O presente trabalho tem os seguintes objetivos: propor uma sistemática para o estudo e a interpretação da estabilidade e adaptabilidade fenotípica, através de duas técnicas de análise multiway (PARAFAC e Tucker3); propor a construção de um gráfico, denominado de Triplot, que possibilita avaliar as relç]oesoes entre os 3 modos (genótipos, locais e anos); implementar uma rotina computacional para a análise de dados, segundo os modelos multiway; implementar uma rotina computacional para a construção do Triplot. Os dados a serem uti- lizados são relativos a experimentos com 13 genótipos de feijão que foram conduzidos em 9 ex- perimentos distintos constituídos pelos anos agrícolas de 2000/2001, 2001/2002 e 2005/2006, pelos municípios de Dourados e Aquidauana, sendo que os experimentos foram instalados na época das águas (Dourados)e também na época da seca (Dourados e Aquidauana). Cada local é constituído de município e uma época de instalação. Os resultados indicaram que o gráfico triplot e joint plot, facilitam o entendimento da interação tripla e traz ao pesquisador informações mais reais sobre a interação tripla, do que a modelagem AMMI de duas entradas; o gráfico triplot, ajuda a identificar genótipos, locais e anos estáveis, dentro de um grande grupo de genótipos, locais e anos; de uma maneira geral recomenda-se, utilizar o triplot e o joint plot juntos, para obter melhores interpretações dos resultados; dentre os genótipos estudados, o genótipo 6 é o que menos contribui para a interação e o os genótipos 12, 9 e 5 são os que mais contribuem para a interação. / The present work has the following objectives: to propose a systematics for the study and the interpretation of the phenotypic stability and adaptability, through several multiway models (PARAFAC and Tucker3); to propose a graphic, called of Triplot, that it makes possible to evaluate the relations between the 3 ways (genotypes, locations and years); to implement a computational routine for the data analysis, according multiway models; to implement a computational routine for the construction of Triplot. The used data are relative the experiments with 13 genotypes of beans that had been lead in 9 experimental distinct ones constituted by agricultural years of 2000/2001, 2001/2002 and 2005/2006, by Dourados and Aquidauana cities, where the experiments had been installed at the time of waters (Dourados) and also at the time of dries (Dourados and Aquidauana). Each location is constituted of city and time of installation. The results indicated that the graphic triplot and joint plot, facilitate the agreement of triple interaction and bring to the researcher more real information about triple interaction, of what AMMI model of two way; the graphic triplot, helps to identify stabels genotypes, locations and years, inside of a great group of genotypes, location and years; in a general recommend to use triplot and joint plot together, to get better interpretations of the results; the genotype 6 is what less contributes for the triple interaction and genotypes 12, 9 and 5 are the that more contribute for the interaction.

Saccharomyces cerevisiae é o organismo de escolha para produção de etanol combustível, apresentando-se adaptadas ao ambiente hostil das dornas de fermentação. Adaptações como floculação, formação de espuma e de biofilme são características fenotípicas indesejáveis ao processo brasileiro. Por isso, a pronta identificação destas características faz-se necessária no intuito de minimizar perdas de rendimento e diminuir o custo da produção. Sabe-se que proteínas codificadas pela família de genes FLO estão relacionadas a estes fenótipos, os quais são regulados pela proteína Flo8p, a qual possui papel fundamental na apresentação destas características fenotípicas indesejáveis. Desta forma, o presente trabalho teve como objetivo analisar o fenótipo e o comportamento metabólico, relacionando-os com a regulação via Flo8p. Os resultados obtidos revelam o papel fundamental de Flo8p na regulação positiva destes fenótipos em linhagem isolada do ambiente industrial (FT02). Verificou-se uma forte tendência da participação do gene FLO11 na formação de espuma, via Flo8p. Além disso, através da deleção do FLO8 da linhagem FT02, observou-se a influência positiva de Flo8p na floculação e hidrofobicidade celular, filamentação e poder invasivo. / Saccharomyces cerevisiae is the chosen organism for fuel ethanol production, presenting adapted to the hostile environment of the fermentation vats. Adaptations like flocculation, d foam formation and biofilm are undesirable phenotypes for the Brazilian process. Hence, the early identification of these characteristics is necessary to minimize yield losses and lower the production cost. Proteins codified by FLO gene family are related with these phenotypes, which are positively regulated by Flo8p protein, showing fundamental role in the expression of the undesirable phenotypes. So, the present work aimed to analyze the phenotypes and the metabolic behavior of an industrial strain isolated directly from Brazilian ethanol production process (FT02), relating them with the Flo8p regulation. Our results showed that the Flo8p plays a fundamental role in the regulation of these characteristics in the industrial strain FT02. We also verified a strong tendency of the FLO11 gene participation in the foam formation, by Flo8p. Moreover, by FLO8 deletion in FT02 strain, we could identify the positive influences of the Flo8p in flocculation and cellular hydrophobicity, filamentation and invasive growth.

Polyploidy is widely acknowledged as a widespread mechanism in the evolution and speciation of the majority of flowering plants. Allopolyploid forms through interspecific hybridization and whole genome duplication. While allopolyploids may display increased vigor relative to their progenitors, they can also face challenges to fertility following hybridization. Genetic changes in allopolyploids result from recombination between the hybridized subgenomes, which can influence phenotype and ultimately determine fitness of future generations. To study dynamic changes that follow allopolyploid formation, Brassica napus lineages were derived by hybridizing Brassica oleracea and Brassica rapa. Two lineages of B. napus were analyzed for genetic and phenotypic changes in the S2, S7, and S12 generations. Although these lineages were genetically identical at the time of hybridization, divergence was apparent by the S2 generation. There was a significant increase in sequence loss across generations within both lineages. Four of six generations from both lineages displayed no significant differences to each other in sequence loss relative to the parental generation. In both lineages, there was a bias towards losing sequences from the B. olereacea subgenome. Some individual plants showed novel phenotypes; however, there was no correlation between the examined genetic changes and selected phenotypes.

WRINKLED1 (AtWRI1) is a key transcription factor in the regulation of plant oil synthesis in seed and non-seed tissues. The structural features of WRI1 important for its function are not well understood. Comparison of WRI1 orthologs across many diverse plant species revealed a conserved 9 bp exon encoding the amino acids “VYL”. Site-directed mutagenesis of amino acids within the ‘VYL’ exon of AtWRI1 failed to restore the full oil content of wri1-1 seeds, providing direct evidence for an essential role of this small exon in AtWRI1 function. Arabidopsis WRI1 is predicted to have three alternative splice forms. To understand expression of these splice forms we performed RNASeq of Arabidopsis developing seeds and queried other EST and RNASeq databases from several tissues and plant species. In all cases, only one splice form was detected and VYL was observed in transcripts of all WRI1 orthologs investigated. We also characterized a phylogenetically distant WRI1 ortholog (EgWRI1) as an example of a non-seed isoform that is highly expressed in the mesocarp tissue of oil palm. The C-terminal region of EgWRI1 is over 90 amino acids shorter than AtWRI1 and has surprisingly low sequence conservation. Nevertheless, the EgWRI1 protein can restore multiple phenotypes of the Arabidopsis wri1-1 loss-of-function mutant, including reduced seed oil, the “wrinkled” seed coat, reduced seed germination, and impaired seedling establishment. Taken together, this study provides an example of combining phylogenetic analysis with mutagenesis, deep-sequencing technology and computational analysis to examine key elements of the structure and function of the WRI1 plant transcription factor.

Alors que la rhinite a un fort impact sur la santé publique, chez l’adulte, il n’existe pas de définition standardisée de la rhinite dans les études épidémiologiques. De plus, les facteurs environnementaux de la rhinite sont mal connus et, en particulier, il existe très peu d'études sur les effets à long terme de la pollution atmosphérique sur la rhinite chez l'adulte. Pour combler ces lacunes, nous avons utilisé les données de deux études épidémiologiques multicentriques européennes ayant des données détaillées sur la santé respiratoire et d'exposition annuelle individuelle à la pollution atmosphérique. Nos résultats ont montré que pour mieux caractériser la rhinite, il faut considérer l’ensemble des caractéristiques des symptômes nasaux, les comorbidités et la sensibilisation allergique, et ne pas limiter la maladie à une question ou à un test de sensibilisation allergique. Nous n'avons trouvé aucune association entre la pollution atmosphérique à long terme et l'incidence de la rhinite, mais nous avons montré que l'exposition à long terme à la pollution était associée à une augmentation de la sévérité de la rhinite, soulignant le besoin de contrôler les niveaux de pollution atmosphérique. / Whereas rhinitis has an important public health impact, in adults there is no standardized definition of rhinitis in epidemiological studies. Furthermore, environmental factors of rhinitis are barely known, and in particular, there are very few studies on the effects of long-term exposure to air pollution on rhinitis in adults. To fill these gaps, we used data from two European multicentre epidemiological studies with extensive data on respiratory health and individual estimated exposures to long-term air pollution. Our findings showed that to better characterize rhinitis, one need to consider together all the characteristics of the nasal symptoms, the comorbidities and the allergic sensitization, and not to restrict the disease to one question or one allergic sensitization test. We found no association between long-term air pollution and incidence of rhinitis, but we showed that long-term exposure to air pollution is associated to an increased severity of rhinitis, emphasising that air pollution needs to be controlled.

In effort to understand and isolate genes that are associated with protein secretion, the Lin-Cereghino laboratory at University of the Pacific created mutant strains of Pichia pastoris using the restriction enzyme mediated integration method. The mutants exhibited an unusual ability to supersecrete beta-galactosidase, due to the effects of a randomly disrupted gene by pREMI-Z. To learn more about the novel effects of the gene disruption, nine beta-galactosidase supersecreters ( bgs ) have been characterized for their phenotypes such as growth rate, cell wall integrity, and ability to produce and secrete various types of recombinant proteins. The mutants showed various population doubling times, which ranged from 1.7 to 2.4 hours. Generally, the mutants with severely diminished growth rates had much lower secretion of the reporter proteins. The mutants also showed different levels of cell wall (osmotic) defect, indicated by moderate to severe leakage of alkaline phosphatase from the vacuole. It was revealed that the cell wall defect was not necessarily associated with increased protein secretion, which suggests that the cell wall may not be a limiting barrier for the secretion of most reporter proteins. The result of the reporter study suggests that the secretion phenotypes of bgs mutants were protein specific and likely to be dependent upon the structure of the secreted protein rather than the size.

Updated: Current version includes the name of Supervisor, and Co-supervisor. Error corrected in preliminary pages. / Background: Emerging evidence suggests that there is both within-disorder heterogeneity and across-disorder overlap in the clinical presentation of children with ASD, ADHD, and OCD. Two prevalent dimensional phenotypes in children with these NDDs that warrant close attention, and are suitable for cross-disorder investigation, are internalizing and externalizing problems. Objectives: The current study uses a data-driven, diagnosis-agnostic approach to identify homogenous clusters that describe behavioural profiles of internalizing and externalizing problems within and across ASD, ADHD, and OCD. Methods: Data on 1565 children (M = 10.76 years) were drawn from the Province of Ontario Neurodevelopmental Disorder (POND) Network. Non-hierarchical clustering approaches were used to empirically derive, distinct behavioural profiles of internalizing and externalizing problems indexed by the Child Behavior Checklist (CBCL). Empirically derived groups were characterized using measures of adaptive functioning indexed by the Adaptive Behaviour Assessment (ABAS-II), and interpreted in relation to original diagnoses. Results: Cluster analyses identified four distinct behavioural profiles that cut across all diagnostic groups: High Internalizing Externalizing (HIE; 15%), High Externalizing (HE; 21%), Low Internalizing Externalizing (LIE; 38%), and Low Externalizing (LE; 26%). Derived clusters had variable levels of adaptive behaviours and reflected different behavioural profiles than the ones defined by the original diagnostic category groups of ASD, ADHD, and OCD. Conclusion: Data-driven, diagnosis-agnostic approaches can inform our understanding of the between and within phenotypic heterogeneity seen in ASD, ADHD, and OCD. Empirical ways of classifying children with homogeneous behavioural profiles may complement existing diagnostic models in our efforts to develop cross-disorder, more personalized interventions for children with neurodevelopmental disorders. / Thesis / Master of Science (MSc) / ASD, ADHD, and OCD are heterogenous neurodevelopmental disorders (NDDs) with some overlapping clinical characteristics and etiological factors. Internalizing and externalizing behavioural problems persist across these three NDDs, and in this study, are used to identify unique behavioural profiles. Study findings reveal four groups with distinct behavioural profiles of internalizing and externalizing problems that are not identified by the original diagnostic groups. This empirical way of classifying children with similar behavioural profiles can be used in combination with diagnostic labels to enhance transdiagnostic interventions that can be tailored to each child’s needs.

INTRODUCTION: Abnormal ventilation is the functional consequence of airway obstruction. In patients with severe asthma, ventilation patterns visualized by magnetic resonance imaging (MRI) exhibit significant inter-patient heterogeneity. Therefore, our objectives were to identify MRI ventilation phenotypes of severe asthma using an unsupervised clustering approach and examine their associated demographic, clinical, physiologic, and inflammatory characteristics. METHODS: This retrospective analysis included 58 adults with severe asthma who underwent hyperpolarized 129Xe ventilation MRI. Nineteen quantitative variables were extracted from ventilation MRI (including ventilation defect percent (VDP), ventilation defect size, and ventilation texture features) and transformed to principal components for hierarchical clustering. Differences in demographics, clinical characteristics, spirometry, inflammatory biomarkers, and computed tomography (CT) measurements between phenotypes were evaluated using one-way ANOVA or Kruskal-Wallis tests. RESULTS: Three ventilation phenotypes of severe asthma were identified. They were significantly different with respect to their age, prevalence of obesity, spirometry, sputum neutrophil percent, sputum cytokines (interleukin-4, interleukin-6, interleukin-15, B-cell activating factor), total lung capacity, CT air-trapping, and CT mucus score (all p<0.05). They were not different with respect to their asthma control or medication requirement, and ~75% of each phenotype reported uncontrolled asthma (ACQ-5≥1.5). Phenotype 1 had normal ventilation (VDP=1.7±0.9%) and predominantly consisted of young, obese females (88% female, 41±11 years old, 63% obese). They had normal-to-moderately reduced FEV1 (80±15%pred), normal post-bronchodilator FEV1/FVC, and reduced total lung capacity (85%pred [57-108]). 25% had intraluminal inflammation (all eosinophilic) and their sputum interleukin-4 levels were elevated. Phenotype 2 had markedly abnormal ventilation (VDP=6.2±3.8%) and was older than Phenotype 1, but also predominantly consisted of obese females (63% female, 54±13 years old, 59% obese). They had mildly-to-severely reduced FEV1 (61±17%pred) and partially reversible obstructive spirometry (72%, post-bronchodilator FEV1/FVC<0.70). 50% had intraluminal inflammation (28% eosinophilic/13% neutrophilic/9% mixed-granulocytic) and their sputum interleukin-6 levels were elevated. Phenotype 3 had severely abnormal ventilation (VDP=24.8±10.2%) and was also older than Phenotype 1 but was gender-balanced and not obese (50% female, 56±12 years old, 11% obese). They had moderately-to-very severely reduced FEV1 (41±12%pred) and partially reversible obstructive spirometry (89%, post-bronchodilator FEV1/FVC<0.70). 73% had intraluminal inflammation (39% eosinophilic/17% neutrophilic/17% mixed-granulocytic) and their sputum interleukin-15 and B-cell activating factor levels were elevated. They had the highest burden of gas-trapping and mucus on CT. CONCLUSION: Three distinct MRI ventilation phenotypes of severe asthma were identified through unbiased analysis, all of which reported uncontrolled asthma. The discordance in ventilation between phenotypes, and their characteristics, suggest different mechanisms that may be driving severe asthma. / Thesis / Master of Science (MSc) / Severe asthma is an airways disease that is characterized by inflamed, twitchy and obstructed airways. There is remarkable clinical heterogeneity between asthma patients due to the various mechanisms of disease. Abnormal ventilation is the functional consequence of abnormal airway pathology in asthma, which can be directly visualized by hyperpolarized 129Xe magnetic resonance imaging (MRI). Each ventilation pattern is unique and there is significant inter-patient variability. Thus, the goal of the thesis was to extract quantitative information from the 129Xe MRI ventilation patterns of patients with severe asthma, identify novel ventilation phenotypes, and determine their clinical relevance. An unsupervised machine learning approach using quantitative ventilation MRI features identified three unique, clinically relevant ventilation phenotypes of severe asthma with distinct clinical, physiological, and biological characteristics. The discordance in ventilation between phenotypes, and their characteristics, suggest different mechanisms that may be driving severe asthma.

BackgroundChronic pain affects 70% of individuals with spinal cord injury (SCI), and up to 53% of individuals with SCI experience neuropathic pain that leads to declines in their health and quality of life. Chronic pain can be categorized by phenotypes as neuropathic or nociceptive. Neuropathic pain arises due to nervous system dysfunction, and nociceptive pain originates in the musculoskeletal system in response to noxious stimuli. Moderate-to-vigorous intensity physical activity (MVPA) and virtual reality (VR) have been effective interventions for pain management, however, the circumstances in which these interventions are efficacious for individuals with SCI are not well understood. The objectives of this study were to: 1) investigate the relationships between MVPA and pain phenotypes, 2) investigate the relationships between VR and pain phenotypes, and 3) explore a practical method of measuring MVPA in individuals with SCI. MethodsFor the first objective, one week of physical activity data was collected in the community from 17 individuals with SCI, and daily pain ratings were obtained. Spearman’s correlation analyses were performed to analyze the relationships between MVPA levels and pain intensity. For the second objective, participants engaged in two bouts of immersive VR, and pain intensity was assessed at baseline and following each bout. Friedman analyses of variance tests were conducted to identify changes in pain intensity across VR exposure time in each pain phenotype. For the third objective, a comparison of summary measures for raw physical activity data was conducted. Spearman’s correlation was used to analyze the relationship between ActiGraph Activity Counts and Monitor Independent Movement Summary (MIMS) Units. ResultsIn neuropathic pain, MVPA and pain intensity are negatively correlated. In nociceptive pain, there was no significant correlation between MVPA and pain intensity. A decrease in neuropathic pain was achieved from VR, however no change in nociceptive pain intensity was observed. Total active minutes summarized from raw accelerometer data by Activity Counts and MIMS Units values were strongly correlated, but minutes of MVPA calculated by Activity Counts were only moderately correlated to minutes of MVPA calculated through personalized thresholds with MIMS Units. The responses to VR for each pain phenotype were different. A decrease in neuropathic pain was achieved within a five-minute bout, and this decrease was maintained at the end of both VR sessions, whereas no change in nociceptive pain level was observed. Lastly, Activity Counts and MIMS Units values were strongly correlated (r = 0.94, p < 0.001). Minutes of MVPA calculated by Activity Counts were moderately correlated (r = 0.44, p < 0.001) to minutes of MVPA calculated through personalized thresholds with MIMS Units. ConclusionPain in each phenotype responded differently to both motor activities that were evaluated. These results may assist in the development of personalized pain management approaches through targeted combinations of interventions. By utilizing data from free-living individuals with SCI in the community, the process of personalized thresholds is translatable and describes MVPA levels more accurately and comprehensively than what can be collected in a laboratory setting. This approach can be implemented to optimize MVPA recommendations for individuals with SCI who experience chronic pain. / Biomedical Sciences

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