High-throughput sequencing technologies are improving in quality, capacity, and costs, providing versatile applications in DNA and RNA research. For small genomes or fraction of larger genomes, DNA samples can be mixed and loaded together on the same sequencing track. This so-called multiplexing approach relies on a specific DNA tag, index, or barcode that is attached to the sequencing or amplification primer and hence accompanies every read. After sequencing, each sample read is identified on the basis of the respective barcode sequence. Alterations of DNA barcodes during synthesis, primer ligation, DNA amplification, or sequencing may lead to incorrect sample identification unless the error is revealed and corrected. This can be accomplished by implementing error correcting algorithms and codes. This barcoding strategy increases the total number of correctly identified samples, thus improving overall sequencing efficiency. Two popular sets of error-correcting codes are Hamming codes and codes based on the Levenshtein distance. Levenshtein-based codes operate only on words of known length. Since a DNA sequence with an embedded barcode is essentially one continuous long word, application of the classical Levenshtein algorithm is problematic. In this thesis we demonstrate the decreased error correction capability of Levenshtein-based codes in a DNA context and suggest an adaptation of Levenshtein-based codes that is proven of efficiently correcting nucleotide errors in DNA sequences. In our adaptation, we take any DNA context into account and impose more strict rules for the selection of barcode sets. In simulations we show the superior error correction capability of the new method compared to traditional Levenshtein and Hamming based codes in the presence of multiple errors. We present an adaptation of Levenshtein-based codes to DNA contexts capable of guaranteed correction of a pre-defined number of insertion, deletion, and substitution mutations. Our improved method is additionally capable of correcting on average more random mutations than traditional Levenshtein-based or Hamming codes. As part of this work we prepared software for the flexible generation of DNA codes based on our new approach. To adapt codes to specific experimental conditions, the user can customize sequence filtering, the number of correctable mutations and barcode length for highest performance. However, not every platform is susceptible to a large number of both indel and substitution errors. The Illumina “Sequencing by Synthesis” platform shows a very large number of substitution errors as well as a very specific shift of the read that results in inserted and deleted bases at the 5’-end and the 3’-end (which we call phaseshifts). We argue in this scenario that the application of Sequence-Levenshtein-based codes is not efficient because it aims for a category of errors that barely occurs on this platform, which reduces the code size needlessly. As a solution, we propose the “Phaseshift distance” that exclusively supports the correction of substitutions and phaseshifts. Additionally, we enable the correction of arbitrary combinations of substitution and phaseshift errors. Thus, we address the lopsided number of substitutions compared to phaseshifts on the Illumina platform. To compare codes based on the Phaseshift distance to Hamming Codes as well as codes based on the Sequence-Levenshtein distance, we simulated an experimental scenario based on the error pattern we identified on the Illumina platform. Furthermore, we generated a large number of different sets of DNA barcodes using the Phaseshift distance and compared codes of different lengths and error correction capabilities. We found that codes based on the Phaseshift distance can correct a number of errors comparable to codes based on the Sequence-Levenshtein distance while offering the number of DNA barcodes comparable to Hamming codes. Thus, codes based on the Phaseshift distance show a higher efficiency in the targeted scenario. In some cases (e.g., with PacBio SMRT in Continuous Long Read mode), the position of the barcode and DNA context is not well defined. Many reads start inside the genomic insert so that adjacent primers might be missed. The matter is further complicated by coincidental similarities between barcode sequences and reference DNA. Therefore, a robust strategy is required in order to detect barcoded reads and avoid a large number of false positives or negatives. For mass inference problems such as this one, false discovery rate (FDR) methods are powerful and balanced solutions. Since existing FDR methods cannot be applied to this particular problem, we present an adapted FDR method that is suitable for the detection of barcoded reads as well as suggest possible improvements.

Clonal plants may be able to cope with spatial heterogeneity due to the physiological integration of ramets. Previous studies demonstrated that benefits of clonal integration increase with patch contrast between individual ramets. However, the same magnitude of contrast may be perceived differently in rich and poor environments. According to the theoretical work of Caraco and Kelly (1991), I expected these benefits to be the greatest in overall poor conditions and high between-patch contrast. To test this hypothesis, I conducted experiments with pairs of ramets of a stoloniferous grass, Agrostis stolonifera, grown in variously nutrient rich conditions. The experiment with pairs of ramet of similar developmental age showed only very weak effect of integration on growth of ramets, although integration significantly improved survival of ramets and also affected root-shoot ratio of ramets. Nevertheless, there were considerable benefits of integration in the experiment with developmentally older mother ramets and their daughter ramets. Contrary to the predictions, the benefits of integration were bigger in rich conditions and they decreased with increasing between-patch contrast. In addition, effect of integration on root-shoot ratio of ramets was opposite to the expected specialization for acquisition...

Introduced populations of Eichhornia crassipes (Pontederiaceae) possess extremely low levels of genetic diversity due to severe bottleneck events and clonal reproduction. While populations elsewhere have been well studied, North American populations of E. crassipes remain understudied. We used Amplified Fragment Length Polymorphism markers to assess genetic diversity and population structure in North American E. crassipes populations. Patterns of diversity over the past fifty years were analyzed using herbarium specimens. Furthermore, we sampled populations across the Gulf Coast of the United States throughout a year to determine contemporary genetic diversity and assess potential seasonal effects. Genetic diversity was found to be scant in the United States without population structure, agreeing with previous studies from other regions. Genetic diversity has remained consistently low over the past fifty years despite significant changes in selection pressure. However, evidence for and against population structure between seasons was found and the consequences of this are discussed.

Plants and microorganisms interact with each other constantly, with some interactions being mutually beneficial and others being detrimental to the plants. The features of the organisms involved in such interactions will determine the characteristics of individual pathosystems. Plants respond readily to pathogen attacks, regardless of the pathosystem; furthermore, variation in the resistance to pathogens within species is common and well documented in many plant species. The variability in pathogen resistance is at the core of genetic improvement programs for disease resistance. True resistance to pathogens in plants is a genetically determined and complex trait that can involve both constitutive and induced mechanisms at different levels of organization. The complexity of this phenomenon makes the study of compatible plant - pathogen interactions challenging, and typically, disease resistance studies focus on specific aspects of a pathosystem, such as field resistance, anatomical or physiological features of resistant plants, or molecular mechanisms of resistance. The Thuja sp. - Didymascella thujina (E.J. Durand) Maire interaction is an important pathosystem in western North America, which has been studied for more than five decades. Western redcedar (Thuja plicata Donn ex D. Don) is very susceptible to cedar leaf blight (D. thujina), a biotroph that affects the tree at all stages, although seedlings are the most sensitive to the pathogen. The characteristics of the Thuja sp. - D. thujina interaction, the wealth of information on the pathosystem and the excellent Thuja sp. genetic resources available from the British Columbia Ministry of Forests, Lands, Natural Resource Operations and Rural Development make this interaction an ideal system to advance the study of disease resistance mechanisms in conifers. This Doctoral project presents a comprehensive investigation of the constitutive and induced resistance mechanisms against D. thujina in T. plicata, Thuja standishii (Gord.) Carrière and a Thuja standishii x plicata hybrid at the phenotypic and gene expression levels, undertaken with the objective of exploring the resistance mechanisms against the biotroph in these conifers. The project also aimed to establish base knowledge for the future development of markers for marker-assisted breeding of T. plicata. The investigations included a combination of histological, chemical and next generation sequencing (NGS) methodologies. NGS data were analyzed, in addition to the traditional clustering analyses, with cutting edge machine learning methods, including grade of membership analysis, dynamic topic modelling and stability selection analysis. The studies were progressively more controlled to narrow the focus on the resistance mechanisms to D. thujina in Thuja sp. Histological characteristics related to D. thujina resistance in Thuja sp. were studied first, along with the relationship between climate of origin and disease resistance. The virulence of D. thujina was also documented early in this project. Chemical and gene expression constitutive and induced responses to D. thujina infection in T. plicata seedlings were studied next. T. plicata clonal lines were then comprehensively studied to shed light on the mechanisms behind known physiologically determined resistance. A holistic investigation of the resistance mechanisms to D. thujina in T. standishii, T. plicata and a T. standishii x plicata hybrid explored the possibility of a gene-for-gene resistance model. Thirty-five T. plicata families were screened during the four field seasons carried out between 2012 and 2015, totalling more than 1,400 seedlings scored for D. thujina severity. Thirteen of those families were used in the five studies performed during the program, along with two T. plicata seedling lines self-pollinated for five generations and three T. plicata clonal lines. One T. standishii clonal line, and one T. standishii x plicata clone were also investigated during the program. A total of 16 histological and anatomical characteristics were studied in more than 750 samples, and more than 270 foliar samples were analyzed for 60 chemical and nutritional compounds. Almost one million transcriptomic sequences in four individually assembled reference transcriptomes were examined during the program. The results of the project support the variability in the resistance to D. thujina in T. plicata, as well as the higher resistance to the pathogen in plants originating from cooler and wetter environments. The data collected also depicted the existence of age-related resistance in T. plicata, and confirmed the full resistance to the disease in T. standishii. Western redcedar plants resistant and susceptible to D. thujina showed constitutive differences at the phenotypic and gene expression levels. Resistant T. plicata seedlings had thicker cuticles, constitutively higher concentrations of sabinene, alpha-thujene, and higher levels of expression of NBS-LRR disease resistance proteins. Resistant clones of T. plicata and T. standishii had higher expression levels of bark storage proteins and of dirigent proteins. Plants from all ages, species and resistance classes studied that were infected with D. thujina showed the accumulation of aluminum in the foliage, and increased levels of sequences involved in cell wall reinforcement. Additional responses to D. thujina infection in T. plicata seedlings included the downregulation of some secondary metabolic pathways, whereas pathogenesis-related proteins were upregulated in clonal lines of T. plicata. The comprehensive approach used here to study the Thuja sp. - D. thujina pathosystem could be applied to other compatible plant-pathogen interactions. / Graduate / 2020-08-31

Invasive exotic species threaten the biodiversity and function of native ecosystems. Existing models, attempting to predict and control successful invaders, often emphasize isolated stages of in their life history and fail to formalize interactions between exotic species and recipient environments. In order to elucidate key mechanisms in the success of select invaders, I investigated the role of dispersal, establishment, proliferation, and persistence in their threat to natural areas. Focusing on Riding Mountain National Park, Manitoba, Canada, I integrated the native climatic range and biological traits of 251 exotic vascular plants reported inside and outside the park. Based on their climatic range in Europe, 155 among 174 exotic plant species absent from the Park were predicted to establish within its boundaries; among these, 40 clonal perennials were considered the highest threat to the Park’s biodiversity. Focusing on smooth brome (Bromus inermis Leyss.), a Eurasian perennial, threatening the structure and function of native prairies throughout the Great Plains, I extended my research to investigate the role of dispersal, establishment, proliferation, and persistence in characterizing its threat to the endemic diversity of northern fescue prairies, protected within Riding Mountain National Park. Patterns of smooth brome invasions were contingent on the type of propagules dispersed. The shallow dispersal gradient of individual florets combined with the steeper gradient of panicles and spikelets suggested that smooth brome is capable of simultaneously invading along dense fronts as well as by establishing isolated foci. While low correlations between the number of dispersed seeds and their recruitment suggested post-dispersal transport, seedling establishment remained contingent on prairie diversity. Seedling biomass increased with declining plant diversity, however, its impact depended on the availability of soil nitrogen. As a result, disturbed areas, preserving the root function of native plants, resisted smooth brome establishment. Even though low nitrogen contributed to a decline in seedling biomass, physiological integration between ramets facilitated their vegetative proliferation in low resource environments. Despite its rapid establishment and proliferation, smooth brome productivity declined at the center of invading clones. Although field and greenhouse observations failed to implicate soilborne pathogens, reasons for the observed decline remain unresolved. My research demonstrates that while Riding Mountain National Park and other natural areas in western Canada will continue to be impacted by exotic plants, integrating key stages in their life history provides an important conceptual framework in predicting their threat to natural areas and prioritizing management. / October 2008

No description available.

Invasive exotic species threaten the biodiversity and function of native ecosystems. Existing models, attempting to predict and control successful invaders, often emphasize isolated stages of in their life history and fail to formalize interactions between exotic species and recipient environments. In order to elucidate key mechanisms in the success of select invaders, I investigated the role of dispersal, establishment, proliferation, and persistence in their threat to natural areas. Focusing on Riding Mountain National Park, Manitoba, Canada, I integrated the native climatic range and biological traits of 251 exotic vascular plants reported inside and outside the park. Based on their climatic range in Europe, 155 among 174 exotic plant species absent from the Park were predicted to establish within its boundaries; among these, 40 clonal perennials were considered the highest threat to the Park’s biodiversity. Focusing on smooth brome (Bromus inermis Leyss.), a Eurasian perennial, threatening the structure and function of native prairies throughout the Great Plains, I extended my research to investigate the role of dispersal, establishment, proliferation, and persistence in characterizing its threat to the endemic diversity of northern fescue prairies, protected within Riding Mountain National Park. Patterns of smooth brome invasions were contingent on the type of propagules dispersed. The shallow dispersal gradient of individual florets combined with the steeper gradient of panicles and spikelets suggested that smooth brome is capable of simultaneously invading along dense fronts as well as by establishing isolated foci. While low correlations between the number of dispersed seeds and their recruitment suggested post-dispersal transport, seedling establishment remained contingent on prairie diversity. Seedling biomass increased with declining plant diversity, however, its impact depended on the availability of soil nitrogen. As a result, disturbed areas, preserving the root function of native plants, resisted smooth brome establishment. Even though low nitrogen contributed to a decline in seedling biomass, physiological integration between ramets facilitated their vegetative proliferation in low resource environments. Despite its rapid establishment and proliferation, smooth brome productivity declined at the center of invading clones. Although field and greenhouse observations failed to implicate soilborne pathogens, reasons for the observed decline remain unresolved. My research demonstrates that while Riding Mountain National Park and other natural areas in western Canada will continue to be impacted by exotic plants, integrating key stages in their life history provides an important conceptual framework in predicting their threat to natural areas and prioritizing management.

Invasive exotic species threaten the biodiversity and function of native ecosystems. Existing models, attempting to predict and control successful invaders, often emphasize isolated stages of in their life history and fail to formalize interactions between exotic species and recipient environments. In order to elucidate key mechanisms in the success of select invaders, I investigated the role of dispersal, establishment, proliferation, and persistence in their threat to natural areas. Focusing on Riding Mountain National Park, Manitoba, Canada, I integrated the native climatic range and biological traits of 251 exotic vascular plants reported inside and outside the park. Based on their climatic range in Europe, 155 among 174 exotic plant species absent from the Park were predicted to establish within its boundaries; among these, 40 clonal perennials were considered the highest threat to the Park’s biodiversity. Focusing on smooth brome (Bromus inermis Leyss.), a Eurasian perennial, threatening the structure and function of native prairies throughout the Great Plains, I extended my research to investigate the role of dispersal, establishment, proliferation, and persistence in characterizing its threat to the endemic diversity of northern fescue prairies, protected within Riding Mountain National Park. Patterns of smooth brome invasions were contingent on the type of propagules dispersed. The shallow dispersal gradient of individual florets combined with the steeper gradient of panicles and spikelets suggested that smooth brome is capable of simultaneously invading along dense fronts as well as by establishing isolated foci. While low correlations between the number of dispersed seeds and their recruitment suggested post-dispersal transport, seedling establishment remained contingent on prairie diversity. Seedling biomass increased with declining plant diversity, however, its impact depended on the availability of soil nitrogen. As a result, disturbed areas, preserving the root function of native plants, resisted smooth brome establishment. Even though low nitrogen contributed to a decline in seedling biomass, physiological integration between ramets facilitated their vegetative proliferation in low resource environments. Despite its rapid establishment and proliferation, smooth brome productivity declined at the center of invading clones. Although field and greenhouse observations failed to implicate soilborne pathogens, reasons for the observed decline remain unresolved. My research demonstrates that while Riding Mountain National Park and other natural areas in western Canada will continue to be impacted by exotic plants, integrating key stages in their life history provides an important conceptual framework in predicting their threat to natural areas and prioritizing management.

Epidermal wound healing requires the coordinated involvement of complex cellular and biochemical processes. In the case of epidermal wounds associated with burns, the healing process may be less than optimal and may take a significant amount of time, possibly resulting in infection and scarring. An innovative method to assist in the repair of the epidermis (the outer layer of skin) is to use an aerosolised apparatus. This method involves taking skin cells from an area of the patient's undamaged skin, culturing the cells in a laboratory, encouraging them to rapidly proliferate, then harvesting and separating the cells from each other. The cells are then sprayed onto the wound surface. We investigate this novel treatment strategy for the healing of epidermal wounds, such as burns. In particular, we model the application of viable cell colonies to the exposed surface of the wound with the intent of identifying key factors that govern the healing process. Details of the evolution of the colony structure are explored in this two-dimensional model of the wound site, including the effect of varying the initial population cluster size and the initial distribution of cell types with different proliferative capacities. During injury, holoclones (which are thought to be stem cells) have a large proliferative capacity while paraclones (which are thought to be transient amplifying cells) have a more limited proliferative capacity. The model predicts the coverage over time for cells that are initially sprayed onto a wound. A detailed analysis of the underlying mathematical models yields novel mathematical results as well as insight into phenomena of healing processes under investigation. Two one-dimensional systems that are simplifications of the full model are investigated. These models are significant extensions of Fisher's equation and incorporate the mixed clonal population of quiescent and active cells. In the first model, an active cell type migrates and proliferates into the wound and undergoes a transition to a quiescent cell type that neither migrates nor proliferates. The analysis yields the identification of the key parameter constraints on the speed of the healing front of the cells on this model and hence the rate of healing of epidermal wounds. Approximations for the maximum cell densities are also obtained, including conditions for a less than optimal final state. The second model involves two active cell types with different proliferative capacity and a quiescent cell type. This model exhibits two distinct behaviours: either both cell types coexist or one of them dies out as the wound healing progresses leaving the other cell type to fill the wound space. Conditions for coexistence are explored.

This work is concerned with the characterization of mesenchymal stem cells (MSC) specifically from bone marrow samples derived from patients with osteoarthritis (OA). The multilineage potential of mesenchymal stem cells as well as their ease of exvivo expansion makes these cells an attractive therapeutic tool for applications such as autologous transplantation and tissue engineering. Bone marrow is considered a source of MSC. However, there is a general assumption that the occurrence of MSCs and their activity in bone marrow diminishes with age and disease. This prompted us to isolate and identify multipotential and self-renewing cells from patients with the degenerative disease osteoarthritis, with the view of using these cells for autologous cell therapies. It is therefore of great potential benefit to investigate the isolation and characterization of stem cell/progenitors from bone marrow samples of patients with osteoarthritis in greater detail. We employed a single cell clone culture method in order to develop clonal cell populations from three bone marrow samples and characterized them based on their proliferation and differentiation capabilities. The clonal populations were grouped into fast-growing and slow-growing clones based on their proliferation rates. The fastgrowing clones displayed 20-30% greater proliferation rate than the slow-growing clones. The study also revealed that the proliferation rates were directly proportional to their differentiation capacities. Most of the fast-growing clones were found to be tripotential for osteogenic, chondrogenic and adipogenic lineages, whereas the slow growing clones were either uni or bipotential. Flow cytometry analysis for the phenotype determination using putative MSC surface markers did not reveal any difference between the two clonal populations indicating a need for further molecular studies. Two approaches were employed to further investigate the molecular processes involved in the existence of such varying populations. In the first method gene expression studies were performed between the fast-growing (n=3) and slow-growing (n=3) clonal populations to identify potential genetic markers associated with cell 'sternness' using the Stem Cell RT2 ProfilerTM PCR Array comprising a series of 84 genes related to stem cell pathways. Ten genes were identified to be commonly and significantly over represented in the fast-growing stem cell clones when compared to slow-growing clones. This included expression of transcripts beyond MSC lineage specification such as SOX2, NOTCH1 and FOXA2 which signified that stem cell maintenance requires a coordinated regulation by multiple signalling pathways. The second study involved an extensive protein expression profiling of the fast growing (n=2) and slow growing (n=2) clonal populations using off-line Two Dimensional Liquid Chromatography (2D-LC)/Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry (MS). A total of 67 proteins were identified, of which 11 were expressed at significantly different levels between the subpopulations. Protein ontology revealed these proteins to be associated with cellular organization, cytokinesis, signal transduction, energy pathways and cell stress response. Of particular interest was the differential presentation of the proteins calmodulin, tropomyosin and caldesmon between fast- and slow-growing clones. Based on their reported roles in the regulation of cell proliferation and maintenance of cell integrity, we draw an association between their expression and the altered status in which the subpopulations exist. Based on our observations, these proteins may be prospective molecular markers to distinguish between the fast-growing and slow-growing subpopulations. In summary, this study demonstrated the existence of potential stem cells of therapeutic importance in spite of a supposedly smaller stem cell compartment in patients with osteoarthritis. Furthermore, the differentially expressed genes between the sub-populations highlight the 'sternness' of the potential clones, an observation supported by the expression of proteins which act as effective modulators in the maintenance of cell integrity and cell cycle regulation. This study provides a basis for more detailed investigations in search of selective cell surface markers