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No description available.

E-selectin is an endothelial adhesion molecule important in the recruitment of leukocytes to the bone marrow and inflammatory tissues; moreover, it has also been implicated in cancer metastasis and as a critical mediator of chemoresistance. This study investigated the effects triggered by the binding of E-selectin on acute myeloid leukemia (AML) cell lines regarding the activation of significant signaling pathways and their impact on biological functions. We observed that upon treatment with a recombinant E-selectin construct on AML cells, there was an activation of the AKT/NF-κB pathways, which are known to be central regulators of many cellular processes, including survival and proliferation. We found that the E-selectin-mediated activation varied in rate and amplitude among AML cell lines spanning differentiation blockage at different stages. Furthermore, we found that E-selectin binding in HL-60 and KG1-a cells sensitized the cells to daunorubicin (DNR), a chemotherapy drug commonly used to treat AML. The observed chemosensitivity could be linked to the decrease of Aldo Keto Reductases (AKR) protein levels upon E-selectin treatment, which is known to metabolize DNR and reduce its cytotoxicity. Additionally, we explored the role of exosomes as a regulator of the generation of therapy resistance by examining the effects treatment with KG1-a derived exosomes, a cell line that exhibits higher chemoresistance compared to other AML cell lines, had on viability in HL-60 cells upon chemotherapy. We found that even in the absence of KG1-a cells, exosomes were sufficient to provide an increase in chemoresistance. Overall, these studies explore properties exerted by AML cells that could lead to further understanding of AML and thus the development of potential therapeutic targets to overcome challenges currently found in the treatment of this disease.

Protein phosphorylation is utilized universally as a mechanism of signal transduction. However, the use of tyrosine phosphorylation by bacteria has been a matter of dispute. Conventional wisdom dictated that "prokaryotic phosphorylation" was typified by phosphorylation of histidine and aspartate residues of proteins, while "eukaryotic phosphorylation" was characterized by modification of serine, threonine, or tyrosine residues. Increasing numbers of reports have emerged challenging the traditional view of "prokaryotic" and "eukaryotic" phosphorlyation. One of the strongest links unifying prokaryotic and eukaryotic protein phosphorylation to date is IphP, a genomically-encoded dual-specificity protein phosphatase from the cyanobacterium Nostoc commune UTEX 584 bearing the active-site signature sequence of eukaryotic tyrosine-specific and dual-specificity protein phosphatases. The catalytic properties and substrate specificity of IphP were examined in detail. The enzyme was able to discriminate among a variety of exogenous peptides and proteins. Kinetic studies revealed that IphP favors protein / peptide substrates over low molecular weight compounds. Heparin effected IphP activity in a substrate-dependent manner. Enzyme activity toward casein (P-Ser) and MAP kinase (P-Thr/P-Tyr) was stimulated in the presence of the polyanion, whereas activity was inhibited by heparin toward other protein substrates. Both stimulation and inhibition by heparin were dose-dependent. The ability to stimulate IphP activity toward select substrates was attributed to the ability of heparin to recruit the enzyme and substrate to the same microenvironment. To facilitate future genetic studies examining the role of tyrosine phosphorylation in cyanobacteria, we searched for evidence of protein tyrosine phosphorylation in Anabaena PCC 7120. In a collaborative effort with the laboratory of Dr. Potts, tyrosine phosphorylated proteins were identified in Anabaena utilizing several approaches, including comparative labelling with alpha- vs gamma-32P-ATP, phosphoamino acid analysis, and selective hydrolysis with a tyrosine specific protein phosphatase. Together, these data unequivocally demonstrate the presence of tyrosine-phosphorylated proteins in Anabaena PCC 7120. Extracts of Anabaena PCC 7120 were examined for protein tyrosine phosphatase activity. An apparent PTP activity was detected, partially purified, and characterized. The protein phosphatase was ~38kDa by SDS-PAGE and sucrose density gradient centrifugation and displayed dual-specificity protein phosphatase (DSP) activity in vitro. The enzyme was localized to the periplasm and was thus assigned the title PAD, for Periplasmic Anabaena DSP. Periplasmic phosphoproteins of ~120 and 55 kDa that had been radiolabelled in vitro were dephosphorylated by partially purified PAD. PAD activity varied in vivo ~5-fold in a rhthymic, seemingly diurnal manner. Periplasmic proteins, including the 55kDa protein, were labelled in vivo and the degree of radiolabel incorporated into these proteins varied inversely with PAD activity. / Ph. D.

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

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

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

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

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

La protéine multifonctionnelle p54nrb est impliquée dans le contrôle transcriptionnel couplée aux phénomènes de maturation des ARN messagers. La transcription et l'épissage sont inhibés en mitose et la phosphorylation de nombreux facteurs participe à ce phénomène. Dans le laboratoire, il a été montré que p54 est hyperphosphorylée en mitose et interagit de façon phosphodépendante avec le régulateur mitotique Pinl (enzyme qui isomérise un lien proline précédé d'un site phosphoryle Thréonine/Sérine). Le rôle de cette interaction est méconnu mais permet de dire que p54 est régulé par phosphorylation en entrée de mitose. La protéine p54 est diffuse dans le nucléoplasme et est également concentrée avec ses partenaires PSF et PSP1 dans les paraspeckles, des compartiments nucléaires à structure dépendante d'un ARN non codant NEAT1. Il a été récemment montré qu'une association entre l'ARN qui structure les paraspeckles et p54nrb permet de maintenir l'intégrité de ces compartiments. Étant donné l'implication de p54nrb dans de nombreux procédés majeurs, l'objectif de ma thèse a été de comprendre le rôle de la phosphorylation mitotique de p54nrb. La localisation, les interactions protéiques et les propriétés de liaison à l'ARN ont été étudiées. Ces expériences ont permis de conclure que p54nrb reste en complexe avec les protéines PSF et PSP1 mais perd son affinité de liaison à l'ARN en mitose via la phosphorylation d'un résidu en amino-terminal (T15). Par contre, la liaison aux homoribopolymères de Guanine (G) ainsi qu'avec l'ARN non codant NEAT1 (riche en G) ne sont pas compromises par la phosphorylation mitotique. Ces résultats ont permis d'établir de nouvelles propriétés fonctionnelles pour la protéine p54nrb. Par ailleurs, j'ai aussi voulu caractériser la phosphatase responsable de sa déphosphorylation en fin de mitose et les premiers résultats impliquent l'action de la phosphatase PPL De plus, le rôle de l'interaction entre Pinl et p54nrb a été étudié dans la possibilité que Pinl puisse être impliqué dans le processus de déphosphorylation de p54nrb. En conclusion, l'ensemble des travaux de cette thèse a permis de mieux caractériser les fonctions de la protéine multifonctionnelle p54nrb notamment dans le cadre de sa phosphorylation mitotique.