Tyrosyl phosphorylation plays an important role in many fundamental cellular processes, including cell growth, differentiation and proliferation. The levels of phosphotyrosine (pY) are regulated by the opposing actions of protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs). A limitation to understanding the roles of PTPs in physiological and pathological cell signaling has been the absence of global proteomic approaches that enable the systematic and comprehensive analysis of PTP expression, regulation and function. This dissertation describes the development and application of novel proteomic methodologies that permit the global analysis of PTP expression (qPTPome), regulation (by oxidation and nitrosylation; q-oxPTPome) and substrates/binding proteins. These methods provide a workflow to begin assessing PTP function at a systems level, rather than its current targeted format. Application of these techniques will provide invaluable information to begin bridging the gap in our understanding of PTP and PTK function in normal and malignant cell signaling.

Several analytical techniques including use case diagrams, process flow diagrams (PFDs), hierarchical task analysis (HTA), failure mode and effects analysis (FMEA), systematic human error reduction and prediction approach (SHERPA), hazard analysis and critical control point (HACCP), heuristics, the Safe Living Guide and the Resident Assessment Instrument – Home Care (RAI-HC) are applied to data obtained from two pilot home visits to determine whether common failure modes, causes, effects and recommendations are yielded by the techniques. The time required to apply each analytical technique to processes uncovered from the pilot data was measured and outputs of the techniques were reviewed for commonality. Of the tools considered, SHERPA was found to return the most failure modes, effects and recommendations, while FMEA was the only human factors tool to yield causes. Additionally, FMEA and SHERPA provided a means of ranking potential failure modes based on severity and probability.

Several analytical techniques including use case diagrams, process flow diagrams (PFDs), hierarchical task analysis (HTA), failure mode and effects analysis (FMEA), systematic human error reduction and prediction approach (SHERPA), hazard analysis and critical control point (HACCP), heuristics, the Safe Living Guide and the Resident Assessment Instrument – Home Care (RAI-HC) are applied to data obtained from two pilot home visits to determine whether common failure modes, causes, effects and recommendations are yielded by the techniques. The time required to apply each analytical technique to processes uncovered from the pilot data was measured and outputs of the techniques were reviewed for commonality. Of the tools considered, SHERPA was found to return the most failure modes, effects and recommendations, while FMEA was the only human factors tool to yield causes. Additionally, FMEA and SHERPA provided a means of ranking potential failure modes based on severity and probability.

Objective: determine whether accreditation through Accreditation Canada is associated with more favorable resident safety in Ontario LTC homes and which facility characteristics are predictive of accreditation. Methods: logistic regression was used to determine predictors of accreditation. To examine the association between accreditation and safety, safety was operationalized as five MDS-RAI quality indicators: prevalence of falls, restraints, catheters, pressure ulcers, and infections. Separate multivariable models were developed for each indicator. Results: the odds of accreditation were approximately six times smaller for municipal (p < 0.001) and non-profit facilities (p < 0.001) relative to for-profits; three times greater for chains relative to non-chains (p < 0.001); and twice as large for urban relative to rural facilities (p = 0.04). Of the five quality indicators examined, only one (falls) was associated with accreditation. After adjusting for confounders, accredited homes were estimated to have 8% lower fall rates than non-accredited homes (p = 0.01).

La problématique du consentement éclairé en santé mentale demeure au coeur des préoccupations des cliniciens, médecins spécialistes et médecins experts. Le travail auprès des cérébrolésés ou des patients souffrant de troubles mentaux graves, tout comme les questions qui me sont adressées depuis près de 20 ans par les médecins spécialistes, juristes ou résidents en psychiatrie, m’ont amenée à y réfléchir davantage. J’ajouterais que le constat personnel d’une compréhension des comportements, attitudes, motivations et jugements des patients vulnérables qui s’est modifiée au fil des ans, et le constat de l’importance de la notion du consentement vs le flou de sa définition et la fragilité des paramètres établis pour l’évaluer et la définir ont ravivé cette réflexion. La présente étude n’a aucunement pour but d’élaborer quelque règle de conduite que ce soit, ni de définir ce que devrait être le consentement éclairé en psychiatrie, mais plutôt d’explorer les dilemmes éthiques et les questionnements cliniques auxquels sont confrontés les médecins psychiatres afin de raviver une réflexion éthique qui semble s’estomper au profit de procédures juridiques et administratives. / In the mental health field, the topic of informed consent has always been among the most important problems to address for clinicians and psychiatrists. My clinical work with head injured patients and patients with severe mental health disorder, as well as all the questions addressed by physicians, residents or lawyers for the last decade lead me to think about it more deeply. Moreover, the personal observation of cognitively impaired patients and the fact that the concept of informed consent is so present compared to the lack of parameters to assess it has lead me to think about it otherwise. This study does not pretend to lead the actions or clinical behaviour, nor as it pretend to find a better definition of the concept of informed consent. It is only a way to explore some aspects of the complexity and clinical difficulties over the legal and administrative frame in which the medical field is evolving.

Master of Science / Department of Clinical Sciences / Thomas Schermerhorn / Tonicity is property of a solution that is defined as the total effective (impermeable) osmole concentration that drives fluid movement across a semipermeable membrane via osmosis. Tonicity is related to but distinct from solution osmolality, which is a summation of all solute concentrations, regardless of the solute membrane permeability. In the mammalian body, tonicity is tightly regulated at both a cellular and systemic level; tonic derangements cause rapid change in cell and tissue volume leading to significant dysfunction. Input from the central nervous, circulatory, endocrine, gastrointestinal, and urinary systems are integral to osmoregulation, so many diseases in veterinary medicine are associated with tonicity disorders. However, because the homeostatic mechanisms that control tonicity overlap with those regulating electrolyte and acid-base balance as well as hydration and vascular volume, tonic consequences of disease can be difficult to isolate. Understanding of disease-associated changes in tonicity is further complicated by the fact that the tonic contributions of many solutes that accumulate in disease are unknown. Additionally, direct assessment of tonicity is difficult because tonicity is not just a physiochemical property, but it implies a physiologic effect. Thus, simple summation of osmole concentrations is an inadequate measurement of tonicity. The following report includes three studies investigating various aspects of tonicity as it applies to veterinary patients. Chapter 2 reports a study that examines the tonic effects of ketoacids and lactate using two different in vitro red blood cell assays. Results demonstrated that the ketoacids, beta-hydroxybutyrate and acetoacetate, behave as ineffective osmoles while the tonic behavior of lactate is variable, implying a more complex cellular handling of this anion. Two additional studies examine whether the mean corpuscular volume difference (dMCV) is a novel clinical marker for hypertonicity in dogs. Results of separate retrospective (Chapter 3) and prospective (Chapter 4) studies provide evidence that dMCV is a useful clinical marker for hypertonicity in dogs.

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Il a été démontré que la sirtuine 1 (Sirt-1), une histone désacétylase de classe III, est surexprimée dans le coeur des rats spontanément hypertendus (SHR). Nous avons récemment montré que les cellules musculaires lisses vasculaires (CMLV) des SHR présentent une expression accrue de Sirt-1 par rapport aux rats Wistar Kyoto (WKY) de même âge qui contribue à l’augmentation de la régulation de la protéine Giα impliquée dans la pathogenèse de l'hypertension. La présente étude a été effectuée pour étudier le rôle de l'augmentation de l'expression de la Sirt-1 dans la pathogenèse de l'hypertension chez les SHR et pour explorer les mécanismes moléculaires impliqués dans cette réponse. Dans cette étude, un inhibiteur sélectif de la Sirt-1, EX-527 (5 mg/kg de poids corporel), a été injecté par voie intrapéritonéale chez des rats SHR adultes de 8 semaines et des rats WKY de même âge, deux fois par semaine pendant 3 semaines. La pression artérielle (PA) et la fréquence cardiaque ont été mesurées deux fois par semaine par la méthode non invasive du brassard autour de la queue. Le traitement avec l’inhibiteur spécifique de la Sirt-1, l'EX-527, a atténué les augmentations de PA (de 76 mmHg) et de fréquence cardiaque chez les rats SHR. La surexpression de Sirt-1 et des protéines Giα dans le coeur, les CMLV et l'aorte a été atténuée au niveau des contrôles par l'inhibiteur de la Sirt-1. L'inhibition de la Sirt-1 a également atténué les niveaux accrus des anions superoxydes, l’activité de la NADPH oxydase et la surexpression des sous-unités de la NADPH oxydase ; les protéines Nox2, Nox4 et P47phox dans les CMLV isolées des SHR traités par l’EX-527. De plus, les niveaux réduits du monoxyde d'azote synthase endothélial (eNOS) et du monoxyde d'azote (NO) et les niveaux accrus de la peroxynitrite (ONOO-) dans les CMLV des SHR ont également été rétablis à des niveaux contrôles par l'inhibiteur de la Sirt-1. Ces résultats suggèrent que l'inhibition de la surexpression de la Sirt-1, en diminuant les niveaux accrus des protéines Giα et du stress nitro-oxydant, atténue la PA élevée chez les rats SHR. Il est donc possible de suggérer que les inhibiteurs de la Sirt-1 puissent être utilisés comme des agents thérapeutiques dans le traitement des complications cardiovasculaires associées à l'hypertension. / Sirtuin-1 (Sirt-1), class III histone deacetylase, has been shown to be overexpressed in hearts from spontaneously hypertensive rats (SHR). We recently showed that vascular smooth muscle cells (VSMC) from SHR exhibit enhanced expression of Sirt-1 as compared to age-matched Wistar Kyoto (WKY) rats, which contributes to the upregulation of Giα protein implicated in the pathogenesis of hypertension. The present study was undertaken to investigate the role of upregulated Sirt-1 expression in the pathogenesis of hypertension in SHR and to explore the underlying molecular mechanisms involved in this response. For this study, a selective inhibitor of Sirt-1, EX-527 (5mg/kg of body weight), was injected intraperitoneally into 8-week-old adult SHR and age-matched WKY rats twice per week for 3 weeks. The blood pressure (BP) and heart rate was measured twice a week by the CODA™ non-invasive tail cuff method. Treatment of SHR with Sirt-1-specific inhibitor, EX-527, attenuated high BP by 76 mmHg and inhibited the augmented heart rate. The overexpression of Sirt-1 and Giα proteins in heart, VSMC and aorta was attenuated to the control levels by Sirt-1 inhibitor. Inhibition of Sirt-1 also attenuated the enhanced levels of superoxide anion, NADPH oxidase activity and the overexpression of NADPH oxidase subunits; Nox2, Nox4 and P47phox proteins in VSMC isolated from EX-527-treated SHR. Furthermore, the decreased levels of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) and increased levels of peroxynitrite (ONOO-) in VSMC from SHR were also restored to control levels by Sirt-1 inhibitor. These results suggest that the inhibition of overexpression of Sirt-1 through decreasing the enhanced levels of Giα proteins and nitro-oxidative stress attenuates the high BP in SHR. It may thus be suggested that inhibitors of Sirt-1 may have the potential to be used as therapeutic agents in the treatment of cardiovascular complications associated with hypertension.

Les plaquettes sont considérées comme l'un des réservoirs les plus importants non seulement des facteurs de croissance, mais aussi des facteurs neurotrophiques qui pourraient contribuer à la réparation des lésions vasculaires et à la prévention de la détérioration neurologique. Parmi ces facteurs, le facteur neurotrophique dérivé du cerveau (Brain-Derived Neurotrophic Factor ou BDNF) – une protéine appartenant à la famille des neurotrophines– est largement exprimée à la fois dans l'hippocampe et au niveau des plaquettes. Les plaquettes constituent un important réservoir de BDNF; cependant, on ne sait que peu de choses sur les facteurs modulant la libération de ce dernier dans la circulation et si les médicaments antiplaquettaires affectent cette sécrétion. Dans le cadre de ce projet, nous avons émis l’hypothèse principale que les différentes voies d’activation plaquettaire peuvent mener à une libération de BDNF, où celle-ci est affectée par les antiplaquettaires. A cette fin, les plaquettes ont été isolées à partir d’échantillons sanguins de volontaires sains (Groupe 1), de patients souffrant de maladies cardiovasculaires stables requérant la prise de médicaments antiplaquettaires [en prévention secondaire et en double thérapie à l’acide acétylsalicylique (ASA ou Aspirine) en association avec un antagoniste du récepteur P2Y12], (Groupe 2) ou en monothérapie à l’ASA (Groupe 3), versus de patients atteints de maladies valvulaires ou de cardiomyopathies ne requérant pas la prise de médicaments antiplaquettaires (Groupe 4). L’agrégation plaquettaire a été étudiée par agrégométrie optique en réponse à des agonistes spécifiques : adénosine diphosphate (ADP), acide arachidonique (AA), épinéphrine, collagène et Thrombin-receptor activated peptide 6 (TRAP-6 amide). Les antiplaquettaires testés sont dirigés contre la cyclo-oxygénase-1 ou COX-1 (ASA), contre le récepteur de P2Y12 de l’ADP (AR-C) et contre le récepteur αIIbβ3 du fibrinogène (Abciximab). La libération du BDNF a été quantifiée par ELISA. La présence du BDNF et de son récepteur Tropomyosin-Related Kinase Receptor type B (TrKB) a été détectée par immunobuvardage. Nous avons montré que l’activation des plaquettes par les différents agonistes testés induit une agrégation plaquettaire de l’ordre de 80% et permet de libérer jusqu’à 5 fois plus de BDNF, passant de 2500 pg / 250 x 106 plaquettes à l’état basal à approximativement 13000 pg / 250 x 106 plaquettes à l’état stimulé. Tous les antiplaquettaires testés réduisent la libération de BDNF par les plaquettes stimulées. Cependant, le niveau d’inhibition et sa significativité dépendent de la nature de l’agoniste; à savoir que l’ASA réduit significativement la sécrétion de BDNF en réponse à l’AA, à l’épinéphrine et au TRAP-6; alors que l’AR-C était plus efficace en réponse à l’ADP, l’AA et l’épinéphrine. L’Abciximab est un antagoniste qui inhibe la sécrétion de BDNF en réponse à tous les agonistes, en inhibant aussi l’agrégation plaquettaire. Notons que la libération de BDNF en réponse au collagène est inhibée par l’ASA et l’AR-C, alors que l’agrégation n’a pas été affectée. Ainsi, aucune corrélation positive et significative entre l’agrégation plaquettaire et la libération de BDNF n’a pu être obtenue. La présence des antiplaquettaires réduits à différents degrés la libération de BDNF chez les différents groupes des patients, malgré que son expression intraplaquettaire était similaire entre les groupes. On remarque que les antiplaquettaires réduisent plus significativement la quantité du BDNF relâchée chez les patients sous mono ou double thérapie antiplaquettaire en comparaison avec les volontaires sains et les patients atteints de maladies valvulaires. Nous avons aussi démontré que le BDNF exogène active les plaquettes isolées et lavées chez les volontaires sains, en induisant une forte agrégation stable et irréversible. Par contre, le BDNF exogène n’arrive pas à agréger les plaquettes en plasma riche en plaquettes. De plus, nos résultats indiquent que la forme tronquée du récepteur BDNF, le TrKB, est exprimée au niveau des plaquettes de volontaires sains. L’inhibition de l’activité kinase du TrKB abolit l’agrégation induite par le BDNF. Ces résultats suggèrent que l’action du BDNF dans les plaquettes lavées pourrait passer par l’intermédiaire du TrKB. Cette étude nous permet de conclure que le BDNF est présent dans les plaquettes et est libéré suite à l’activation plaquettaire et que cette libération est réduite par les antiplaquettaires. Cependant, l’agrégation plaquettaire ne semble pas être associée directement à la sécrétion du BDNF, ce qui suggère que d’autres mécanismes sous-jacents pourraient intervenir dans le contrôle de cette sécrétion. Les antiplaquettaires réduisent la libération de BDNF et il semble que l’action pro-agrégante du BDNF sur les plaquettes lavées passe par l’intermédiaire du TrKB, sans exclure la possibilité que d’autres types de récepteurs plaquettaires soient impliqués dans le signal déclenché par le BDNF. L’implication physiopathologique du BDNF libéré suite à l’activation plaquettaire ou sa biodisponibilité en présence des antiplaquettaires au niveau cardiovasculaire reste à être élucidée afin de révéler son potentiel diagnostique ou thérapeutique. / Platelets are considered one of the most important reservoirs not only of growth factors but also of neurotrophic factors that may contribute to the repair of vascular lesions and prevention of neurological deterioration. Among these factors, the Brain-Derived Neurotrophic Factor (BDNF), a protein belonging to the neurotrophin family, is largely expressed in both the hippocampus and platelets. In fact, platelets constitute an important reservoir of BDNF; however, little is known about the factors controlling its release into the circulation and whether antiplatelet drugs affect this secretion. Henceforth, the main hypothesis of this project is that platelet activation pathways lead to BDNF release which is affected by antiplatelet agents. For this purpose, platelets were isolated from the blood of four groups of human subjects following their consent. Group 1 consisted of healthy volunteers; Group 2 and Group 3 consisted of patients with stable cardiovascular disease on, respectively, dual antiplatelet therapy (aspirin + P2Y12 receptor antagonist) or monotherapy (aspirin) as secondary prevention; and Group 4 consisted of patients with valvular disease or cardiomyopathy who are not on antiplatelet therapy. Platelet aggregation was studied by optical aggregometry in response to the following agonists: adenosine diphosphate (ADP), arachidonic acid (AA), epinephrine, collagen, and thrombin-receptor activated peptide 6 (TRAP-6 amide). The antiplatelet agents that were tested antagonize cyclooxygenase-1 (COX-1) (acetylsalicylic acid (ASA) or aspirin), ADP P2Y12 receptor (AR-C), and fibrinogen receptor αIIbβ3 (Abciximab). BDNF release was quantified by ELISA. BDNF protein and its Tropomyosin-Related Kinase Receptor Type B (TrKB) receptor were detected by immunoblotting. Our results show that platelet activation in response to several agonists tested induced 80% platelet aggregation and augmented BDNF release by 5 folds, from 2500 pg / 250 x 106 platelets at baseline to approximately 13000 pg / 250 x 106 after stimulation. Moreover, all the tested antiplatelet agents reduced the release of BDNF by stimulated platelets. However, the level of reduction varied differentially between platelet antagonists depending on the platelet agonist used. Indeed, ASA significantly reduced BDNF secretion in response to AA, epinephrine, and TRAP-6, whereas AR-C was more effective in response to ADP, AA, and epinephrine. Abciximab inhibited BDNF secretion as well as platelet aggregation in response to all agonists. Noteworthy, the release of BDNF in response to collagen was inhibited by ASA and AR-C, while platelet aggregation was not affected. Accordingly, no significant correlation between platelet aggregation and BDNF release could be obtained. Although intra-platelet expression was similar in the different groups, the presence of antiplatelet agents reduced the release of BDNF to varying degrees between groups. As such, antiplatelet agents reduced BDNF release more significantly in patients on dual or mono antiplatelet therapy (Groups 2 and 3) as compared to healthy volunteers (Group 1) and valvular disease patients (Group 4). We have also shown that exogenous BDNF activated isolated/washed platelets from healthy volunteers, inducing strong, stable, and irreversible aggregation. In contrast, exogenous BDNF could not induce aggregation of platelets in platelet-rich plasma. In addition, our results indicate that the truncated form of the BDNF receptor, TrKB, is expressed in platelets of healthy volunteers. Hence, the inhibition of TrKB kinase activity abolished BDNF-induced aggregation. These results suggest that the action of BDNF in washed platelets might ensue through TrKB. We conclude from this study that BDNF is present in platelets and released following platelet activation, and its release is reduced by antiplatelet agents. However, platelet aggregation does not appear to be directly associated with BDNF secretion, suggesting that other underlying mechanisms may be involved in controlling its secretion. Antiplatelet agents reduce the release of BDNF, and it appears that the pro-aggregating action of BDNF on washed platelets ensues, non-exclusively, through TrKB, which means that other types of platelet receptors may also be involved in BDNF signaling. The pathophysiological implication of BDNF released following platelet activation or its bioavailability in the presence of antiplatelet agents in the cardiovascular system thus remain to be elucidated in order to reveal its diagnostic or therapeutic potential.