Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: This study describes: 1. The isolation and the purification of tissue type plasminogen activator and urokinase plasminogen activator in bovine milk. 2. The biochemical characterisation of tissue type plasminogen activator in bovine milk. 3. An investigation of the influence of the addition of purified tissue type plasminogen activator to ultra high temperature milk, Gouda cheese and yoghurt. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf: 1. Die isolering en suiwering van weefseltipe-plasminogeenaktiveerder en urokinase-plasminogeenaktiveerder in beesmelk. 2. Die biochemiese karakterisering van weefseltipe-plasmingeenaktiveerder in beesmelk. 3. `n Ondersoek na die invloed van die byvoeging van gesuiwerde weefseltipe-plasminogeenaktiveerder by ultra hoë temperatuur melk, Gouda kaas en joghurt.

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Class of 2006 Abstract / Objectives: To determine the cost-effectiveness of urokinase (UK) and alteplase (recombinant tissue plasminogen activator, rt-PA) when used intra-arterially for the treatment of acute peripheral arterial occlusions. Methods: A decision analytic model was constructed using TreeAge Pro 2005 Suite to determine the cost- effectiveness of these therapies. Data for costs and probabilities were collected from published literature, as well as other references. Average and incremental cost-effectiveness ratios were calculated with a 95% confidence interval. A two-dimensional (sampling plus trials) Monte Carlo analysis with 5,000 patients was performed, along with a sensitivity analysis of the costs and variables. The costs measured were direct medical costs from the perspective of the healthcare institution. The primary outcome variable assessed in this model was 30-day survival. Results: The Monte Carlo microsimulation indicated that average cost-effectiveness (C/E) ratio for rt-PA was $54,141 (95th CI: 44,647 to 62,832) per successful treatment, while the average C/E ratio for UK was $65,515 (95th CI: 56,286 to 76,135). The ICER for rt-PA versus UK as the baseline was calculated to be $284,170 per additional survival over 30 days (95th CI: 186,097 to 418,443). Neither strategy was dominant. Conclusions: This study found rt-PA to be less costly but also slightly less efficacious than UK for patients treated for acute arterial occlusions. Neither therapy was indicated to be dominant over the other in terms of 30-day survival. Further long-term outcome data is necessary to more extensively assess the benefits of each therapy.

La dépression majeure représente l’une des affections les plus lourdes dans le monde, touchant plus de 350 millions depersonnes. La 5e édition du Diagnostic and Statistical Manual of Mental Disorders (DSM-V) est la référence mondiale utiliséepour poser le diagnostic de la pathologie chez l’humain. Bien que très nombreux, les antidépresseurs prescrits à ce jour restentencore malheureusement inefficaces pour 30% des patients. Dans ce contexte, il est fondamental de développer de nouvellesstratégies thérapeutiques pour soigner les patients. Des études récentes suggèrent, sans toutefois le démontrer véritablement,l’implication de l’axe « activateur tissulaire du plasminogène / inhibiteur de l’activateur tissulaire du plasminogène de type 1 »(axe tPA/PAI-1) dans la pathogenèse de la dépression majeure.La première partie de mes travaux a été consacrée à la mise au point d’un nouveau système d’évaluation comportementalede la dépression majeure chez la souris en modélisant de manière exhaustive et standardisée les symptômes cliniques du DSMV.La seconde partie de mes travaux a consisté à étudier les mécanismes d’action potentiels de l’axe tPA/PAI-1 dans ladépression majeure. Pour ce faire, j’ai tout d’abord caractérisé le phénotype comportemental de souris déficientes en tPA (souristPA-/-) et en PAI-1 (souris PAI-1-/-), ainsi que de leurs homologues de type sauvage, grâce au système fonctionnel d’évaluationinitialement mis en place. Par ailleurs, du fait de la forte comorbidité entre anxiété et dépression, les comportements de typeanxieux ont également été analysés chez ces animaux. Mes expériences ont révélé un phénotype de type dépressif, indépendantdu tPA, chez les souris déficientes en PAI-1, associé à des diminutions des concentrations de deux monoamines (sérotonine etdopamine) dans des structures cérébrales connues pour être impliquées dans la dépression majeure (hippocampe et noyau du litde la strie terminale). De surcroît, l’enrichissement modéré de l’environnement n’amenuise pas les symptômes de type dépressifdes souris PAI-1-/- mais conduit cependant à la disparition des troubles anxieux dépendants, quant à eux, de l’axe tPA/PAI-1.La troisième partie de ma thèse a été dédiée à des manipulations pharmacologiques visant à tester l’efficacitéd’antidépresseurs de type inhibiteurs de la recapture de la sérotonine. L’escitalopram produit un effet anxiolytique chez les sourisdéficientes en PAI-1, sans toutefois contrebalancer le phénotype dépressif chez ces mêmes sujets. Qui plus est, la fluoxétine, àla même dose que l’escitalopram, est toxique pour ces souris.Les résultats de ma thèse apportent ainsi la première démonstration de l’implication de PAI-1 dans la dépression majeurepar un mécanisme indépendant de son interaction avec le tPA. Ces travaux démontrent également que la souris PAI-1-/- constitueun outil essentiel et innovant pour étudier les mécanismes cellulaires et moléculaires sous-jacents à la dépression majeure, ainsique pour la recherche de cibles thérapeutiques visant à améliorer l’efficacité des traitements. / Major depressive disorder is one of the heaviest mental disorders in the world, affecting more than 350 people worldwide.It is in the fifth edition of Diagnostic and Statistical Manual of Mental Disorders (DSM-V) that the basis for an internationallyadmitted diagnosis was laid. Albeit diverse, existing antidepressants still remain ineffective for 30% of the patients. Under suchcircumstances, the necessity of developing new therapeutical strategies has arisen. Recent studies tend to suggest, withoutabsolute demonstration, the implication of the axis "Tissue Plasminogen Activator / Plasminogen Activator Inhibitor type-1"(tPA/PAI-1 axis) in the pathogenesis of major depressive disorders.The first section of my works has been devoted to the development of a new system of behavioural assessment in micefor depressive-like disorders, through a comprehensive and standardised modelling of clinical symptoms of DSM-V.The second section of my works has consisted in studying the potential action mechanisms of the tPA/PAI-1 axis in theemergence of depressive-like disorders. To do so, I first had to identify the behavioural phenotype of mice having from a tPA(tPA-/- mice) and PAI-1 (PAI-1-/-mice) deficiency as well as their wild-type counterparts through the system of assessment setup in the beginning of my research. In addition, due to the significant comorbidity between anxiety and depression, anxious-likebehaviours have been analysed as well. Among PAI-1-deficient mice, my experiments have disclosed a depressive-likephenotype, independent of tPA, and correlated with a decrease in the concentration of two monoamines (serotonin and dopamine)in brain structures known to be involved in major depressive disorder (hippocampus and bed nucleus of the stria terminalis).Besides, the moderate enrichment of the environment does not reduce the depressive-like symptoms of PAI-1-/- mice, yet inducesthe dissipation of dependent-tPA/PAI-1 axis anxious disorders.The third section of my PhD has been devoted to pharmacological experiments meant to assess the effectiveness ofantidepressants classified among selective serotonin reuptake inhibitors (SSRIs). Escitalopram produces anxiolytic falloutsamong PAI-1-deficient mice without for all that offsetting the depressive phenotype among these same mice. Moreover,fluoxetine administered in the same concentration as escitalopram has proven to be toxic for these mice.The results of these doctoral experiments have therefore demonstrated for the first time the implication of PAI-1 in theprocess of major depressive disorder through a mechanism independent from its interaction with tPA. These works have alsodemonstrated that PAI-1-/- mice make up a fundamental and cutting edge tool to study the cellular and molecular mechanismsunderlying major depressive disorder as well as to develop competent therapeutical targets intended to improve the efficiency oftreatments.

L’activateur tissulaire du plasminogène (tPA) est une sérine protéase initialement découverte dans le sang pour sa capacité à convertir le plasminogène en plasmine, une enzyme capable de dégrader les chaînes de fibrine des caillots sanguins. Pour cette fonction pro-fibrinolytique, le tPA est le seul traitement pharmacologique aujourd’hui utilisé dans la phase aiguë de l’accident vasculaire cérébral (AVC) de type ischémique, même s’il présente plusieurs limites. Outre son rôle dans la fibrinolyse, le tPA est aussi capable de moduler différents phénomènes physiologiques et pathologiques au sein du système nerveux central et de l’unité neurovasculaire, tels que la mémoire, l’excitotoxicité ou encore le couplage neurovasculaire comme décrit plus récemment. Plusieurs fonctions du tPA impliquent son interaction avec les récepteurs N-Methyl-D-Aspartate (NMDA), qui permet de potentialiser leur signalisation. Sur le plan structurel, deux formes du tPA ont été identifiées : une forme simple chaîne (sc-tPA) et une forme double chaîne (tc-tPA). Ces deux formes, dont les proportions peuvent varier dans la solution administrée aux patients pour la thrombolyse post-AVC ischémique, partagent certaines fonctions communes mais peuvent aussi avoir des actions différentes. Le premier objectif de nos travaux visait à mieux comprendre l’implication du tPA dans le couplage neurovasculaire, un phénomène essentiel au fonctionnement cérébral permettant aux régions en activité de bénéficier d’un apport accru en sang afin de subvenir à la demande énergétique des neurones. Dans une seconde partie, nous nous sommes intéressés aux effets des formes sc-tPA et tc-tPA utilisées lors de la thrombolyse dans un modèle murin d’AVC ischémique thromboembolique.Premièrement, nos résultats mettent en évidence la capacité du tPA vasculaire à augmenter l’hyperhémie fonctionnelle dans le cadre du couplage neurovasculaire. En effet, nous montrons chez la souris que le tPA vasculaire peut interagir avec les récepteurs NMDA présents à la surface des cellules endothéliales des artères et artérioles, et augmenter leur dilatation lors d’une activité neuronale. D’autre part, dans le cadre de l’ischémie cérébrale, nos résultats indiquent que lorsqu’ils sont utilisés pour la thrombolyse précoce, le sc-tPA et le tc-tPA ont des effets différents et parfois opposés. Le sc-tPA permet de réduire les volumes de lésion et d’améliorer la récupération fonctionnelle, alors que le tc-tPA est moins efficace pour réduire la lésion et ne diminue pas les déficits fonctionnels. De fait, nos données montrent que le tc-tPA aggrave l’altération de l’intégrité de la barrière hématoencéphalique par rapport au sc-tPA. Dans l’ensemble, ces données permettent d’améliorer les connaissances sur les mécanismes d’actions du tPA dans des phénomènes physiologiques et pathologiques importants. Nos travaux soulignent également la nécessité de prendre en compte les différences entre les formes de tPA dans l’amélioration du traitement actuel des AVC et dans l’élaboration de futures stratégies thérapeutiques impliquant cette molécule. / The tissue-type plasminogen activator (tPA) is a serine protease initially discovered in the blood for its ability to convert plasminogen into plasmin, an enzyme capable of degrading fibrin chains of blood clots. tPA is the only pharmacological treatment currently used for the acute phase of ischemic stroke, although it has several limitations. Besides its role in fibrinolysis, tPA also modulates various physiological and pathological phenomena within the central nervous system and neurovascular unit, such as memory, excitotoxicity and neurovascular coupling, which has been described recently. Several functions of tPA involve its interaction with N-Methyl-D-Aspartate (NMDA) receptors, which leads to an increase in NMDA signaling. Structurally, two forms of tPA have been identified: a single chain form (sc-tPA) and a double chain form (tc-tPA). These two forms, whose proportions vary in the solution administered for thrombolysis during ischemic stroke, share some common functions but may also differ in their therapeutic action. The first objective of our work was to better understand the implication of tPA in neurovascular coupling, which is an essential phenomenon for cerebral functioning that allows active brain regions to benefit from increased blood supply in order to meet local energy demands. In the second part of our work, we investigated the effects of sc-tPA and tc-tPA in a murine model of ischemic thromboembolic stroke.Our results establish a role for vascular tPA in increasing functional hyperemia in neurovascular coupling. We show that vascular tPA interacts with NMDA receptors present at the surface of endothelial cells of arteries and arterioles to increase their dilation during neuronal activity. In the context of cerebral ischemia, our results indicate that when administered during early thrombolysis, sc-tPA and tc-tPA have different and sometimes opposite effects. tc-tPA is less effective than sc-tPA in reducing lesion volume and protecting against functional impairment. In fact, our data show that tc-tPA worsens the integrity of the blood-brain barrier compared to sc-tPA. Overall, these data improve our knowledge of the mechanisms of action of tPA in important physiological and pathological phenomena. Our work underlines the need to take into account differences between sc-tPA and tc-tPA when trying to improve the current treatment for stroke and in the development of future therapeutic strategies involving this molecule.

L'ischémie cérébrale est une pathologie complexe impliquant une cascade de mécanismes cellulaires qui conduisent, entre autres, à une augmentation de l’autophagie dans les neurones. Bien que l’activation de l’autophagie dans l’AVC ischémique soit aujourd’hui un fait avéré, le rôle de l'activateur tissulaire du plasminogène (tPA ; médicament utilisé dans la phase aigüe de l’AVC ischémique et neuromodulateur du système nerveux central) n’a jamais été décrit. Le tPA est une sérine protéase initialement découverte dans le compartiment vasculaire jouant un rôle important dans la fibrinolyse. Mais le tPA est aussi exprimé dans le parenchyme cérébral où il intervient dans le système glutamatergique, la plasticité synaptique et la survie neuronale. Afin de mieux comprendre les effets moléculaires du tPA dans l’autophagie, nous avons utilisé un modèle in vitro d'ischémie cérébrale consistant à sevrer en oxygène et en glucose (OGD) puis à réoxygéner des neurones corticaux primaires murins avec ou sans tPA. Nous avons confirmé, dans un premier temps, que l’OGD induit une autophagie délétère via une diminution de l’axe PI3K/Akt/mTORC1. Nous avons ensuite étudié l’effet du tPA sur l’autophagie induite par l’OGD. Nos résultats démontrent que le tPA protège les neurones de la mort induite par l’OGD en réduisant l’autophagie via l’activation du récepteur du facteur de croissance à l'insuline (IGF-1R, un récepteur tyrosine kinase) et de la voie PI3K/Akt/mTOR. Ce travail de thèse a donc permis de décrire le rôle neuroprotecteur et anti-autophagique du tPA, et d’identifier un nouveau récepteur cible du tPA : IGF-1R. / Cerebral ischemia is a complex pathology involving a cascade of cellular mechanisms leading, among other things, to an increase of neuronal autophagy. The activation of autophagy in ischemic stroke conditions is now well accepted, but the role of tissue-type plasminogen activator (tPA, a drug used in the acute phase of ischemic stroke, and a neuromodulator) on this pathway has never been studied. tPA is a serine protease originally discovered in the vascular compartment, that plays an important role in fibrinolysis. Interestingly, tPA is also expressed in the cerebral parenchyma where it is involved in the glutamatergic neurotransmission, synaptic plasticity and neuronal survival. To better understand molecular effects of tPA on autophagy, we used an in vitro model of cerebral ischemia consisting in an oxygen and glucose deprivation (OGD) followed by reoxygenation, on murine primary cortical neurons with or without tPA. First we reported that OGD enhances deleterious autophagy through the decrease of PI3K/Akt/mTOR pathways. Then, we investigated the effect of tPA on OGD-induced autophagy. Our results demonstrate that tPA protects neurons from OGD-induced death by reducing autophagy through Insulin Growth Factor Receptor (IGF-1R, a tyrosine kinase receptor) and an increase of PI3K/Akt/mTOR pathways. This thesis has made it possible to describe the neuroprotective and anti-autophagic effect of tPA, and to identify a new target receptor for tPA: IGF-1R.

PURPOSE: Hemothorax, the collection of blood in the intrapleural space, commonly arises in patients suffering from thoracic trauma. Fluid collections in this space can compromise cardiac and respiratory function and if left untreated, can result in hypovolemic crisis. Fluid is often successfully drained via a tube thoracostomy, in which an intercostal drain is inserted into the pleural space. If residual blood remains, however, clotting may occur and result in a retained hemothorax (RH). Intrapleural administration of tissue plasminogen activator (tPA), a fibrinolytic drug typically utilized in ischemic stroke, has been shown to be both a safe and effective technique to hydrolyze RH clots and reduce the need for more invasive surgical interventions. The present study aims to evaluate the safety and efficacy of tPA administration at Boston Medical Center (BMC) and compare this data to those of prior studies. This study will also investigate if tPA as a definitive treatment for RH, could reduce the need for additional interventions such as surgical procedures including Video-Assisted Thoracoscopic Surgery (VATS) and/or invasive thoracaotomy. Hospital/intensive care unit (ICU) lengths of stay (LOS), ventilator time, and complication rates will be used to determine if tPA may allow for a significant decrease in patient cost and burden of care versus surgery. An analysis of patient demographics and injury data will be used to determine the individual factors that could be used to predict the success of tPA as a definitive treatment. Using evidence-based treatment protocols, the aforementioned data will be critically evaluated to determine the appropriate timing and sequential positioning of tPA administration in the treatment algorithm for retained hemothorax. METHODS: A single-institution retrospective chart review was conducted of patients treated for traumatic pneumohemothorax by the Department of Acute Care and Trauma Surgery at Boston Medical Center. A study on predictive factors of the development of retained hemothorax included all such patients that presented to the emergency department (ED) between May 2014 and June 2016. Demographic and injury characteristics were analyzed to determine if patients from specific groups or with specific injuries are more prone to develop RH. To evaluate the safety of intrapleural tissue plasminogen activator, the incidence of complications such as post-trauma infection and mortality were determined in patients that were administered tPA to resolve retained hemothorax between May 2014 through December 2016. Next, utilizing an expanded data set, the efficacy of tPA was evaluated by determining the percentage of cases in which tPA was able to definitively resolve RH. Secondary efficacy data including average hospital length of stay, average ICU length of stay, average mechanical ventilation time, and rate of readmission were compared between various interventions as well. Finally, to elucidate the risk factors for RH and independent predictors of tPA as a definitive treatment, demographic data including age, ethnicity, and gender as well as injury data including mechanism of injury, the presence or absence of multisystem trauma, and the presence or absence of specific injuries such as rib fracture, pulmonary contusion, or diaphragmic insult were collected. RESULTS: A statistically significant positive correlation was observed between the likelihood of developing RH and both abdominal alimentary tract and extremity injuries, indicating that these injuries may serve as predictive factors for RH development. In a study investigating the safety of intrapleural tPA, there was no statistically significant difference in post-trauma infection rates between individuals treated with tPA and those who were not. Additionally, tPA treatment was associated with a lower mortality rate. Efficacy studies revealed that tPA therapy was associated with a statistically significant decrease in mechanical ventilation time, as compared to surgical intervention, however, tPA carried a RH resolution rate of just 43% with one patient experiencing a major adverse systemic reaction to the drug. Finally, demographic and injury data were analyzed to determine predictive factors of tPA success, but no statistically significant relationships were observed between any of these characteristics and the outcome of tPA therapy. CONCLUSION: Intrapleural tPA is a safe and effective alternative to more invasive surgical procedures. The success rate of tPA therapy in the present study was less than previous studies have indicated, however, the potential decreased ventilation time is important for preventing ventilator associated pneumonia (VAP) and the high rate of mortality it carries. Although the success rate is lower than expected, tPA should still be considered in the RH treatment protocol, prior to surgery, to decrease required ventilation time and potentially prevent the need for more invasive interventions with higher costs, morbidity, mortality rates, and patient burden.

Angioedema is a localized, non-pitting, non-dependent, submucosal, and subcutaneous swelling resulting from the extravasation of fluid into the interstitium due to the increased production of plasma kinins and histamine. It can present with urticaria or anaphylaxis and is usually associated with angiotensin-converting enzyme inhibitors (ACEis), complement deficiencies, or the side effects of tissue plasminogen activator (tPA). Orolingual angioedema following tPA for acute ischemic stroke is a transient, self-resolving hemifacial swelling contralateral to neurological deficits that can rarely progress to the airway, compromising it and leading to a life-threatening situation if not managed promptly.

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