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Cells respond to their environment through dynamic posttranslational modification of their existing proteins. There are more than 20 posttranslational modifications that occur on eukaryotic proteins. Several of these modifications, with phosphorylation being the hallmark, participate in signal transduction. Generally, glycosylation is not thought to participate directly in signaling. Complex N-and 0-linked glycosylation occurs on membrane-bound or secreted proteins that are synthesized in the endoplasmic reticulum and Golgi apparatus. The lumenal or extracellular localization ofthese glycans restricts their potential for dynamic responsiveness to signals. In contrast, 0-GlcNAc is a simple monosaccharide modification that is abundant on serine or threonine residues ofnucleocytoplasmic proteins. An 0-GlcNAc site consensus motif has not yet been identified. However, many attachment sites are identical to those used by serine/threonine) kinases, and a neural network program has been developed to predict 0GlcNAc sites. The dynamic glycosylation of serine or threonine residues on nuclear and cytosolic proteins by 0-linked beta-N-acetylglucosamine (0-GlcNAc) is abundant in all multicellular eukaryotes. On several proteins, 0-GlcNAc and 0-phosphate alternatively occupy the same or adjacent sites, leading to the hypothesis that one function of this saccharide is to transiently block phosphorylation. Many proteins have been identified that carry this modification, including transcription factors, cytoskeletal proteins, nuclear pore proteins, oncogene products, and tumor suppressors. 0-GlcNAc appears to modify a large number of nucleocytoplasmic proteins· One of important regulatory proteins on which this project concentrates is β-catenin. Here, we examined where does this type ofposttranslational modification takes place on the protein. Our results indicated that P-catenin is 0-glycosylated on both the N-terminus and Cterminus, but not at the ARMADILLO segment. Further, we show that the known phosphorylation sites located at theN-terminal "destruction box" of this protein are not involved in 0-glycosylation. Furthermore, we demonstrated that the threonines adjacent to phosphorylation-site Threonin41 are not essential in 0-glycosylation process. In addition, treatment ofprostate cancer lines with PUGNAc, a non-cytotoxic reversible inhibitor ofOGlcNAcase, caused a decrease in the expression oftransfected P-catenin in the nucleus with increasing cellular 0-glycosylation ofthe protein suggesting that 0-glycosylation was hindering P-catenin's nuclear translocation. Additional studies showed that 0-glycosylation of P-catenin decreased transcriptional activity of a TopFlash reporter plasmid. In summary, our results show that P-catenin is 0-glycosylated on theN-and C-terminus, but not on ARMADILLO segment, and that phosphorylation sites are not the critical for 0-glycosylation. Furthermore, our data show that 0-glycosylation of P-catenin may represent a novel mechanism important in the regulation of the nuclear localization and transcriptional activity of P-catenin. / Thesis / Master of Science (MSc)

Hemophilia B is a debilitating and life-threatening disorder caused by a deficiency in or dysfunction of factor IX (FIX), a complex plasma glycoprotein required for the formation and maintenance of blood clots. Treatment of hemophilia B involves infusion of replacement FIX currently derived from two sources: FIX purified from pools of human plasma (pd-FIX) and a single recombinant FIX product generated in genetically engineered Chinese hamster ovary (CHO) cells. Both of these FIX products are prohibitively expensive, limiting of the treatment options of hemophiliacs worldwide. As a result, a more abundant and affordable FIX product would greatly improve the life prospects for hemophiliacs. The biological activity of FIX is dependent upon its numerous post-translational modifications (PTMs), including gamma-carboxylation, proteolytic maturation, phosphorylation, sulfation, and glycosylation. Of these PTMs, those known to be vital for activity are gamma-carboxylation of multiple glutamate residues near the N-terminus and proteolytic cleavage of the FIX propeptide. When expressed at a high rate in exogenous expression systems, however, the ability of current systems to effect the necessary PTMs is severely rate limited, restricting the production of active FIX. The transgenic pig bioreactor represents a promising source for the production of large quantities biologically active FIX due to its demonstrated ability to perform the required FIX PTMs. It was the goal of this study to characterize the PTM structure and the resulting function of recombinant FIX when expressed at 1-3 mg/ml in the transgenic pig mammary epithelium (tg-FIX). It was found that the expressed tg-FIX is comprised of a heterogeneous mixture of FIX PTM isoforms. This mixture represents a spectrum of tg-FIX molecules of varying gamma-carboxyglutamic acid (Gla) and propeptide content, indicating that rate limitations in effecting these PTMs are present. A purification process was developed utilizing heparin-affinity chromatography to purify the total population of tg-FIX from pig milk, a complex multi-phase feedstock. Subsequently, a process was developed to fractionate the total population of tg-FIX into subpopulations based upon the extent of post-translational modification. Q ion-exchange chromatography was utilized to fractionate tg-FIX based upon molecular acidity which was found to be correlated to both biological activity and Gla content. The resulting biologically active tg-FIX population contained an average of 7 of the 12 Gla residues found in pd-FIX. Immuno-affinity chromatography was subsequently utilized to further fractionate tg-FIX into mature tg-FIX and propeptide-containing tg-FIX populations. The isolated FIX PTM populations were subjected to functional analysis by investigating in vitro clotting activity, activation by factor XIa, and in vivo pharmacokinetics. From this analysis it was found that mature tg-FIX with an average 7 Gla residues, representing approximately 9% of the total tg-FIX produced, exhibits wild-type in vitro clotting activity and normal activation by factor XIa. The remainder of the tg-FIX produced, characterized by either a lower Gla content or the presence of the propeptide, was found to be inactive and displayed less efficient activation by factor IXa. In an in vivo pharmacokinetic study in the hemophilia B mouse model, biologically active tg-FIX was found to possess altered circulating properties. Tg-FIX was characterized by a lower recovery, approximately one-sixth that of pd-FIX, but an extended circulation half-life. From this study it was found that the mean residence time of tg-FIX after injections is approximately twice that observed for pd-FIX. These altered pharmacokinetic properties are likely linked to the unique tg-FIX PTM structure, perhaps through altered endothelial cell binding characteristics caused by the reduced Gla content. / Ph. D.

The epigenome carries dynamic information that controls gene expression and maintains cell identity during both disease and normal development. The inherent plasticity of the epigenome paves new avenues for developing diagnostic and therapeutic tools for human diseases. Microfluidic technology has improved the sensitivity and resolution of epigenomic analysis due to its outstanding ability to manipulate nanoliter-scale liquid volumes. In this thesis, I report three projects focusing on low-input, cell-type-specific and spatially resolved histone modification profiling on microfluidic platforms. First, I applied Microfluidic Oscillatory Washing-based Chromatin Immunoprecipitation followed by sequencing (MOWChIP-seq) to study the effect of culture dimensionality, hypoxia stress and bacterium infection on histone modification landscapes of brain tumor cells. I identified differentially marked regions between different culture conditions. Second, I adapted indexed ChIPmentation and introduced mu-CM, a low-input microfluidic device capable of performing 8 assays in parallel on different histone marks using as few as 20 cells in less than 7 hours. Last, I investigated the spatially resolved epigenome and transcriptome of neuronal and glial cells from coronal sections of adult mouse neocortex. I applied unsupervised clustering to identify distinct spatial patterns in neocortex epigenome and transcriptome that were associated with central nervous system development. I demonstrated that our method is well suited for scarce samples, such as biopsy samples from patients in the context of precision medicine. / Doctor of Philosophy / Epigenetic is the study of alternations in organisms not caused by alternation of the genetic codes. Epigenetic information plays pivotal role during growth, aging and disease. Epigenetic information is dynamic and modifiable, and thus serves as an ideal target for various diagnostic and therapeutic strategies of human diseases. Microfluidics is a technology that manipulates liquids with extremely small volumes in miniaturized devices. Microfluidics has improved the sensitivity and resolution of epigenetic analysis. In this thesis, I report three projects focusing on low-input, cell-type-specific and spatially resolved histone modification profiling on microfluidic platforms. Histone modification is one type of epigenetic information and regulates gene expression. First, we studied the influence of culture condition and bacterium infection on histone modification profile of brain tumor cells. Second, we introduced mu-CM, combining a low-input microfluidic device with indexed ChIPmentation and is capable of performing 8 assays in parallel using as few as 20 cells. Last, we investigated spatial variations in the epigenome and transcriptome across adult mouse neocortex, the outer layer of brain involving in higher-order function, such as cognition. I identified distinct spatial patterns responsible for central nervous system development using machine learning algorithm. Our method is well suited for studying scarce samples, such as cells populations isolated from patients in the context of precision medicine.

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

Yes / Protein O-GlcNAcylation is a reversible post-translational modification of serines/threonines on nucleocytoplasmic proteins. It is cycled by the enzymes O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (O-GlcNAcase or OGA). Genetic approaches in model organisms have revealed that protein O-GlcNAcylation is essential for early embryogenesis. Drosophila melanogaster OGT/supersex combs (sxc) is a polycomb gene, null mutants of which display homeotic transformations and die at the pharate adult stage. However, the identities of the O-GlcNAcylated proteins involved, and the underlying mechanisms linking these phenotypes to embryonic development, are poorly understood. Identification of O-GlcNAcylated proteins from biological samples is hampered by the low stoichiometry of this modification and limited enrichment tools. Using a catalytically inactive bacterial O-GlcNAcase mutant as a substrate trap, we have enriched the O-GlcNAc proteome of the developing Drosophila embryo, identifying, amongst others, known regulators of Hox genes as candidate conveyors of OGT function during embryonic development. / Wellcome Trust Investigator Award (110061); MRC grant (MC_UU_12016/5); and Royal Society Research Grant.

Ce mémoire porte sur l'évolution de la dynamique des marais intertidaux du moyen estuaire du Saint-Laurent. Depuis la fin des années 1980, des recherches en géomorphologie côtière suggèrent une compréhension partielle des facteurs intrinsèques et extrinsèques influençant l'évolution des marais dans le contexte de changements climatiques. Ce mémoire examine la fiabilité des prédictions d'érosion généralisée annoncées dans ces études en analysant le cas de l'évolution des marais intertidaux de Montmagny. La problématique de l'étude est de comprendre pourquoi, malgré les prédictions d'érosion linéaire, la superficie totale des marais de Montmagny a augmenté depuis les années 1980. L'hypothèse proposée suggère qu'il y a une autocyclicité hydrosédimentaire entre les processus d'érosion vus sur le schorre supérieur et l'accrétion du schorre inférieur. Cette interconnectivité a été bien étudiée en Europe, mais ignorée dans la plupart des études portant sur le Saint-Laurent. L'objectif principal du mémoire est de reconstituer l'évolution morphostratigraphique des marais intertidaux de Montmagny, étudier la dynamique sédimentaire actuelle ainsi qu'identifier et expliquer les changements ayant affecté la superficie totale des marais. La méthodologie privilégiée dans cette étude inclut une analyse critique de la littérature scientifique portant sur les facteurs intrinsèques et extrinsèques influençant l'évolution des marais, une analyse morphostratigraphique et sédimentologique du marais au cours du dernier siècle et une analyse multidate d'une série de photographies aériennes du secteur à l'étude afin de connaître l'évolution des limites du marais. Les résultats appuient l'hypothèse d'une autocyclicité hydrosédimentaire. Ces résultats permettront de revoir la manière dont l'évolution des marais intertidaux et leur avenir sont interprétés. Les résultats mettront en lumière une interconnectivité entre les zones biomorphologiques du haut et du bas schorre avec la slikke. Ces observations suggèrent une interconnectivité entre les différentes zones du schorre et de la slikke qui jouent un rôle important, mais encore mal compris, dans l'évolution des marais. Cette étude permettra d'appliquer, au Québec, des concepts de géomorphologie côtière qui sont évoqués dans la littérature internationale, telle que ceux d'autocyclicité, d'interconnectivité, et de la résilience des marais face aux changements climatiques. Mots clés : Marais intertidal, estuaire moyen, sédimentation, érosion, autocyclicité sédimentaire, géomorphologie côtière, paradigme scientifique.

Abstract: It is well established that ionizing radiation induces a variety of damage in DNA by direct effects that are mediated by one-electron oxidation and indirect effects that are mediated by the reaction of water radiolysis products, e.g., hydroxyl radicals (•OH). In cellular DNA, direct and indirect effects appear to have about an equal effect toward DNA damage. We have shown that ϒ-(gamma) ray irradiation of aqueous solutions of DNA, during which •OH is the major damaging ROS can lead to the formation several lesions. On the other hand, the methylation and oxidative demethylation of cytosine in CpG dinucleotides plays a critical role in the gene regulation. The C5 position of cytosine in CG dinucleotides is frequently methylated by DNA methyl transferees (DNMTs) and constitutes 4-5% of the total cytosine. Here, my PhD research work focuses on the analysis of oxidative base modifications of model compounds of methylated and non methylated oligonucleotides, isolated DNA (calf-thymus DNA) and F98 cultured cell by gamma radiation. In addition, we identified a series of modifications of the 2-deoxyribose moiety of DNA arising from the exposure of isolated and cellular DNA to ionizing radiation. We also studied one electron oxidation of cellular DNA in cultured human HeLa cells initiated by intense nanosecond 266 nm laser pulse irradiation, which produces cross-links between guanine and thymine bases (G*-T*). To achieve these goals, we developed several methods based on mass spectrometry to analyze base modifications in isolated DNA and cellular DNA. / Résumé : Les radiations ionisantes induisent une variété de dommages à l'ADN selon des effets directs, correspondant à une oxydation suite à l’éjection d’un électron, et indirecte, médiés par une réaction avec les produits issus de la radiolyse de l’eau environnante, tels que les radicaux hydroxyles (•OH). Au sein d’une cellule, l’importance relative des effets directs et indirects semble être quantitativement similaire en ce qui concerne les dommages induits à l'ADN cellulaire. Nous avons démontré que l'irradiation par rayons Υ-(gamma) de solutions aqueuses d'ADN, dont l’action délétère est principalement véhiculé e par l’intermédiaire des radicaux hydroxyles, peut induire sur l’ADN la formation de toute une palette de modifications. D'autre part, la méthylation et la déméthylation oxydative de la cytosine au sein de couples de dinucléotides CpG jouent un rôle essentiel dans la régulation des gènes. La position C5 de cette cytosine se retrouve fréquemment méthylée par les méthyltransférases (DNMTs) et constitue alors 4-5% de l’ensemble de la cytosine présente au sein de l’ADN. Mon projet de recherche est centralisé autour de l'analyse de la modification des bases de l’ADN suite à leur oxydation dans des composés modèles constitués d'oligonucléotides méthylés et non-méthylés, puis dans l'ADN isolé (extrait de cellules de thymus de veau) et enfin au sein de cultures cellulaires F98 ayant subies une irradiation par rayons Υ-(gamma). De plus, nous avons identifié une série de modifications spécifiques au groupement fonctionnel 2-désoxyribose de l'ADN résultant de l'exposition de l'ADN isolé et cellulaire aux rayonnements ionisants. Nous avons également étudié les conséquences de l’irradiation par des impulsions lasers nanoseconde à 266 nm de cultures cellulaires de lignée humaine (HeLa). Responsable d’une réaction d’oxydation suite à l’éjection d’un électron, l’identification des modifications induites à l’ADN cellulaire suite à l’irradiation laser a permis de mettre en évidence des pontages ADN-ADN caractéristiques entre les bases guanine et thymine (G*-T*). Pour atteindre ces objectifs, nous avons développé plusieurs méthodes d’analyse des modifications de bases au sein de l’ADN isolé et de l'ADN cellulaire basées sur la spectroscopie de masse.