Au cours des dernières années, les tests unitaires sont devenus un élément essentiel de tout projetlogiciel sérieux afin de vérifier son bon fonctionnement.Cependant, les tests sont fastidieux et coûteux pour l'industrie: il est difficile d'évaluer le retour surinvestissement.Pour surmonter ce problème, la recherche étudie l'automatisation de la création de tests.Même si ces suites de tests générées automatiquement ont une très bonne qualité, il existe toujours desobstacles à l'adoption de telles techniques par l'industrie.Cela s'explique par les difficultés à comprendre, intégrer et gérer la suite de tests générée.L'objectif de cette thèse est de remédier à ces difficultés en proposant une nouvelle technique quiproduit des nouveaux tests basés sur les tests existants. Cette technique s'appelle "amplification detests". Par construction, ces nouveaux tests sont donc proches des tests écrits par des humains, et doncsont faciles à comprendre, à intégrer et à gérer. Cette approche est implémentée sous forme d'un outilappelé DSpot. / In recent years, unit testing has become an essential part of any serious software project to verify itsproper functioning.However, the tests are tedious and expensive for the industry: it is difficult to evaluate the return oninvestment.To overcome this problem, the research is investigating the automation of test creation.Although these automatically generated test suites are of high quality, there are still barriers to theindustry adoption of such techniques.This is because of the difficulty in understanding, integrating, and managing the generated test suite.The objective of this thesis is to overcome these difficulties by proposing a new technique thatproduces new tests based on existing tests. This technique is called "test amplification". Byconstruction, these new tests are close to the tests written by humans, and therefore are easy tounderstand, integrate and manage. This approach is implemented as a tool called DSpot.

A limiting factor in the widespread deployment of LMDS systems is the limited distance of current systems. Rain attenuation and limited transmitter power are the primary causes of the limited distance. Adaptive digital predistortion is presented as a method of increasing effective transmitter power. A background on LMDS link design, non-linear amplification, and predistortion is presented to assist the reader. A developed simulation uses AM-AM and AM-PM characteristics obtained from laboratory measurements of a 28 GHz amplifier to determine the effect of several predistortion implementation options and to confirm the feasibility of the proposed architecture. The potential impact of this predistortion architecture on LMDS system design is considered. The presented multi-stage predistortion architecture is found to be capable of implementation at Msymbol/second rates utilizing a FPGA or custom IC and a moderate speed digital signal processor. / Master of Science

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<p> Nonlinear amplification schemes for hearing aids have been developed to deal primarily with the problem of loudness recruitment. The most commonly used form of nonlinear amplification is wide-dynamic-range compression (WDRC). Unfortunately, finding WDRC characteristics that satisfactorily deal with loudness recruitment while maintaining good speech intelligibility has proven difficult. An alternative nonlinear scheme, Advanced Dynamic Range Optimization (ADRO), has been shown in several studies to provide better speech intelligibility and listening comfort than fast-acting WDRC. ADRO uses a set of fuzzy-logic rules to make gain changes to optimize audibility, comfort, protection against loud sound, and noise attenuation. The "hearing protection" gain rule acts instantaneously, whereas the audibility and comfort rules adjust the gain slowly, such that ADRO provides linear amplification most of the time.</p> <p> The goal of this study is to examine the physiological basis for the relative performance of linear amplification, WDRC, and ADRO. Sentences from the TIMIT Speech Database were processed by each algorithm. In the case of WDRC, both single-channel and multi-channel schemes with fast and slow dynamics were tested. Speech signals were presented at 52, 62, 74, and 82 dB SPL (sound pressure level) with various noise levels and types, to simulate real-life environments. The simulations first use an auditory-periphery model to generate a "neurogram" of the auditory nerve's representation of the test speech material. The spectral and temporal modulations in the neurogram are then analyzed by a model of cortical speech processing. The effects of the background noise, the presentation level, the hearing loss and the amplification scheme are evaluated by comparing the cortical model response for a given condition (the "test" response) to the cortical model response to the same TIMIT sentence presented in quiet at 65 dB SPL to the normal-hearing model (the "template" response). From the difference between the test and template responses, a spectro-temporal modulation index (STMI) value is calculated. High STMI values predict good speech intelligibility, while low values predict poor intelligibility. Results show that ADRO is better at restoring the neural representation of speech than the other algorithms tested, even when the WDRC algorithms utilize slow time constants. In the case of no background noise, all the algorithms perform similarly well. However, when background noise is added, STMI values for higher SPLs drop notably for all the algorithms except for ADRO, which sustains a stable value throughout the range of SPLs test.</p> / Thesis / Master of Engineering (MEngr)

Yes / Polymerase chain reaction (PCR) has become a powerful tool for detecting various diseases due to its high sensitivity and specificity. However, the long thermocycling time and the bulky system have limited the application of PCR devices in Point-of-care testing. Herein, we have proposed an efficient, low-cost, and hand-hold PCR microdevice, mainly including a control module based on water-cooling technology and an amplification module fabricated by 3D printing. The whole device is tiny and can be easily hand-held with a size of about 110 mm × 100 mm × 40 mm and a weight of about 300 g at a low cost of about $170.83. Based on the water-cooling technology, the device can efficiently perform 30 thermal cycles within 46 min at a heating/cooling rate of 4.0/8.1 ℃/s. To test our instrument, plasmid DNA dilutions were amplified with this device; the results demonstrate successful nucleic acid amplification of the …

Le parasite Leishmania est un protozoaire de l’ordre des Kinetoplastidae et de la famille des Trypanosomatidae, responsable de maladies appelées leishmanioses. En tant qu’eucaryote unicellulaire, Leishmania possède de nombreux mécanismes d’adaptation aux stress environnementaux. En effet, plusieurs mécanismes de résistance peuvent être développés par le parasite pour répondre au stress et sont basés sur la modulation du génome, comme l’amplification génique ou l’introduction de mutations. De fait, l’étude de l’intégrité génomique et des mécanismes sous-jacents l’acquisition de la résistance sont des éléments clés de la compréhension du fonctionnement de ce parasite. L’amplification génique est rendue possible par la présence de nombreuses séquences répétées distribuées à travers le génome de Leishmania et qui permettent la formation d’amplicons circulaires et linéaires contenant des gènes essentiels à l’acquisition de la résistance. Les mécanismes sous-jacents la formation des amplicons restent encore à élucider mais les protéines de réparation de l’ADN semblent jouer un rôle majeur dans l’amplification génique. Dans le laboratoire, il a été démontré que l’amplification circulaire reposait essentiellement sur la protéine de réparation de l’ADN RAD51. Cependant, les acteurs impliqués dans la formation d’amplicon linéaire restaient inconnus. Les travaux de cette thèse ont permis de caractériser les fonctions de la protéine MRE11 chez Leishmania, protéine majeur de la réparation de l’ADN par recombinaison homologue, et qui possède une activité de liaison à l’ADN ainsi qu’une activité nucléase. MRE11 semble impliquée dans l’amplification linéaire puisque son inactivation conduit à une diminution du nombre d’amplicons linéaires dans la cellule. De plus, inhiber l’activité nucléase de MRE11 conduit à une augmentation de l’amplification circulaire, suggérant ainsi un rôle prépondérant de cette activité nucléase dans la formation d’amplicons. Cette étude a également permis d’étudier l’implication des protéines de réparation de l’ADN dans le maintien de l’intégrité du génome. / Nous avons pu établir un lien entre MRE11 et RAD50, deux protéines agissant au sein d’un même complexe MRN (MRE11-RAD50-NBS1), et l’instabilité génomique. En effet, l’inactivation de MRE11 et RAD50 a conduit à des réarrangements chromosomiques qui semblent être survenus par micro-homologie. Ces travaux ont donc permis de mettre en lumière qu’en l’absence des protéines majeures de la recombinaison homologue, un mécanisme de réparation par micro-homologie indépendant de MRE11 se met en place dans la cellule. De plus, l’inactivation du gène RAD50 dans une souche sauvage s’est révélée impossible alors qu’elle l’était dans une souche préalablement inactivée pour MRE11, suggérant une certaine hiérarchie dans le complexe MRN. Cette thèse présente également l’étude de l’acquisition de mutations ponctuelles dans le gène du transporteur de miltéfosine qui conduit à la résistance des parasites à cette drogue. La technologie de séquençage à profondeur élevée (« deep-sequencing ») nous a permis de séquencer ce gène à différents stades intermédiaires de la résistance, afin de déterminer la fréquence d’acquisition et la stabilité de ces mutations. Nous avons pu observer que les mutations ponctuelles dans le gène du transporteur de miltéfosine ne sont pas présentes dans les premiers stades de la résistance mais acquises à des concentrations plus élevées. L’analyse du gène a également démontré un plus grand nombre de mutations au fur et à mesure que la pression de sélection s’intensifie ainsi qu’une certaine stabilité des mutations après retrait de la pression de drogue, laissant entrevoir un maintien de la résistance. Ces résultats mettent en évidence l’implication des protéines de réparation de l’ADN dans la stabilité génomique chez Leishmania, mais surtout un rôle dans l’amplification extra-chromosomique liée à la résistance. L’utilisation des nouvelles technologies de séquençage nous a également permis d’étudier la dynamique d’acquisition de mutations ponctuelles impliquées dans la résistance.

Tetraploidy – the presence of four copies of the haploid chromosome complement – is common in cancer. There is evidence that ~40% of tumors pass through a tetraploid stage at some point during their development, and tetraploid cells injected in mice are more tumorigenic than their diploid counterparts. However, the reason for this increased tumorigenicity of tetraploid cells is not well established. Most routes by which cells may become tetraploid also confer cells with double the number of centrosomes, the small membraneless organelle that organizes the cell's microtubule cytoskeleton and mitotic spindle apparatus. Centrosome number homeostasis is crucial for health, and recent studies have shown inducing extra centrosomes in cells can induce tumor formation in mice. This has led some researchers to propose that the extra centrosomes that arise together with tetraploidy may be the reason that tetraploid cells are more tumorigenic. However, several anecdotal reports have found that tetraploid clones generated and grown in vitro appear to lose their extra centrosomes. Here, I investigate the population dynamics of the loss of extra centrosomes in newly formed tetraploid cells generated via cytokinesis failure. I uncover the mechanism driving the process and build a mathematical model that captures the experimentally observed dynamics. Next, I investigate karyotypic heterogeneity in newly formed tetraploid cells and their counterparts that are grown for 12 days under standard culture conditions and find that karyotypic heterogeneity has increased after 12 days of growth after tetraploidization. The day 12 'evolved' population with increased heterogeneity formed larger colonies in soft agar than newly formed tetraploid cells or diploid parental precursors and karyotype analysis of the largest soft agar colonies revealed recurrent aneuploidies shared by a subset of colonies. Finally, I investigate the effects of different culture conditions - meant to mimic various conditions in the tumor microenvironment - on the evolution of centrosome and chromosome number in newly formed tetraploid cells and identify a small subset of conditions that altered centrosome homeostasis or the fitness of tetraploid cells. / Doctor of Philosophy / The genetic information in cancer cells is often drastically altered compared to normal cells in the body. As one important example of this, the number and structure of chromosomes - the DNA structures that hold the genetic information - is often abnormal in cancer cells. Abnormal chromosome number is closely linked with cancer development, but the details of why this leads to more cancer are not clear. One important kind of chromosome number change is when a cell undergoes incomplete cell division, and the resulting cell acquires double the number of chromosomes compared to a normal cell (known as tetraploidy). Tetraploidy occurs in close to 40% of cancers and is linked with the most aggressive cases. The abnormal cell divisions that cause tetraploidy also lead to other cellular changes. One important change is that tetraploid cells also acquire double the number of the structures that organizes cell division (centrosomes). The centrosome organizes the mitotic spindle, the major apparatus that is responsible for equally distributing chromosomes to two daughter cells during the cell division process. Extra centrosomes in cells are closely linked with cancer and can lead to additionally chromosome number changes. Researchers have believed that the extra centrosomes that are acquired with doubled chromosome number may be the major reason that tetraploid cells are linked to more aggressive cancer. However, recent studies have suggested that tetraploid cells may lose their extra centrosomes, calling into question the details of the relationship between tetraploidy and tumor formation. Here, I use human cells grown in culture to understand how extra centrosomes are lost from tetraploid cells. I find that extra centrosomes in newly formed tetraploid cells promote abnormal 'multipolar' cell divisions, in which chromosomes are segregated unevenly to three or more partitions. Such divisions are often fatal to daughter cells. In some cases, the extra centrosomes can cluster to form bipolar spindles that segregate chromosomes into two equal partitions (as is normal). When forming bipolar spindles, extra centrosomes can cluster asymmetrically (three centrosomes at one pole, one at the other) or symmetrically (two centrosomes at each pole). Tetraploid cells with a normal number of centrosomes emerge when extra centrosomes cluster asymmetrically in a bipolar spindle, yielding one tetraploid daughter cell with a normal number of centrosomes. Such cells have a fitness advantage over cells with extra centrosomes, which over time are very likely to undergo fatal multipolar divisions. Thus, cells with a normal centrosome number 'take over' the population. Next, I investigate the cancer-like properties of tetraploid cells without their extra centrosomes and find that they display increased tumor-like behavior even in the absence of extra centrosomes. Finally, I investigate whether changing the conditions in which cells are grown (in ways meant to mimic different conditions that may be experienced in the body) affects whether tetraploid cells lose their extra centrosomes. We identify a small number of conditions that do influence loss of extra centrosomes. Together, these studies illuminate important details of the relationship between tetraploidy and tumor formation. This work lays the foundation to further explore and understand the relative roles of tetraploidy, extra centrosomes, and tissue environment in cancer.

MLL/KMT2A amplifications and translocations are prevalent in infant, adult and therapy-induced leukemia. However, the molecular contributor(s) to these alterations are unclear. Here we demonstrate that histone H3 lysine 9 mono- and di-methylation (H3K9me1/2) balance at the MLL/KMT2A locus regulates these amplifications and rearrangements. This balance is controlled by the cross-talk between lysine demethylase KDM3B and methyltransferase G9a/EHMT2. KDM3B depletion increases H3K9me1/2 levels and reduces CTCF occupancy at the MLL/KMT2A locus, and in turn, promotes amplification and rearrangements. Depleting CTCF is also sufficient to generate these focal alterations. Furthermore, the chemotherapy Doxorubicin (Dox), which associates with therapy-induced leukemia and promotes MLL/KMT2A amplifications and rearrangements, suppresses KDM3B and CTCF protein levels. KDM3B and CTCF overexpression rescues Dox-induced MLL/KMT2A alterations. G9a inhibition in human cells or mice also suppresses MLL/KMT2A events accompanying Dox treatment. Therefore, MLL/KMT2A amplifications and rearrangements are controlled by epigenetic regulators that are tractable drug targets, which has clinical implications. The data presented in this thesis were published in Cell in 2023. / Biomedical Sciences

Cette thèse de doctorat porte dans l’ensemble une étude approfondie sur une technologie émergente pour l’amplification isotherme des acides nucléiques appelée recombinase polymerase amplification (RPA). L’introduction porte une description détaillée sur la RPA. Cette revue de littérature documente et discute les diverses applications de la RPA en soulignant les connaissances actuelles concernant les applications diagnostiques. Malgré la composition complexe de la RPA (6 à 7 protéines dans le même mélange réactionnel), cette dernière s’avère une technologie rapide (générant des résultats &lt; 20 min), spécifique et sensible (détection de l’ordre de quelques copies de génome), et largement appliquée dans différentes disciplines. Ces avantages nous permettent de croire que la RPA possède la flexibilité nécessaire pour être utilisée comme outil de diagnostic rapide des maladies infectieuses en réduisant le temps d’obtention des résultats à moins d’une heure au lieu de 2 à 3 jours avec les tests de cultures standards. En conséquence, il sera possible d’intégrer la RPA dans des plateformes microfluidiques ou laboratoire sur puce qui permettent la préparation d’échantillons, l’amplification et la détection des acides nucléiques des microbes causant des infections. En premier lieu, les travaux de cette thèse ont généré des lignes directrices additionnelles pour la conception des amorces/sondes RPA. En second lieu, nos travaux ont permis de développer un essai diagnostic RPA pour la détection des streptocoques du groupe B, responsables de la septicémie et la méningite chez les nouveau-nés. Cet essai fut le premier à évaluer la performance de la RPA avec des échantillons cliniques humains. Ce test diagnostic RPA a été comparé à une méthode de référence, la réaction en chaîne par polymérase (PCR). Cette démonstration sur des échantillons cliniques nous à inciter à pousser notre étude pour réaliser le dernier objectif de ce projet qui consistait à automatiser la RPA par intégration dans un système microfluidique miniaturisé centripète. Une collaboration avec des experts en génies et en matériaux a permis de générer un dispositif microfluidique appelé blade ainsi de l’instrument impliqué dans l’opération des différentes tâches mécanistiques. Ces résultats préliminaires suggèrent qu’il sera important d’offrir un système automatisé complet applicable au chevet du patient. Par conséquence, il sera possible d’exécuter une analyse complète des agents infectieux en moins d’une heure sans le besoin des procédures complexes de préparation et de transport des échantillons cliniques ni le recours à du personnel qualifié. / This dissertation consists of an exhaustive study on an emerging technology for isothermal amplification of nucleic acids called recombinase polymerase amplification (RPA). The introduction of this thesis is a detailed description of the RPA. This review documents and discusses the various applications of this technology by pointing to the current knowledge about RPA for diagnostic applications. Despite the complex composition of RPA (6 to 7 proteins in the same reaction mixture), the latter was shown to be rapid (generating results in &lt; 20 min), specific and sensitive (detecting few target genome copies), and applied widely in different fields. Based on these advantages, we assume that RPA has a flexibility allowing it to be used for the rapid diagnosis of infectious diseases thus reducing time-to-result to less than an hour. Consequently, it will be possible to integrate RPA in microfluidic platforms providing a lab-on chip system. The first part of this doctoral project generated additional guidelines for RPA primers/probes design to develop specific RPA diagnostic assays. Second, we developed an RPA diagnostic test for the detection of group B streptococci, responsible for sepsis and meningitis in newborns. This assay was the first to evaluate RPA with human clinical samples. This diagnostic test was compared to a reference method, the polymerase chain reaction (PCR). This demonstration with clinical samples served to carry out the final objective of this project that was to automate RPA in a miniaturized microfluidic centripetal system. Collaboration with engineers and experts in materials has generated the microfluidic device called "blade" and the instrument involved in the operation of various mechanistic tasks. These preliminary results suggested that it will be important to provide an automated system applicable at bedside. Consequently, it will be possible to perform a complete analysis of infectious agents in less than an hour without the need for complex procedures for the preparation and transport of clinical specimens or the assistance of qualified personnel.