Zn, and trivalent Al appear to accumulate in gill and kidney tissues. Metal contents of organ tissues in Anodonta sp. vary with size class. Organ metal content varies among size classes, thus requiring consideration of size in biomonitoring studies. Shucked zebra and quagga mussel tissues, exhibited similar size class to Al content trends. In addition they reflected the Al content trends of top (approximately 10 cm) most sediments in the Twelve Mile Creek watershed. Quagga mussels appear to have higher Al concentrations than zebra mussels, thus suggesting that quagga mussels may be better passive biomonitors of AI. Cd content in zebra mussel tissues, seemed to increase with size class trends. This was not demonstrated in the quagga mussel tissues. This suggests that Cd may be regulated by quagga mussels and not by zebra mussels, and that zebra mussels may be better passivebiomonitors of Cd than are quagga mussels. Zebra mussel, quagga mussel, Anodonta sp., and Elliptio sp. were used in a two part, active (translocated) biomonitoring study of the Twelve Mile Creek watershed. There was no statistical difference in death rates between zebra and quagga mussels after 65 days of biomonitoring. However there does appear to be a difference of death rates between sites. Unfortunately the data base did not permit us to differentiate between sites. Relative to Port Colborne Harbour (Port Colborne, Ontario), the Twelve Mile Creek watershed appears to be elevated in bioavailable AI. An area near the terminus of the Twelve Mile Creek appears to be an area of environmental concern since mussels seemed to have accumulated relatively large concentrations of Cd, Zn, and Pb. In addition to possible metal loading from a nearby outfalls, or possible upstream outfalls, road salt runoff from storm sewers may have contributed to metal accumulation through cation exchanges processes. Similar trends in cumulative quagga mussel metal concentrations during the two time periods (65 and 159 days), suggest that quagga mussels may reach equilibrium within 65 days of translocation. Differences in bioaccumulated metal concentrations of the two dreissenid species demonstrate that active biomonitoring studies must use a variety of organisms to adequately assess the environmental situation of specific waterways and/or bodies.

An examination of transcriptional and translational regulation of the 70kDa heat shock proteins following amputation of the tail and forelimb in the newt Notophthalmus viridescens

Several stresses to tissues including hyperthermia, ischemia, mechanical trauma and heavy metals have been demonstrated to affect the regulation of a subset of the family of heat shock proteins of70kOa (hsp70). In several organisms following some of these traumas, the levels of hsp70 mRNA and proteins are dramatically upregulated. However, the effects of the stress on limb and tail amputation in the newt Notophthalmus viridescens, involving mechanical tissue damage, have not adequately been examined. In the present study, three techniques were utilized to quantitate the levels of hsp70 mRNA and protein in the tissues of the forelimbs and tails of newts during the early post-traumatic events following surgical resection of these:: appendages. These included quantitative Western blotting of proteins separated by both one and twodimensional SDS-polyacrylamide gel electrophoresis and quantitative Northern blot analysis of total RNA. In tissues of both the limb and tail one hour after amputation, there were no significant differences in the levels of hsp70 protein measured by one-dimensional SOSPAGE followed by Western blotting, when compared to the levels measured in the unamputated limb. A 30 minute heat shock at 35°C failed to elicit an increase in the levels of hsp70 protein in these tissues. Further analysis using the more sensitive 20 PAGE separation of stump tissue proteins revealed that at least some of the five hsp70 isoforms of the newt may be differentially regulated in limbs and tails in response to trauma. It appears also that amputation of the tail and limb tissues leads to slight 3 elevation in the levels of HSP70 mRNA when compared to those of their respective unstressed tissues.

A Pragmatic Approach to Ethical Inquiry on Transhuman Athletes and Gene Doping in Sport

Gene doping is the most recent addition to the list of banned practices formulated by the World Anti-doping Agency. It is a subset of doping that utilizes the technology involved in gene therapy. The latter is still in the experimental phase but has the potential to be used as a type of medical treatment involving alterations of a patient‘s genes. I apply a pragmatic form of ethical inquiry to evaluate the application of this medical innovation in the context of sport for performance-enhancement purposes and how it will affect sport, the individual, society and humanity at large. I analyze the probable ethical implications that will emerge from such procedures in terms of values that lie at the heart of the major arguments offered by scholars on both affirmative and opposing sides of the debate on gene doping, namely fairness, autonomy and the conception of what it means to be human.

Delineation of within-site terroir effects using soil and vine water measurements in Riesling vineyards within the Niagara Peninsula

The major focus of this dissertation was to explain terroir effects that impact wine varietal character and to elucidate potential determinants of terroir by testing vine water status (VWS) as the major factor of the terroir effect. It was hypothesized that consistent water status zones could be identified within vineyard sites, and, that differences in vine performance, fruit composition and wine sensory attributes could be related to VWS. To test this hypothesis, ten commercial Riesling vineyards representative of each Vintners Quality Alliance sub-appellation were selected. Vineyards were delineated using global positioning systems and 75 to 80 sentinel vines per vineyard were geo-referenced for data collection. During the 2005 to 2007 growing seasons, VWS measurements [midday leaf water potential ('l')] were collected from a subset of these sentinel vines. Data were collected on soil texture and composition, soil moisture, vine performance (yield components, vine size) and fruit composition. These variables were mapped using global information system (GIS) software and relationships between them were elucidated. Vines were categorized into "low" and "high" water status regions within each vineyard block and replicate wines were made from each. Many geospatial patterns and relationships were spatially and temporally stable within vineyards. Leaf'l' was temporally stable within vineyards despite different weather conditions during each growing season. Generally, spatial relationships between 'l', soil moisture, vine size, berry weight and yield were stable from year to year. Leaf", impacted fruit composition in several vineyards. Through sorting tasks and multidimensional scaling, wines of similar VWS had similar sensory properties. Descriptive analysis further indicated that VWS impacted wine sensory profiles, with similar attributes being different for wines from different water status zones. Vineyard designation had an effect on wine profiles, with certain sensory and chemical attributes being associated from different subappellations. However, wines were generally grouped in terms of their regional designation ('Lakeshore', 'Bench', 'Plains') within the Niagara Peninsula. Through multivariate analyses, specific sensory attributes, viticulture and chemical variables were associated with wines of different VWS. Vine water status was a major contributor to the terroir effect, as it had a major impact on vine size, berry weight and wine sensory characteristics.

Regulation of Systemic Acquired Resistance through the Interaction of Arabidopsis thaliana Transcription factors TGAI and TGA2 with NPRI

Arabidopsis thaliana is an established model plant system for studying plantpathogen interactions. The knowledge garnered from examining the mechanism of induced disease resistance in this model system can be applied to eliminate the cost and danger associated with current means of crop protection. A specific defense pathway, known as systemic acquired resistance (SAR), involves whole plant protection from a wide variety of bacterial, viral and fungal pathogens and remains induced weeks to months after being triggered. The ability of Arabidopsis to mount SAR depends on the accumulation of salicylic acid (SA), the NPRI (non-expressor of pathogenesis related gene 1) protein and the expression of a subset of pathogenesis related (PR) genes. NPRI exerts its effect in this pathway through interaction with a closely related class of bZIP transcription factors known as TGA factors, which are named for their recognition of the cognate DNA motif TGACG. We have discovered that one of these transcription factors, TGA2, behaves as a repressor in unchallenged Arabidopsis and acts to repress NPRI-dependent activation of PRJ. TGA1, which bears moderate sequence similarity to TGA2, acts as a transcriptional activator in unchallenged Arabidopsis, however the significance of this activity is J unclear. Once SAR has been induced, TGAI and TGA2 interact with NPRI to form complexes that are capable of activating transcription. Curiously, although TGAI is capable of transactivating, the ability of the TGAI-NPRI complex to activate transcription results from a novel transactivation domain in NPRI. This transactivation domain, which depends on the oxidation of cysteines 521 and 529, is also responsible for the transactivation ability of the TGA2-NPRI complex. Although the exact mechanism preventing TGA2-NPRI interaction in unchallenged Arabidopsis is unclear, the regulation of TGAI-NPRI interaction is based on the redox status of cysteines 260 and 266 in TGAl. We determined that a glutaredoxin, which is an enzyme capable of regulating a protein's redox status, interacts with the reduced form of TGAI and this interaction results .in the glutathionylation of TGAI and a loss of interaction with NPRl. Taken together, these results expand our understanding of how TGA transcription factors and NPRI behave to regulate events and gene expression during SAR. Furthermore, the regulation of the behavior of both TGAI and NPRI by their redox status and the involvement of a glutaredoxin in modulating TGAI-NPRI interaction suggests the redox regulation of proteins is a general mechanism implemented in SAR.

Educator Evaluation of Academic and Social Competence in Students with Acquired Brain Injury (ABI) Relative to Assessed Performance and Sense of Belonging

Acquired brain injury (ABI) is the leading cause of death and disability amongst children and adolescents andpresents itself with challenges associated in cognitive, social, emotional, and behavioural domains. These changes may interfere with academic performance and social inclusion, influencing self-esteem and personal success. The current study examined a subset of data to capture the sense of academic and social belonging for students with ABI as a function of the classroom teachers’ subjective perception of ability, their ABI knowledge, and student identification. Overall, a discrepancy was found between educators’ subjective ratings of student performance and students’ neurocognitive capacity. Educator knowledge and identification of ABI influenced student success in academic and social domains independent of teaching approach. This research has implications for the identification of ABI in the classroom and related challenges students experience. Educators are underprepared for the reintegration of students returning to school and lack appropriate knowledge and strategies to accommodate individual needs.

A Survey of hMLH1 mRNA Splicing Profiles in Human Cell Lines: Comparing Primary Cultured Fibroblasts Before and After Oxidative Stress and Transiently versus Stably Demethylated Colon Cancer Derived Cell Lines

Most human genes undergo alternative splicing and loss of splicing fidelity is associated with disease. Epigenetic silencing of hMLH 1 via promoter cytosine methylation is causally linked to a subset of sporadic non-polyposis colon cancer and is reversible by 5-aza-2' -deoxycytidine treatment. Here I investigated changes in hMLHI mRNA splicing profiles in normal fibroblasts and colon cancer-derived human cell lines. I established the types and frequencies of hMLHI mRNA transcripts generated under baseline conditions, after hydrogen peroxide induced oxidative stress, and in acutely 5-aza-2' -deoxycytidine-treated and stably derepressed cancer cell lines. I found that hMLHI is extensively spliced under all conditions including baseline (50% splice variants), the splice variant distribution changes in response to oxidative stress, and certain splice variants are sensitive to 5- aza-2' -deoxycytidine treatment: Splice variant diversity and frequency of exon 17 skipping correlates with the level of hMLHI promoter methylation suggesting a link between promoter methylation and mRNA splicing.

Heuristics for the Critical Node Detection Problem in Large Complex Networks

Complex networks have recently attracted a significant amount of research attention due to their ability to model real world phenomena. One important problem often encountered is to limit diffusive processes spread over the network, for example mitigating pandemic disease or computer virus spread. A number of problem formulations have been proposed that aim to solve such problems based on desired network characteristics, such as maintaining the largest network component after node removal. The recently formulated critical node detection problem aims to remove a small subset of vertices from the network such that the residual network has minimum pairwise connectivity. Unfortunately, the problem is NP-hard and also the number of constraints is cubic in number of vertices, making very large scale problems impossible to solve with traditional mathematical programming techniques. Even many approximation algorithm strategies such as dynamic programming, evolutionary algorithms, etc. all are unusable for networks that contain thousands to millions of vertices. A computationally efficient and simple approach is required in such circumstances, but none currently exist. In this thesis, such an algorithm is proposed. The methodology is based on a depth-first search traversal of the network, and a specially designed ranking function that considers information local to each vertex. Due to the variety of network structures, a number of characteristics must be taken into consideration and combined into a single rank that measures the utility of removing each vertex. Since removing a vertex in sequential fashion impacts the network structure, an efficient post-processing algorithm is also proposed to quickly re-rank vertices. Experiments on a range of common complex network models with varying number of vertices are considered, in addition to real world networks. The proposed algorithm, DFSH, is shown to be highly competitive and often outperforms existing strategies such as Google PageRank for minimizing pairwise connectivity.

Feature Selection and Classification Using Age Layered Population Structure Genetic Programming

The curse of dimensionality is a major problem in the fields of machine learning, data mining and knowledge discovery. Exhaustive search for the most optimal subset of relevant features from a high dimensional dataset is NP hard. Sub–optimal population based stochastic algorithms such as GP and GA are good choices for searching through large search spaces, and are usually more feasible than exhaustive and deterministic search algorithms. On the other hand, population based stochastic algorithms often suffer from premature convergence on mediocre sub–optimal solutions. The Age Layered Population Structure (ALPS) is a novel metaheuristic for overcoming the problem of premature convergence in evolutionary algorithms, and for improving search in the fitness landscape. The ALPS paradigm uses an age–measure to control breeding and competition between individuals in the population. This thesis uses a modification of the ALPS GP strategy called Feature Selection ALPS (FSALPS) for feature subset selection and classification of varied supervised learning tasks. FSALPS uses a novel frequency count system to rank features in the GP population based on evolved feature frequencies. The ranked features are translated into probabilities, which are used to control evolutionary processes such as terminal–symbol selection for the construction of GP trees/sub-trees. The FSALPS metaheuristic continuously refines the feature subset selection process whiles simultaneously evolving efficient classifiers through a non–converging evolutionary process that favors selection of features with high discrimination of class labels. We investigated and compared the performance of canonical GP, ALPS and FSALPS on high–dimensional benchmark classification datasets, including a hyperspectral image. Using Tukey’s HSD ANOVA test at a 95% confidence interval, ALPS and FSALPS dominated canonical GP in evolving smaller but efficient trees with less bloat expressions. FSALPS significantly outperformed canonical GP and ALPS and some reported feature selection strategies in related literature on dimensionality reduction.

Feature Selection and Classification Using Age Layered Population Structure Genetic Programming

The curse of dimensionality is a major problem in the fields of machine learning, data mining and knowledge discovery. Exhaustive search for the most optimal subset of relevant features from a high dimensional dataset is NP hard. Sub–optimal population based stochastic algorithms such as GP and GA are good choices for searching through large search spaces, and are usually more feasible than exhaustive and determinis- tic search algorithms. On the other hand, population based stochastic algorithms often suffer from premature convergence on mediocre sub–optimal solutions. The Age Layered Population Structure (ALPS) is a novel meta–heuristic for overcoming the problem of premature convergence in evolutionary algorithms, and for improving search in the fitness landscape. The ALPS paradigm uses an age–measure to control breeding and competition between individuals in the population. This thesis uses a modification of the ALPS GP strategy called Feature Selection ALPS (FSALPS) for feature subset selection and classification of varied supervised learning tasks. FSALPS uses a novel frequency count system to rank features in the GP population based on evolved feature frequencies. The ranked features are translated into probabilities, which are used to control evolutionary processes such as terminal–symbol selection for the construction of GP trees/sub-trees. The FSALPS meta–heuristic continuously refines the feature subset selection process whiles simultaneously evolving efficient classifiers through a non–converging evolutionary process that favors selection of features with high discrimination of class labels. We investigated and compared the performance of canonical GP, ALPS and FSALPS on high–dimensional benchmark classification datasets, including a hyperspectral image. Using Tukey’s HSD ANOVA test at a 95% confidence interval, ALPS and FSALPS dominated canonical GP in evolving smaller but efficient trees with less bloat expressions. FSALPS significantly outperformed canonical GP and ALPS and some reported feature selection strategies in related literature on dimensionality reduction.