Organophosphate hydrolase (OPH, EC 3.1.8.1) is a bacterial enzyme with a broad spectrum of potential substrates that include organophosphorus pesticides, herbicides, and chemical warfare agents. OPH has been expressed successfully in bacterial, fungal, and insect cell culture systems; however, none of these systems produces amounts of enzyme suitable for applications outside of the research laboratory. Therefore, a transgenic Zea mays L. (maize) system was developed to express OPH as an alternate to the current OPH expression systems. The bacterial gene encoding the OPH protein was optimized for transcriptional and translational expression in maize. The optimized gene was inserted into the maize genome under the control of embryo specific, endosperm specific, and constitutive plant promoters. Select transformants were analyzed for the expression of OPH. Expression was observed in the seeds of plants transformed with each of the three constructs with the highest expression observed with the embryo specific and constitutive promoter constructs. The highest OPH expressing lines of transgenic maize had expression levels higher than those reported for the E. coli expression system. OPH was purified from transgenic maize seed and analyzed for posttranslational modification and kinetic properties. OPH was observed to undergo a glycosylation event when expressed in maize that yielded at least two forms of OPH homogolous dimer. The glycosylated form of OPH bound tightly to the Concanavalin A sepharose and remained active after months of storage at room temperature. OPH activity was checked against a number of organophosphate herbicides. Enzymatic activity was observed against the herbicide Amiprophos-methyl and kinetic properties were measured. Enzymatic activity was also tested against the organophosphate Haloxon. Transgenic maize callus, leaf, and seed tissue could be screened for the presence of the optimized opd gene by enzymatic activity. Comparison of the growth of transgenic and control callus on media containing organophosphates showed that the transgenic callus was resistant to the herbicidal effects of haloxon. Transgenic plants expressing OPH were also resistant to the herbicide bensulide when compared to control plants. This indicates that OPH can be used as a screenable marker in plant systems and may be a potential scorable marker system as well.

Organophosphate hydrolase (OPH, EC 3.1.8.1) is a bacterial enzyme with a broad spectrum of potential substrates that include organophosphorus pesticides, herbicides, and chemical warfare agents. OPH has been expressed successfully in bacterial, fungal, and insect cell culture systems; however, none of these systems produces amounts of enzyme suitable for applications outside of the research laboratory. Therefore, a transgenic Zea mays L. (maize) system was developed to express OPH as an alternate to the current OPH expression systems. The bacterial gene encoding the OPH protein was optimized for transcriptional and translational expression in maize. The optimized gene was inserted into the maize genome under the control of embryo specific, endosperm specific, and constitutive plant promoters. Select transformants were analyzed for the expression of OPH. Expression was observed in the seeds of plants transformed with each of the three constructs with the highest expression observed with the embryo specific and constitutive promoter constructs. The highest OPH expressing lines of transgenic maize had expression levels higher than those reported for the E. coli expression system. OPH was purified from transgenic maize seed and analyzed for posttranslational modification and kinetic properties. OPH was observed to undergo a glycosylation event when expressed in maize that yielded at least two forms of OPH homogolous dimer. The glycosylated form of OPH bound tightly to the Concanavalin A sepharose and remained active after months of storage at room temperature. OPH activity was checked against a number of organophosphate herbicides. Enzymatic activity was observed against the herbicide Amiprophos-methyl and kinetic properties were measured. Enzymatic activity was also tested against the organophosphate Haloxon. Transgenic maize callus, leaf, and seed tissue could be screened for the presence of the optimized opd gene by enzymatic activity. Comparison of the growth of transgenic and control callus on media containing organophosphates showed that the transgenic callus was resistant to the herbicidal effects of haloxon. Transgenic plants expressing OPH were also resistant to the herbicide bensulide when compared to control plants. This indicates that OPH can be used as a screenable marker in plant systems and may be a potential scorable marker system as well.

Streptococcus gordonii, a commensal oral bacterium, is considered a good candidate to function as a live oral vaccine vector. The introduction of vaccine antigen genes into S. gordonii relies on the use of antibiotic resistance genes as selectable markers, which is undesirable. In this study, we used auxotrophic complementation (deletion of an essential gene from the chromosome and insertion into a plasmid) as a means to create an antibiotic marker-free gene delivery system in S. gordonii. S. gordonii ?thyA was created and complemented by an antibiotic marker-free expression plasmid containing the intact thyA gene, pDL276/thyAdelkan. Transformation of pDL276/thyAdelkan into the mutant gave an unexpected 100-fold increase in transformation efficiency as compared to pDL276. The transformants arose from both single and double crossing over. The increase in transformation efficiency suggests that a highly efficient antibiotic marker-free system to deliver genes to the chromosome has been created using thyA complementation.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

The demand for faster data transmission has been increasing since the wide acceptance of the Internet. To meet this ever increasing demand, optical communication systems use Wavelength Division Multiplexing (WDM) to transport more information per fiber by utilizing multiple wavelength channels. Multi-wavelength laser sources are one of the most important components of such WDM systems. In this thesis a multi-wavelength laser source is proposed. A fiber Sagnac loop filter is used to produce a grid pattern with peaks at a certain set of wavelengths. Then any desired channel is chosen from the grid by using an adjustable Fiber Bragg Grating.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

La dihydrofolate réductase humaine (DHFRh) est une enzyme essentielle à la prolifération cellulaire. Elle réduit le dihydrofolate en tétrahydrofolate, un co-facteur impliqué dans la biosynthèse des purines et du thymidylate. La DHFRh est une cible de choix pour des agents de chimiothérapie comme le méthotrexate (MTX), inhibant spécifiquement l’enzyme ce qui mène à un arrêt de la prolifération et ultimement à la mort cellulaire. Le MTX est utilisé pour le traitement de plusieurs maladies prolifératives, incluant le cancer. La grande utilisation du MTX dans le milieu clinique a mené au développement de mécanismes de résistance, qui réduisent l’efficacité de traitement. La présente étude se penche sur l’un des mécanismes de résistance, soit des mutations dans la DHFRh qui réduisent son affinité pour le MTX, dans le but de mieux comprendre les éléments moléculaires requis pour la reconnaissance de l’inhibiteur au site actif de l’enzyme. En parallèle, nous visons à identifier des variantes plus résistantes au MTX pour leur utilisation en tant que marqueurs de sélection en culture cellulaire pour des systèmes particuliers, tel que la culture de cellules hématopoïétiques souches (CHS), qui offrent des possibilités intéressantes dans le domaine de la thérapie cellulaire. Pour étudier le rôle des différentes régions du site actif, et pour vérifier la présence d’une corrélation entre des mutations à ces régions et une augmentation de la résistance au MTX, une stratégie combinatoire a été dévelopée pour la création de plusieurs banques de variantes à des résidus du site actif à proximité du MTX lié. Les banques ont été sélectionnées in vivo dans un système bactérien en utilisant des milieux de croissance contenant des hautes concentrations de MTX. La banque DHFRh 31/34/35 généra un nombre considérable de variantes combinatoires de la DHFRh hautement résistantes au MTX. Les variantes les plus intéressantes ont été testées pour leur potentiel en tant que marqueur de sélection dans plusieurs lignées cellulaires, dont les cellules hématopoïétiques transduites. Une protection complète contre les effets cytotoxiques du MTX a été observée chez ces cellules suite à leur infection avec les variantes combinatoires. Pour mieux comprendre les causes moléculaires reliées à la résistance au MTX, des études de structure tridimensionnelle de variantes liées au MTX ont été entreprises. La résolution de la structure de la double variante F31R/Q35E lié au MTX a révélé que le phénotype de résistance était attribuable à d’importantes différences entre le site actif de la double variante et de l’enzyme native, possiblement dû à un phénomème dynamique. Une compréhension plus générale de la reconnaissance et la résistance aux antifolates a été réalisée en comparant des séquences et des structures de variantes de la DHFR résistants aux antifolates et provenant de différentes espèces. En somme, ces travaux apportent de nouveaux éléments pour la comprehension des intéractions importantes entre une enzyme et un ligand, pouvant aider au développement de nouveaux antifolates plus efficaces pour le traitement de diverses maladies. De plus, ces travaux ont généré de nouveaux gènes de résistance pouvant être utilisés en tant que marqueurs de sélection en biologie cellulaire. / Human dihydrofolate reductase (hDHFR) is an enzyme that is essential to cell proliferation. It reduces dihydrofolate to tetrahydrofolate, an important cofactor involved in purine and thymidylate biosynthesis. hDHFR is a choice target for chemotherapeutic drugs like methotrexate (MTX), which specifically inhibits the enzyme, stopping cell proliferation and leading to cellular death. MTX is used for the treatment of many proliferative diseases, including cancers. Widespread use of MTX has lead to the development of resistance mechanisms appear which impair treatment efficiency. The present work focuses on a mechanism of resistance, namely mutations in hDHFR that reduce its affinity for MTX, to better understand the underlying mechanisms of inhibitor recognition at the active site of the enzyme. In parallel, we aim at identifying the most MTX-resistant variants to offer novel selectable markers for particular cell culture systems, such as hematopoietic cell culture, which offer important perspectives for cellular therapy. To study the role of different regions of the hDHFR active site, and to verify if a correlation exists between mutations in these regions and increased resistance to MTX, a combinatorial strategy was developed enabling the creation of several hDHFR variant libraries at active site residues located in proximity to bound MTX. The libraries were selected in vivo in a bacterial system using culture media containing high concentration of the inhibitor. One library in particular, hDHFR 31/34/35, yielded a considerable number of highly MTX-resistant combinatorial hDHFR variants. The most interesting candidates were tested for their potential as selectable markers in various cell lines, including transduced hematopoietic cells. Complete protection from MTX-cytotoxicity was obtained for these cells following infection with the combinatorial variants. To better understand the molecular causes of MTX resistance, resolution of the crystal structures of variant proteins in presence of MTX was attempted. Resolution of the F31R/Q35E double variant revealed that the resistance phenotype was related to important differences in the active site relative to WT, possibly attributable to a dynamic motion effect. A more general comprehension of antifolate recognition and resistance was achieved by sequence and structural comparison of antifolate-resistant DHFR variants from different species. Overall, our work contributes to the better understanding of enzyme-inhibitor interactions, which could provide new insights into the development of more efficient clinical therapies. In addition, this work has yielded novel drug-resistance genes useful as selectable markers for cellular biology.

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