From genotype to phenotype : diversity and population structure of Flavobacterium psychrophilum

Flavobacterium psychrophilum is the etiological agent of bacterial cold-water disease (BCWD) causing high fish mortalities and significant economic losses to the freshwater salmonid aquaculture industry around the world. Today BCWD outbreaks are mainly treated with environmentally hazardous antimicrobial agents and alternative preventative measures are urgently needed in order to ensure the well-being of animals and the sustainability of aquaculture. The diversity of pathogenic bacteria challenges the development of universal control strategies and in many cases the pathogen population structure, i.e. the total genetic diversity of the species must be taken into account. This work integrates the tools of modern molecular biology and conventional phenotypic microbiology to gain knowledge about the diversity and population structure of F. psychrophilum. The present work includes genetic characterization of a large collection of isolates collected from diverse origins and years, from aquaculture in a whole region including different countries, and provides the first international validation of a universal multilocus sequence typing (MLST) approach for unambiguous genetic typing of F. psychrophilum. Population structure analyses showed that the global F. psychrophilum population is subdivided into pathogenic species-specific clones, of which one particular genetic lineage, clonal complex CC-ST2, has been responsible for the majority of BCWD outbreaks in rainbow trout (Oncorhynchus mykiss) in European aquaculture facilities over several decades. Genotypic and phenotypic population heterogeneity affecting antimicrobial resistance in F. psychrophilum within BCWD outbreaks was discovered. Specific genotypes were associated with severe infections in farmed rainbow trout and Atlantic salmon (Salmo salar), and in addition to high adherence, antimicrobial resistance was strongly associated with outbreak strains. The study brought additional support for the hypothesis of an epidemic F. psychrophilum population structure, where recombination is an important force for the generation and maintenance of genetic diversity, and a significant contribution towards mapping the genetic diversity of this important fish pathogen. Evidence indicating dissemination of virulent strains with commercial movement of fish and fish products was strengthened.

Follicular helper T cell in immunity and autoimmunity

The traditional concept that effector T helper (Th) responses are mediated by Th1/Th2 cell subtypes has been broadened by the recent demonstration of two new effector T helper cells, the IL-17 producing cells (Th17) and the follicular helper T cells (Tfh). These new subsets have many features in common, such as the ability to produce IL-21 and to express the IL-23 receptor (IL23R), the inducible co-stimulatory molecule ICOS, and the transcription factor c-Maf, all of them essential for expansion and establishment of the final pool of both subsets. Tfh cells differ from Th17 by their ability to home to B cell areas in secondary lymphoid tissue through interactions mediated by the chemokine receptor CXCR5 and its ligand CXCL13. These CXCR5+ CD4+ T cells are considered an effector T cell type specialized in B cell help, with a transcriptional profile distinct from Th1 and Th2 cells. The role of Tfh cells and its primary product, IL-21, on B-cell activation and differentiation is essential for humoral immunity against infectious agents. However, when deregulated, Tfh cells could represent an important mechanism contributing to exacerbated humoral response and autoantibody production in autoimmune diseases. This review highlights the importance of Tfh cells by focusing on their biology and differentiation processes in the context of normal immune response to infectious microorganisms and their role in the pathogenesis of autoimmune diseases.

The effect of calculators on the numerical problem-solving skills and attitudes of primary students towards mathematics /

The purpose of this study was to determine the effect that calculators have on the attitudes and numerical problem-solving skills of primary students. The sample used for this research was one of convenience. The sample consisted of two grade 3 classes within the York Region District School Board. The students in the experimental group used calculators for this problem-solving unit. The students in the control group completed the same numerical problem-solving unit without the use of calculators. The pretest-posttest control group design was used for this study. All students involved in this study completed a computational pretest and an attitude pretest. At the end of the study, the students completed a computational posttest. Five students from the experimental group and five students from the control group received their posttests in the form of a taped interview. At the end of the unit, all students completed the attitude scale that they had received before the numerical problem-solving unit once again. Data for qualitative analysis included anecdotal observations, journal entries, and transcribed interviews. The constant comparative method was used to analyze the qualitative data. A t test was also performed on the data to determine whether there were changes in test and attitude scores between the control and experimental group. Overall, the findings of this study support the hypothesis that calculators improve the attitudes of primary students toward mathematics. Also, there is some evidence to suggest that calculators improve the computational skills of grade 3 students.

Coevolution of Heliconius spp. and Passiflora spp. : a phylogenetic comparison

Although a substantial amount of research has been done on all aspects ofHeliconius biology and their ecological interactions with Passiflora, there has not hitherto been a phylogenetic examination of this association for coevolution. To test the HeliconiuslPassilfora association for coevolutionary congruence, phylogenies for each group were established and compared. The phylogeny for 14 species ofHeliconiinae from Costa Rica was based on combined sequence data from rRNA ITS 2 and partial EF-1a gene regions. For the Passifloraceae, 17 host plant species were utilized to establish a phylogeny based on tRNALeucine and ITS 1/5.8S1 ITS 2 sequence data. The phylogenies for both groups were largely in agreement with current classification (for Passifloraceae) and previously established phylogenies. Associations with the large subgenera Passiflora and Decaloba correspond with the two major Advanced Radiation groups in Heliconius. Although strict congruence above subgenus level was not observed, broad scale congruence was evident. One main host shift as well as other possible explanations for lack of strict congruence are suggested.

Size regulation of double and half embryos in the mice musculus

Genetic chimeras made by aggregating early mouse embryos have many uses in developmental biology and have also provided insights into embryonic growth regulation. There is an indication that the embryo can regulate for an increase in size because although aggregation chimeras are twice as big as normal embryos when made, they are born of normal size. Upward regula..... tion of size reduced embryos is also possible. Half embryos made by the isolation or destruction of one of the blastomeres of a 2-cell embryo are also born of normal size. Little is known about the timing or the mechanism of this size regulation. In this study, the timing of size regulation in double and half embryos was clearly established by comparison of cell numbers derived from serial reconstruction of light microscope sections of control and experimental embryos. It was shown that size regulation in double embryos occurred around 6dl6h and in half embryos by 7dOh. Size regulation occurred in all tissues at the same time indicating a single control mechanism for the entire embryo. More detailed examination of the growth of double embryos revealed that size regulation occurred by alteration in cell cycle length~ No excessive cell death was found in double embryos compared to the controls and continuous labelling with [3H] thymidine showed no large non-dividing cell population in double embryos. However, a comparison of the mitotic index of double and control embryos after colcemid treatment, revealed a large difference between the two around 5dl6h to 6d16h. During this period, control embryos underwent a proliferative burst not shown by the double embryos. The mechanism for cell cycle control is not clear; it may be intrinsic to the embryo or determined by the uterine environment. Evidence was found suggesting that differentiation in the postimplantation embryo was cell number dependent. The timing of differentiative events was examined in half, double and control embryos. Proamnion formation, which occurs prior to size regulation, occurs at the same cell number but at different times in the three groups of embryos. However mesoderm which appears after size regulation was seen at the same time in all grollps of embryos.

Motivational differences in science course enrolment shown by males and females in grades 9 through OAC

A sample of 1,345 students enrolled in advanced-level science courses from Grades 9 through OAe was surveyed in order to gain perspective into the existence of motivational differences attributing to science course enrolment by gender. Records of enrolment were examined in order to detect patterns and trends. A questionnaire was devised and piloted. It measured five motivational variables - demographics, science and science-related experiences, science ability and attitudes, impressions about women in science, and importance of science and science-related skills. The students also provided some impressions about the image of scientists. Results of the questionnaire were analyzed for frequency of responses and for significant gender differences using the chi-square. Differences were found to exist in the areas of science anxiety as it relates to testing and oral participation; in motivation generated by the performance of extra-curricular science and science-related activities, and by the classroom environment; in impressions of women in science; in the importance of science skills, and in the area of teacher influence. The study also showed a differential enrolment of females, with an emphasis on biology and chemistry. The males were enrolled in courses of physics and chemistry. The findings lead to numerous suggested strategies and programs for encouraging the participation of females in science education and careers.

Evolutionary synthesis of stochastic gene network models using feature-based search spaces

A feature-based fitness function is applied in a genetic programming system to synthesize stochastic gene regulatory network models whose behaviour is defined by a time course of protein expression levels. Typically, when targeting time series data, the fitness function is based on a sum-of-errors involving the values of the fluctuating signal. While this approach is successful in many instances, its performance can deteriorate in the presence of noise. This thesis explores a fitness measure determined from a set of statistical features characterizing the time series' sequence of values, rather than the actual values themselves. Through a series of experiments involving symbolic regression with added noise and gene regulatory network models based on the stochastic 'if-calculus, it is shown to successfully target oscillating and non-oscillating signals. This practical and versatile fitness function offers an alternate approach, worthy of consideration for use in algorithms that evaluate noisy or stochastic behaviour.

Automatic Structure Generation using Genetic Programming and Fractal Geometry

Three dimensional model design is a well-known and studied field, with numerous real-world applications. However, the manual construction of these models can often be time-consuming to the average user, despite the advantages o ffered through computational advances. This thesis presents an approach to the design of 3D structures using evolutionary computation and L-systems, which involves the automated production of such designs using a strict set of fitness functions. These functions focus on the geometric properties of the models produced, as well as their quantifiable aesthetic value - a topic which has not been widely investigated with respect to 3D models. New extensions to existing aesthetic measures are discussed and implemented in the presented system in order to produce designs which are visually pleasing. The system itself facilitates the construction of models requiring minimal user initialization and no user-based feedback throughout the evolutionary cycle. The genetic programming evolved models are shown to satisfy multiple criteria, conveying a relationship between their assigned aesthetic value and their perceived aesthetic value. Exploration into the applicability and e ffectiveness of a multi-objective approach to the problem is also presented, with a focus on both performance and visual results. Although subjective, these results o er insight into future applications and study in the fi eld of computational aesthetics and automated structure design.

Evolutionary Origin and Maintenance of Sociality in the Small Carpenter Bees

Many arthropods exhibit behaviours precursory to social life, including adult longevity, parental care, nest loyalty and mutual tolerance, yet there are few examples of social behaviour in this phylum. The small carpenter bees, genus Ceratina, provide important insights into the early stages of sociality. I described the biology and social behaviour of five facultatively social species which exhibit all of the preadaptations for successful group living, yet present ecological and behavioural characteristics that seemingly disfavour frequent colony formation. These species are socially polymorphic with both / solitary and social nests collected in sympatry. Social colonies consist of two adult females, one contributing both foraging and reproductive effort and the second which remains at the nest as a passive guard. Cooperative nesting provides no overt reproductive benefits over solitary nesting, although brood survival tends to be greater in social colonies. Three main theories explain cooperation among conspecifics: mutual benefit, kin selection and manipulation. Lifetime reproductive success calculations revealed that mutual benefit does not explain social behaviour in this group as social colonies have lower per capita life time reproductive success than solitary nests. Genetic pedigrees constructed from allozyme data indicate that kin selection might contribute to the maintenance of social nesting -, as social colonies consist of full sisters and thus some indirect fitness benefits are inherently bestowed on subordinate females as a result of remaining to help their dominant sister. These data suggest that the origin of sociality in ceratinines has principal costs and the great ecological success of highly eusociallineages occurred well after social origins. Ecological constraints such as resource limitation, unfavourable weather conditions and parasite pressure have long been considered some of the most important selective pressures for the evolution of sociality. I assessed the fitness consequences of these three ecological factors for reproductive success of solitary and social colonies and found that nest sites were not limiting, and the frequency of social nesting was consistent across brood rearing seasons. Local weather varied between seasons but was not correlated with reproductive success. Severe parasitism resulted in low reproductive success and total nest failure in solitary nests. Social colonies had higher reproductive success and were never extirpated by parasites. I suggest that social nesting represents a form of bet-hedging. The high frequency of solitary nests suggests that this is the optimal strategy when parasite pressure is low. However, social colonies have a selective advantage over solitary nesting females during periods of extreme parasite pressure. Finally, the small carpenter bees are recorded from all continents except Antarctica. I constructed the first molecular phylogeny of ceratinine bees based on four gene regions of selected species covering representatives from all continents and ecological regions. Maximum parsimony and Bayesian Inference tree topology and fossil dating support an African origin followed by an Old World invasion and New World radiation. All known Old World ceratinines form social colonies while New World species are largely solitary; thus geography and phylogenetic inertia are likely predictors of social evolution in this genus. This integrative approach not only describes the behaviour of several previously unknown or little-known Ceratina species, bu~ highlights the fact that this is an important, though previously unrecognized, model for studying evolutionary transitions from solitary to social behaviour.

STUDYING ABERRANT METHYLATION AND miRNA PROFILING FOR THE DETECTION OF LUNG CANCER BIOMARKERS

Lung cancer is a major chronic disease responsible for the highest mortality rate, among other types of cancer, and represents 29% of all deaths in Canada. The clinical diagnosis of lung carcinoma still requires a standard diagnostic approach, as there are no symptoms in its early stage. Therefore, it is usually diagnosed at a later stage, when the survival rate is low. With the recent advancement in molecular biology and biotechnology, a molecular biomarker approach for the diagnosis of early lung cancer seems to be a potential option. In this study, we aimed to investigate and standardize a promising Lung ,Cancer Biomarker by studying the aberrant methylation of two tumour suppressor genes, namely RASSFIA and RAR-B, and the miRNA profiling of four . commonly deregulated miRNA (miR-199a-3p, miR-182, miR-lOO and miR-221). Four lung cancer cell lines were used (two SCLC and two NSCLC), with comparisons being made with normal lung cell lines. Our results, we found that none of these genes were methylated. We then evaluated TP53, and found the promoter of this gene to be methylated in the cancer cell lines, as compared to the normal cell lines, indicating gene inactivation. We carried out miRNA profiling of the cancer cell lines and reported that 80 miRNAs are deregulated in lung cancer cell lines as compared to the normal cell lines. Our study was the first of its kind to indicate that hsa-mir-4301, hsa-mir-4707-5p and hsa-mir-4497 (newly discovered miRNAs) are deregulated in lung cancer cell lines. We also investigated miR-199a-3p, mir-lOO and miR-182, and found that miR-199a -3p and mir-l00 were down-regulated in cancer lines, whereas miR-182 was up-regulated in the cancer cell lines. In the final part of the study we observed that mir-221 could be a putative biomarker to distinguish between the two types of lung cancer because it was down-regulated in SCLC, and up-regulated in the NSCLC cell lines. In conclusion, we found four miRNA molecular biomarkers that possibly could be used in the early diagnosis of the lung cancer. More studies are still required with larger numbers of samples to effectively establish these as molecular biomarkers for the diagnosis of lung cancer

DNA-Directed DNA Polymerases Evolve From Reverse Transcriptase

Scientists have been debating for decades the origin of life on earth. A number of hypotheses were proposed as to what emerged first RNA or DNA; with most scientists are in favour of the "RNA World" hypothesis. Assuming RNA emerged first, it fellow that the RNA polymerases would've appeared before DNA polymerases. Using recombinant DNA technology and bioinformatics we undertook this study to explore the relationship between RNA polymerases, reverse transcriptase and DNA polymerases. The working hypothesis is that DNA polymerases evolved from reverse transcriptase and the latter evolved from RNA polymerases. If this hypothesis is correct then one would expect to find various ancient DNA polymerases with varying level of reverse transcriptase activity. In the first phase of this research project multiple sequence alignments were made on the protein sequence of 32 prokaryotic DNA-directed DNA polymerases originating from 11 prokaryotic families against 3 viral reverse transcriptase. The data from such alignments was not very conclusive. DNA polymerases with higher level of reverse transcriptase activity were non-confined to ancient organisms, as one would've expected. The second phase of this project was focused on conditions that may alter the DNA polymerase activity. Various reaction conditions, such as temperature, using various ions (Ni2+, Mn2+, Mg2+) were tested. Interestingly, it was found that the DNA polymerase from the Thermos aquatics family can be made to copy RNA into DNA (i.e. reverse transcriptase activity). Thus it was shown that under appropriate conditions (ions and reactions temperatures) reverse transcriptase activity can be induced in DNA polymerase. In the third phase of this study recombinant DNA technology was used to generate a chimeric DNA polymerase; in attempts to identify the region(s) of the polymerase responsible for RNA-directed DNA polymerase activity. The two DNA polymerases employed were the Thermus aquatic us and Thermus thermophiles. As in the second phase various reaction conditions were investigated. Data indicated that the newly engineered chimeric DNA polymerase can be induced to copy RNA into DNA. Thus the intrinsic reverse transcriptase activity found in ancient DNA polymerases was localized into a domain and can be induced via appropriate reaction conditions.

DFT Studies of the Organocatalytic Aldol Reaction and a Frustrated Lewis Pair

The computational study, and in particular the density functional theory (DFT) study of the organocatalytic α-chlorination-aldol reaction and the chiral backbone Frustrated Lewis Pair (FLP) system served as a valuable tool for experimental purposes. This thesis describes methods to consider different transition states of the proline- catalyzed α-chlorination aldol reaction to determine the reasonable transition state in the reaction between the enamine and α-chloro aldehydes. Moreover, the novel intramolecular Frustrated Lewis pair based on a chiral backbone for the asymmetric hydrogenation of imines and enamines was designed and the ability of hydrogen splitting by this new FLP system was examined by computational modeling and calculating the hydrogen activation energy barrier.

Multi-Prover and parallel repetition in non-classical interactive games

Depuis l’introduction de la mécanique quantique, plusieurs mystères de la nature ont trouvé leurs explications. De plus en plus, les concepts de la mécanique quantique se sont entremêlés avec d’autres de la théorie de la complexité du calcul. De nouvelles idées et solutions ont été découvertes et élaborées dans le but de résoudre ces problèmes informatiques. En particulier, la mécanique quantique a secoué plusieurs preuves de sécurité de protocoles classiques. Dans ce m´emoire, nous faisons un étalage de résultats récents de l’implication de la mécanique quantique sur la complexité du calcul, et cela plus précisément dans le cas de classes avec interaction. Nous présentons ces travaux de recherches avec la nomenclature des jeux à information imparfaite avec coopération. Nous exposons les différences entre les théories classiques, quantiques et non-signalantes et les démontrons par l’exemple du jeu à cycle impair. Nous centralisons notre attention autour de deux grands thèmes : l’effet sur un jeu de l’ajout de joueurs et de la répétition parallèle. Nous observons que l’effet de ces modifications a des conséquences très différentes en fonction de la théorie physique considérée.

Identification et caractérisation de gènes différemment exprimés dans les tubules proximaux de reins diabétiques et impliqués dans le développement de la néphropathie diabétique

La néphropathie diabétique est une maladie rénale caractérisée par un syndrome néphrotique et de la glomérulosclérose. Celle-ci est reliée à l’angiopathie de capillaires suite au diabète. Il s’agit d’une importante cause d’insuffisance rénale en Amérique. Or, les anomalies tubulaires comme l’apoptose ou le détachement de tubules des glomérules sont reconnues comme étant de bons marqueurs de progression de cette maladie. Ainsi, il a été proposé au cours des travaux reliés à cette thèse d’étudier les différents mécanismes moléculaires reliés à l’apoptose des tubules proximaux, en particulier dans un thème de relation avec les dommages reliés aux espèces réactives oxygénées (ROS). Une des hypothèses développée au cours de précédents travaux faisait état que l’une des sources initiales qui entrainent le développement de dommages tubulaires soit régulée à travers la production de ROS dérivés des NADPH oxydases. Ainsi, une des premières séries d’expériences entreprises au cours de cette thèse a été effectuée sur un modèle animal de diabète de type 2, la souris db/db. Suite à la caractérisation des différentes pathologies rénales et leur réduction par la surexpression de l’enzyme antioxydante catalase dans les tubules proximaux, des expériences de micro-puces d’expression génétiques furent effectuées. À l’aide de cet outil et par des analyses bioinformatiques, il a été possible d’établir un profilage de gènes reliés à différentes voies de signalisation modulées par le diabète et la catalase. Ainsi, il a été possible d’effectuer de plus amples études sur des gènes reliés à l’apoptose surexprimé dans les tubules proximaux de souris diabétiques. Un des gènes pro-apoptotique mieux caractérisé durant cette thèse fut le gène Bmf, un membre de la famille des régulateurs de Bcl-2 impliqués dans l’apoptose via le relâchement de cytochrome c de la mitochondrie. Ainsi, il a été déterminé que ce gène est surexprimé dans les tubules proximaux de souris diabétiques, et que celui-ci était augmenté dans différents modèles in vitro de diabète. Cela a permis de conclure que Bmf joue sans doute un rôle important la régulation de l’apoptose et de l’atrophie des tubules proximaux. Une autre étude effectuée dans le cadre de cette thèse était reliée avec l’utilisation d’un modèle transgénique afin de mieux définir le rôle que jouent les dommages reliés au stress oxydatif dans la progression des pathologies rénales reliées à l’induction du système rénine-angiotensine. Les résultats obtenus ont permis de déterminer que la surexpression de l’enzyme antioxydante catalase a permis de réduire les différentes pathologies rénales observées dans les souris transgéniques, ce qui permet de conclure que les espèces réactives oxygénées jouen un rôle important dans le développement de l’hypertension et des dommages rénaux.

Cell wall composition regulates cell shape and growth behaviour in pollen tubes

L’une des particularités fondamentales caractérisant les cellules végétales des cellules animales est la présence de la paroi cellulaire entourant le protoplaste. La paroi cellulaire joue un rôle primordial dans (1) la protection du protoplaste, (2) est impliquée dans les mécanismes de filtration et (3) est le lieu de maintes réactions biochimiques nécessaires à la régulation du métabolisme et des propriétés mécaniques de la cellule. Les propriétés locales d’élasticité, d’extensibilité, de plasticité et de dureté des composants pariétaux déterminent la géométrie et la forme des cellules lors des processus de différentiation et de morphogenèse. Le but de ma thèse est de comprendre les rôles que jouent les différents composants pariétaux dans le modelage de la géométrie et le contrôle de la croissance des cellules végétales. Pour atteindre cet objectif, le modèle cellulaire sur lequel je me suis basé est le tube pollinique ou gamétophyte mâle. Le tube pollinique est une protubérance cellulaire qui se forme à partir du grain de pollen à la suite de son contact avec le stigmate. Sa fonction est la livraison des cellules spermatiques à l’ovaire pour effectuer la double fécondation. Le tube pollinique est une cellule à croissance apicale, caractérisée par la simple composition de sa paroi et par sa vitesse de croissance qui est la plus rapide du règne végétal. Ces propriétés uniques font du tube pollinique le modèle idéal pour l’étude des effets à courts termes du stress sur la croissance et le métabolisme cellulaire ainsi que sur les propriétés mécaniques de la paroi. La paroi du tube pollinique est composée de trois composantes polysaccharidiques : pectines, cellulose et callose et d’une multitude de protéines. Pour comprendre les effets que jouent ces différents composants dans la régulation de la croissance du tube pollinique, j’ai étudié les effets de mutations, de traitements enzymatiques, de l’hyper-gravité et de la gravité omni-directionnelle sur la paroi du tube pollinique. En utilisant des méthodes de modélisation mathématiques combinées à de la biologie moléculaire et de la microscopie à fluorescence et électronique à haute résolution, j’ai montré que (1) la régulation de la chimie des pectines est primordiale pour le contrôle du taux de croissance et de la forme du tube et que (2) la cellulose détermine le diamètre du tube pollinique en partie sub-apicale. De plus, j’ai examiné le rôle d’un groupe d’enzymes digestives de pectines exprimées durant le développement du tube pollinique : les pectate lyases. J’ai montré que ces enzymes sont requises lors de l’initiation de la germination du pollen. J’ai notamment directement prouvé que les pectate lyases sont sécrétées par le tube pollinique dans le but de faciliter sa pénétration au travers du style.