Context: Mutation testing is a fault-based technique for measuring the effectiveness of a test set in terms of its ability to detect faults. Mutation testing seeds artificial faults into an application and checks whether a test suite can detect them. If these faults are not found, the test suite is still not considered to be 'good enough'. However, there are also mutations which keep the program semantics unchanged and thus cannot be detected by any test suite. Finding a way to assess these mutations is also known as the equivalent mutant problem (EMP). Objectives: The main objective of this thesis is to conduct a systematic literature review in the field of mutation testing, to identify and classify existing methods for equivalent mutants detection. In addiction, other objectives are: analyze possibilities to improve existing methods for equivalent mutant detection, implement new or improved method and compare it with existing ones. Methods: Based on the systematic literature review method we have went over publications from six electronic databases and one conference proceedings. Standard method was extended by scanning lists of references and some alternative sources: searching in Google Scholar, checking personal websites of relevant authors and contacting all of them. We have performed all the systematic literature review steps such as the protocol development, initial selection, final selection, quality assessment, data extraction and data synthesis. In the second part of this thesis - an experiment, we have implemented four second order mutation testing strategies and compared them each other from four different perspectives: mutants reduction, equivalent mutants reduction, fault detection loss and mutation testing process time reduction. Results: The search identified 17 relevant techniques in 22 articles. Three categories of techniques can be distinguished: detecting (DEM), suggesting (SEM) and avoiding equivalent mutants generation (AEMG). Furthermore, for each technique current state of development and some ideas on how to improve it are provided. The experiment proved that DifferentOperators strategy gives the best results in all four investigated areas. In addition, time for manual mutants classification against equivalence was measured. Assessing one first order mutant takes 11 minutes 49 seconds, while for the second order mutants classification time is 9 minutes 36 seconds in average. Conclusions: After three decades of studies, results obtained for techniques from the DEM group are still far from perfection (best one is detecting 47,63% of equivalent mutants). Thus, new paths for the solution have been developed - SEM and AEMG group. Methods from both categories help in dealing with EMP, however from SEM provide only mutants likely to equivalent, while from AEMG cause some loss of test effectiveness. The conclusion from the experiment is that DifferentOperators strategy gives the best results among all proposed.

No description available.

Forward genetic screen was performed in Arabidopsis thaliana to isolate novel genes involved in leaf development. The tarani (tni) mutant was selected for further study based on its unique cup-shaped lamina with +ve Gaussian curvature. We show that the larger size of tni leaves is due to rapid growth rate due to excess and prolonged cell division. We monitored the front of the receding cell division zone as a function of time and showed that the shape of the front is more concave compared to wild type, leading to positive curvature. Application of gibberellic acids (GA) synthesis inhibitor rescued the positive curvature of tni suggesting a role for GA in maintaining leaf flatness. Overexpression of cell cycle inhibitor KRP2 also flattened the leaf, confirming a role of cell division. The floral organs and seed are also larger in the tni mutant. Besides growth, tni trichomes are hyper-branched which usually happens when there is more endoreduplication. We found that the nuclei of tni trichomes are larger than wild type nuclei, suggesting increased DNA content. Genetic interaction studies showed that TNI works independent of other trichome branching genes such as with TRYPTICHON and FURCA1. Map-based cloning showed that tni is positioned on left arm of the 3rd chromosome. Using molecular markers, we narrowed down to interval to a 65 kb region, which codes for 19 genes. Sequencing several of them revealed a G→A transition at the 3rd intron - 4th exon junction of At3g20630 gene. RT-PCR analysis showed the presence of an additional full-length transcript with extra un-spliced 3rd intron. Overexpression of this un-spliced variant in wild type plants produced phenotypes like hyperbranched trichomes and cup-shaped leaves; plus additional phenotypes like organ fusion and organ polarity defects. Complementation and allelic tests confirmed that TNI codes for AtUBP14, an ubiquitin protease. The tni plants have longer stem and roots which grow at faster rate compared to wild type. Confocal microscopic analysis of mature embryos showed that both shoot (SAM) and root apical meristems (RAM) of tni plants are larger in size. In RAM, the numbers of quiescent center (QC) cells and stem cells have increased in tni plants. The tni inflorescence and flowers are bigger than wild type in size. Also the degree of axillary shoots has increased in the tni plants. Overexpression of the splice variant of TNI produced undifferentiated callus-like structures in the shoot apex and in hypocotyl. All these phenotypes show that TNI is involved in meristem proliferation. The tni siliques produced many un-fertilized ovules and shrunken and malformed seeds suggesting gametic and/or embryo lethality. We observed that tni embryos were mis-patterned at various stages of development. Following the cell division pattern shows that cells arising from the ‘basal cell’ of the embryo take apical cell fate in tni embryos. The topmost cell of the suspensor, which is also the precursor cell of RAM, is not specified as hypophysial cell in several tni embryos. In the forward genetic screen, we isolated another mutant called tooth (tth), which has deeper serrations at the leaf margin and narrower leaves compared to wild type. It has been mapped to the longer arm of the 2nd chromosome. Genetic interaction studies show that tth is not allelic to other serration mutants such as serrate and mir164a.

L'acide ascorbique (AsA, vitamine C) est l'un des composés parmi les plus importants chez les eucaryotes. En raison de son potentiel antioxydant élevé, l'AsA représente un trait important de la qualité nutritionnelle des végétaux. Au-delà de sa valeur bénéfique pour la santé, une augmentation de la teneur en AsA des fruits bénéficierait probablement à la qualité post-récolte et à la résistance aux pathogènes. Pour mieux comprendre ces régulations chez les plantes et leurs impacts sur la qualité des fruits, une collection de tomates EMS hautement mutée (cv. Micro-Tom) a été criblée pour identifier des mutants dont les fruits sont enrichis en AsA. Cette stratégie de génétique directe associant le criblage à une approche de cartographie par séquençage devait permettre d’identifier de nouveaux gènes liés au caractère AsA+. L'un des mutants, noté P21H6, présentait un enrichissement en AsA de 2 à 4 fois supérieur à celui du WT, et fut le premier à être génétiquement caractérisé. Cette étude a permis de mettre en évidence une nouvelle classe de photorécepteurs impliqués dans la détection de la lumière bleue, appelée SlPLP, en tant que régulateur négatif de l'accumulation d'AsA dans la tomate. Le rôle de PLP dans le phénotype AsA+ du fruit a été confirmé par une stratégie de mutagenèse dirigée, avant d’entreprendre sa caractérisation fonctionnelle. Nous avons démontré que SlPLP interagit avec SlGGP (GDP-L-galactose phosphorylase), une enzyme clé de la voie du L-Galactose, sous contrôle de la lumière bleue et que cette interaction a lieu dans le cytoplasme et le noyau. Nos résultats renforcent le rôle central du GGP dans la biosynthèse de l'AsA et suggèrent un nouveau mécanisme de régulation par la lumière bleue de la fonction du GGP, en plus de son activité métabolique. Parallèlement, nous avons entrepris la caractérisation d’un autre mutant, le P17C5-3, qui présentait le plus fort taux d'AsA (jusqu'à 10 fois le WT). Outre le phénotype AsA+, le mutant P17C5 présentait de fortes altérations morphologiques, notamment l’absence de graines, rendant la mise en place de la stratégie de cartographie difficile. Grâce à un croisement avec la variété commerciale M82, la mutation causale pu être identifiée dans un ORF cis-régulateur en amont de GGP. Ce résultat confirme le rôle clé de GGP dans la voie L-Galactose. Des études préliminaires liées au phénotype parthénocarpique suggèrent un problème de stérilité mâle associé aux processus de développement du pollen. Enfin, dans l’étude de la qualité des fruits après la récolte, des expériences de stress froid effectuées avec les fruits P21H6 semblent démontrer que l’augmentation de la teneur en AsA améliore la durée de conservation et la capacité de maturation des fruits. Dans l'ensemble, nos résultats confirment la position clé de la protéine GGP dans la voie de biosynthèse de l'AsA, et fournissent des outils et du matériel végétal précieux pour décortiquer la régulation de l'AsA et son rôle physiologique dans la qualité des fruits et les caractères post-récolte. / Ascorbic acid (AsA, vitamin C) is one of the most important biochemical in living organisms. Due to its high antioxidant potential, AsA represents an important trait of nutritional quality in fruits and vegetables. In addition to its beneficial health value in fruit consumption, increasing fruit AsA content would likely affect postharvest quality and resistance to pathogens. Thus, understanding the regulation of AsA accumulation in order to improve crop species of agronomical interest is an important issue in plant breeding for many fleshy fruit species. To get a better understanding of the regulation of AsA level in plants and its impact on fruit quality, a highly mutagenized EMS tomato collection (cv. Micro-Tom) was screened for AsA+ fruit mutants. This forward genetic strategy combined with a mapping-by-sequencing approach, had allowed identifying new genes related to the AsA+ trait. One of the mutant line named P21H6, displayed an AsA-enrichment 2 to 4 fold that of the WT, and was the first to be genetically characterized. It allowed highlighting a new class of photoreceptor involved in blue light sensing named SlPLP as a negative regulator of AsA accumulation in tomato. We confirmed the role of the PLP in the fruit AsA+ phenotype using a directed mutagenesis strategy, undertaking its functional characterization. We demonstrate that PLP interacts with GGP (GDP-L-galactose phosphorylase), a key enzyme of the L-Galactose pathway, under blue light control and that this interaction takes place in the cytoplasm and the nucleus. Our results strengthen the central role of GGP in the AsA biosynthesis and suggest a new regulation mechanism by blue light of the GGP function in addition to its metabolic activity. Besides we started the characterization another mutant, the P17C5-3, which displayed the highest level of AsA (up to 10 times the WT). Beyond its AsA+ content, the P17C5 mutant showed strong morphological alterations including a seedless phenotype making the mapping difficult at first. Thanks to the crossing with the commercial M82 tomato cultivar, the causal mutation was identified in a cis-acting ORF, upstream of the GGP gene. This result confirmed the key role of GGP in the L-Galactose pathway. Preliminary studies related to the parthenocarpic phenotype suggest a problem of male sterility associated with pollen development processes. Finally, in the study of the post-harvest fruit quality, chilling stress experiments carried out with the P21H6 fruits seem to demonstrate that increasing AsA content improve the fruit shelf life and its maturation capacity. Taken as a whole, our results confirmed the key position of the GGP protein in the AsA biosynthesis pathway and they provided precious tools and plant material for deciphering the regulation of AsA and its physiological role in fruit quality and post-harvest traits.

La protéine multidomaine Pr55Gag de VIH-1 joue un rôle crucial dans les étapes finales de la réplication virale, notamment lors de la reconnaissance et la sélection de l’ARN génomique ainsi que lors de la production de nouvelles particules virales. Outre son rôle structural, Pr55Gag chaperonne aussi les séquences d’acides nucléiques, une propriété cruciale pour la dimérisation de l’ARN génomique et l’hybridation de l’amorce ARNt à l’ARN génomique. Des partenaires cellulaires comme la protéine ribosomale RPL7 sont supposées être recrutées par Pr55Gag afin d’augmenter son potentiel chaperon. Afin d’étudier le mécanisme d’hybridation des acides nucléiques par Gag et RPL7, nous avons examiné leur effet sur la réaction d’hybridation entre dTAR, la version ADN de l’élément de transactivation virale et sa séquence complémentaire cTAR. Nos résultats révèlent que Gag et RPL7 présentent des mécanismes différents pour promouvoir l’hybridation cTAR/dTAR. Utilisés de concert, RPL7 peut aider Gag à chaperonner des séquences stables de l’ARN génomique que Gag seule pourrait difficilement chaperonner. Ce renforcement par RPL7 de l’activité chaperonne de Gag jouerait un rôle critique dans l’assemblage du virus. / The multidomain Pr55 Gag protein of HIV-1 plays a crucial role during late stages of viral replication, notably for the recognition and selection of genomic RNA as well as for the production of new viral particles. In addition to its structural role, Pr55 Gag also chaperones nucleic acid sequences, a property which is crucial for genomic RNA dimerization and annealing of the primer tRNA to the genomic RNA. Cellular partners like ribosomal protein RPL7 are thought to be recruited by Pr55 Gag to enhance its chaperoning potential. To investigate the nucleic acid annealing mechanism of Gag and RPL7, we examined their effect on the annealing reaction between dTAR, the DNA version of the viral transactivation element and its complementary cTAR sequence taken as relevant model HIV-1 sequences. Our data show that Gag and RPL7 exhibit different mechanisms for promoting the cTAR/dTAR annealing. When used together, RPL7 can help Gag to chaperone stable sequences of the genomic RNA that Gag would hardly be able to chaperone alone. This RPL7-driven boost in Gag chaperone activity is thought to be critical in the viral assembly process.

Actuellement, plus de 70% des biomédicaments commercialisés sont des glycoprotéines recombinantes. Les coûts élevés de production de ces biomédicaments ont poussé les scientifiques à développer des organismes de production alternatifs. Récemment, les microalgues ont été proposées en tant que potentiel système de production compte-tenu de leur rapidité de croissance et de leurs faibles coûts de production. Cependant, avant de produire des biomédicaments industriels chez les microalgues, il est impératif de s’assurer que les modifications post-traductionnelles, comme la N-glycosylation, soit conservées et compatibles avec une utilisation thérapeutique. Dans ce contexte, l’étude de la Nglycosylation de deux microalgues modèles, Chlamydomonas reinhardtii (microalgue verte) et Phaeodactylum tricornutum (diatomée) a été réalisée. Dans un premier temps, l’ingénierie de la N-glycosylation de C. reinhardtii a été initiée en exprimant une Nacétylglucosaminyltransférase I (GnT I) hétérologue. Les résultats obtenus ont permis de réévaluer les voies de N-glycosylation de C. reinhardtii et de montrer que cette microalgue synthétise une structure glycannique linéaire qui n’est pas substrat de la GnT I. Dans un second temps, un protocole d’extraction et de caractérisation des précurseurs glycanniques de C. reinhardtii et P. tricornutum a été développé et appliqué pour déterminer la structure des précurseurs glycanniques dans ces espèces. Enfin, la caractérisation de deuxxylosyltransférases potentielles (XTA et XTB) de C. reinhardtii a été menée en utilisant des mutants d’insertion et des analyses des N-glycannes par spectrométrie de masse. Cette étude a confirmé les rôles spécifiques de XTA et XTB dans la voie de N-glycosylation de C. reinhardtii. / Currently, more than 70% of the commercialized biopharmaceuticals are glycoproteins. The high production costs lead scientists to develop alternative organisms suitable for such production. Recently, microalgae emerged as a potential interesting production system thanks to their quick growth rate and low production costs. However, prior to start industrial glycoproteins production in microalgae, protein post-translational modifications like Nglycosylation, must be carefully controlled. This PhD thesis focused on the analysis of the Nglycosylation pathway of two different microalgae, Chlamydomonas reinhardtii (greenmicroalgae) and Phaeodactylum tricornutum (diatom). In order to start N-glycan engineering, heterologous N-acetylglucosaminyltransferase I (GnT I) sequences were expressed in C.reinhardtii. This study demonstrated that C. reinhardtii synthetize a linear N-glycan unsuitable for GnT I activity and allows the reinvestigation of the C. reinhardtii N-glycosylation pathway. A second chapter of this work focus on the optimization of a protocol suitable for analyzing the structure of the Dolichol N-linked precursors of C. reinhardtii and P. tricornutum. Lastly, two potential xylosyltransferases (XTA and XTB) from C. reinhardtii were characterized using insertional mutants and N-glycomic analyses by mass spectrometry approaches. This work allows us to propose specific involvement of XTA and XTB in the xylosylation processing of C.reinhardtii N-glycans.

No description available.

The regulation of pyrimidine biosynthesis was studied in Pseudomonas putida. The biosynthetic and salvage pathways provide pyrimidine nucleotides for RNA, DNA, cell membrane and cell wall biosynthesis. Pyrimidine metabolism is intensely studied because many of its enzymes are targets for chemotheraphy. Four aspects of pyrimidine regulation are described in this dissertation. Chapter I compares the salvage pathways of Escherichia coli and P. putida. Surprisingly, P. putida lacks several salvage enzymes including nucleoside kinases, uridine phosphorylase and cytidine deaminase. Without a functional nucleoside kinase, it was impossible to feed exogenous uridine to P. putida. To obviate this problem, uridine kinase was transferred to P. putida from E. coli and shown to function in this heterologous host. Chapter II details the enzymology of Pseudomonas aspartate transcarbamoylase (ATCase), its allosteric regulation and how it is assembled. The E. coli ATCase is a dodecamer of two different polypeptides, encoded by pyrBI. Six regulatory (PyrI) and six catalytic (PyrB) polypeptides assemble from two preformed trimers (B3) and three preformed regulatory dimers (I2) in the conserved 2B3:3I2 molecular structure. The Pseudomonas ATCase also assembles from two different polypeptides encoded by pyrBC'. However, a PyrB polypeptide combines with a PyrC. polypeptide to form a PyrB:PyrC. protomer; six of these assemble into a dodecamer of structure 2B3:3C'2. pyrC' encodes an inactive dihydroorotase with pyrB and pyrC' overlapping by 4 bp. Chapter III explores how catabolite repression affects pyrimidine metabolism. The global catabolite repression control protein, Crc, has been shown to affect pyrimidine metabolism in a number of ways. This includes orotate transport for use as pyrimidine, carbon and nitrogen sources. Orotate is important because it interacts with PyrR in repressing the pyr genes. Chapter IV describes PyrR, the positive activator of the pyrimidine pathway. As with other positive activator proteins, when pyrimidine nucleotides are depleted, PyrR binds to DNA thereby enhancing expression of pyrD, pyrE and pyrF genes. When pyrimidine nucleotides are in excess, the PyrR apoprotein binds to orotate, its co-repressor, to shut down all the pyrimidine genes. Like many positive activators, PyrR is subject to autoregulation and has catalytic activity for uracil phosphoribosyltransferase inducible by orotate.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Neurodegenerative disorders (NDD) are an ever-increasing burden on healthcare; consequently, elucidating the mechanisms underlying neurodegeneration (ND) is critical for the development of effective treatments for these diseases. In order to unravel the molecular pathways underlying movement disorders and identify new genes involved in ND, two ataxic mouse mutants characterised by cell death in the cerebellum were studied in detail using a combination of in vitro and in vivo techniques. The robotic mouse demonstrated the key role of a transcription factor, Af4, in Purkinje cell (PC) survival and how only small changes in the levels of a single transcriptional cofactor could deleteriously affect normal cerebellum function. Expression array studies of the robotic PCs revealed the first confirmed targets of Af4-mediated transcription, including insulin-like growth factor 1 (Igf-1). It was demonstrated that Igf-1 is critical for PC survival, highlighting the role of the IGF-1 signalling pathway as a potential therapeutic target for the treatment of cerebellar ataxia in humans. Detailed analysis of the bella mutant demonstrated that ataxia and apoptotic cerebellar degeneration is caused by loss of the oxidative resistance 1 (Oxr1) gene. In vitro modelling experiments went on to show that the levels of this previously uncharacterised gene are critical for controlling the sensitivity of neuronal cells to oxidative stress (OS). Moreover, this study showed that Oxr1 was up-regulated both in human and pre-symptomatic mouse models of amyotrophic lateral sclerosis (ALS), demonstrating that Oxr1 was an early marker of ROS defence, prior to pathology, and potentially a novel neuroprotective factor in NDD. Preliminary interaction studies show that Oxr1 is likely to be a multi-functional protein that forms complexes with proteins known to be mutated in NDD. Thus, the study of both the robotic and the bella mouse has demonstrated the value of the phenotype-driven approach to investigate novel neurodegenerative pathways.