The role of plasmin and plasminogen activators (PA) in bovine lactation and porcine ovulation has been examined. There is no difference in the activation pattern of plasminogen to plasmin throughout the whole range of somatic cell counts (SCC) and from third to ninth month in lactation. The ratio of (plasminogen + plasmin)/plasmin, which serves as an index of the activation process, was 7.27 during early (first and second month) and 4.23 during late lactation (tenth month) and both values are different (p $<$ 0.01) from all the other ratios throughout the whole range of SCC and from third to ninth month in lactation suggesting limited and increased activation of plasminogen to plasmin during early and late lactation, respectively. Macrophages produce but they do not secrete urokinase-PA, suggesting a minor role in influencing milk plasmin. Somatotropin administration resulted in a suppression of milk plasmin in vivo. Insulin-like growth factor-1 (IGF-1), the most likely mediator of the effects of somatotropin on the bovine mammary gland, inhibited the induction of tissue-PA (t-PA) production which is observed when mammary epithelial cells are cultured in the absence of IGF-1. Plasmin and t-PA increased while PA inhibitor-1 decreased in porcine granulosa, theca interna cells and follicular fluid just prior to the time of expected ovulation suggesting a role for plasmin in follicle rupture.

Atherosclerotic cardiovascular disease is the leading cause of death of adults in North America. Diabetes is a classical risk factor for atherosclerotic cardiovascular disease. Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of fibrinolysis. Elevated levels of PAI-1, oxidized low-density lipoprotein (oxLDL) and glycated LDL (glyLDL) were detected in patients with diabetes. Increased oxidative stress is associated with diabetic cardiovascular complications. Previous studies in our laboratory demonstrated that oxLDL or glyLDL increased the production of PAI-1 or reactive oxygen species (ROS) in vascular endothelial cells (EC). This study was undertaken to investigate transmembrane signaling mechanisms involved in oxLDL or glyLDL-induced upregulation of PAI-1 in cultured vascular EC. Further, we examined the mechanism for oxLDL or glyLDL-induced oxidative stress in EC. The results of the present studies demonstrated novel transmembrane signaling pathway for oxLDL-induced PAI-1 production in vascular EC. We demonstrated that lectin-like oxLDL receptor-1, H-Ras, a small G-protein and Raf-1/ERK-1/2 mediate oxLDL-induced PAI-1 expression in cultured EC. GlyLDL may activate EC via a distinct transmembrane signaling pathway. The results of the present study demonstrated that receptor for advanced glycation end products, NADPH oxidase and H-Ras/Raf-1 are implicated in the upregulation of heat shock factor-1 or PAI-1 in vascular EC under diabetes-associated metabolic stress. We investigated the effects of oxLDL or glyLDL on mitochondrial function in EC. Treatment with oxLDL or glyLDL significantly impaired the activities of electron transport chain (ETC) enzymes and also increased mitochondria-associated ROS in EC. The findings suggest that oxLDL or glyLDL attenuated activity of ETC and increased ROS generation in EC, which potentially contributes to oxidative stress in vasculature. In conclusion, diabetes-associated lipoproteins may upregulate stress response mediators and PAI-1 production via distinct transmembrane signaling pathways. OxLDL or glyLDL may increase ROS production via NOX activation and the impairment of mitochondrial ETC enzyme activity in EC. The understanding and identification of the regulatory mechanisms involved in diabetes-associated lipoprotein-induced signaling may help pharmacological design for the management of diabetic cardiovascular complications.

The objectives were: (1) to determine the pre-ovulatory changes in plasminogen activator (PA) and (PA) inhibitor (PAI) activities in the porcine follicle, and, (2) to determine if changes in the PA/plasmin system associated with ovulation were prostaglandin (PG)-dependent. PA activity (change in absorbance/h/mg wet tissue weight, three gilts per treatment group) was elevated in both granulosa cells (GC) and theca interna cells (TIC) prior to human chorionic gonadotropin (hCG) administration (0.582 $ pm$ 0.171 and 0.718 $ pm$ 0.221, respectively) but returned to basal levels in these two compartments (0.023 $ pm$ 0.013 and 0.052 $ pm$ 0.024, respectively) at 29 h post-hCG. PA activity remained basal thereafter in GC but increased approximately ten-fold in the TIC (0.549 $ pm$ 0.239) at the time of ovulation (three gilts at 41 h and one of three gilts at 38 h). PAI activity did not change in TIC over the pre-ovulatory period but increased in GC as ovulation approached. PAI activity in GC peaked at 38 h (being significantly different (p $<$ 0.05) to all other times except 41 h). Although indomethacin (INDO) effectively inhibited both PG synthesis (1.1 $ pm$ 0.2 vs. 9.2 $ pm$ 0.9 ng/ml in controls) and ovulation (0 vs. 27-61% in controls), elevated PA activity (0.801 and 0.349) was detected in the TIC of two out of nine INDO-treated gilts. Levels were basal (0.074 $ pm$ 0.028) in the other gilts. These inconclusive results are believed to reflect the occurrence of ovulation earlier than predicted, in as many as 40% of control gilts, and the short duration of increased PA activity at this time. In conclusion, elevated PA activity, in GC and TIC prior to ovulation induction, may play a role in follicular development. Elevated TIC PA activity may play an important role in the ovulatory process, but is probably PG-independent.

Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor traditionally regarded as a regulator of fibrinolysis and extracellular matrix degradation. More recently, PAI-2 has been implicated in diverse processes such as keratinocyte differentiation, cell death and viral pathogenesis. Although PAI-2s limited pattern of expression in vivo generates significant interest in this molecule, little is known about the underlying mechanisms controlling its cell specific regulation. In this thesis, the function that the previously identified PAI-2 gene silencer (Antalis et al., 1996) plays in the regulation of PAI-2 gene expression was investigated. The PAI-2 upstream silencer element 1 (PAUSE-1) is located approximately 1800bp upstream of the PAI-2 transcription initiation site. By employing electrophoretic mobility shift assays and transient transfection assays with mutant PAUSE-1 sequences, the sequence that defines PAUSE-1 was identified as TCT N3 AGA N3 T4. This element was shown to bind a number of protein complexes of similar electrophoretic mobility from various cultured cell lines. Transient transfection assays with the cervical adenocarcinoma, HeLa S3 and the macrophage-like, U937 cell lines, showed that PAUSE-1 repressed transcription by approximately 2.5 fold when cloned into the SV40 promoter or the minimal PAI-2 promoter. Ultraviolet (UV)-crosslinking analyses determined that the PAUSE-1 binding protein (BP) was approximately 67kDa. Examination of several similar DNA promoter sequences, such as the human IFNb and insulin promoters, suggested that PAUSE-1 might be an example of a universal silencer with the consensus sequence TCT Nx AGA, where x=4. The PAUSE-1 sequence shows significant homology to the binding sequence of the transcriptional regulators Ski, Smad3 and Smad4. EMSAs incorporating anti-Ski, -Smad3 and -Smad4 antibodies suggested that each are members of the PAUSE-1 BP complex in HeLa S3 cells. The PAUSE-1 BP complex has been purified by employing DNA affinity chromatography using streptavidin labelled magnetic beads. Approximately nine PAUSE-1 associated proteins from HeLa S3 extracts were visualised. Amino-terminal protein sequencing identified the first eight amino acids of the PAUSE-1 BP as EIQQRAAQ. The PAUSE-1 BP fails to show significant sequence similarity to any known protein and therefore potentially represents a novel DNA binding protein.

Thesis (Ph.D.)--University of Wollongong, 2006. / Typescript. Includes bibliographical references: leaf 148-160.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [137]-148).

Submitted by Erika Demachki (erikademachki@gmail.com) on 2017-02-16T17:00:17Z No. of bitstreams: 2 Dissertação - Edilânia Gomes Araújo Chaves - 2013.pdf: 3289607 bytes, checksum: 174bfb3cce0a9109022321f4adf3368d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2017-02-17T16:53:18Z (GMT) No. of bitstreams: 2 Dissertação - Edilânia Gomes Araújo Chaves - 2013.pdf: 3289607 bytes, checksum: 174bfb3cce0a9109022321f4adf3368d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-02-17T16:53:18Z (GMT). No. of bitstreams: 2 Dissertação - Edilânia Gomes Araújo Chaves - 2013.pdf: 3289607 bytes, checksum: 174bfb3cce0a9109022321f4adf3368d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2013-03-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Paracoccicoidioides is the etiological agent of paracoccidioidomycosis (PCM), a disease considered one of the main causes of mortality among systemic mycoses in Brazil. The success in establishing of the infection is related with the ability of fungus to adhere and degrade components of the extracellular matrix (ECM). Human plasminogen (hPlg) is a protein of blood plasma of the host that presents fibrinolytic activity when activated into plasmin. Many pathogens are able to subvert the plasminogen/plasmin system using linker molecules and promote the degradation of tissue barriers. In this work, we identified through Far Western and proteomic analysis, a total of 15 proteins secreted of Paracoccidioides that are plasminogen binding proteins. Those proteins are probable targets of the interaction of the fungus with the host and could contribute to the invasiveness of the fungus. The fructose 1,6-biphosphate aldolase was described in other organisms such as plasminogen binder and presentes participation in the adherence of Paracoccidioides to host cells. This protein selected for validation tests and their presence was observed on the surface and secretory vesicles of the fungus. FBA confirm the ability to convert plasminogen to plasmin in the presence of tissue plasminogen activator (tPA) and this interaction promoted the degradation of fibrin. In infection assays, the addition of antibodies blocking the FBA binding site reduced the interaction of the fungus with macrophages and the interaction of FBA with Plg increased the rate of cell invasion. These data suggest that the FBA can contribute to adhesion, invasion and spread process of the fungus. / Paracoccicoidioides é o agente etiológico da paracoccidioidomicose (PCM), uma doença considerada como primeira causa de mortalidade entre as micoses sistêmicas no Brasil. O sucesso no estabelecimento da infeção está relacionado com a capacidade do fungo de aderir e degradar componentes da matriz extracelular (MEC). O plasminogênio humano (hPlg) é uma proteína presente no plasma, a qual possui atividade fibrinolítica quando é ativado em plasmina. Muitos micro-organismos patogênicos são capazes de subverter o sistema plasminogênio/plasmina através de moléculas ligantes e promovem a degradação de barreiras teciduais. Neste trabalho foram identificadas, através de Far-Western e análise proteômica, um total de 15 proteínas secretadas por Paracoccidioides com habilidade de ligação ao plasminogênio. Essas proteínas são prováveis alvos da interação do patógeno com o hospedeiro, que contribui para o potencial invasivo do fungo. A frutose 1,6-bifosfato aldolase (FBA) foi descrita em outros organismos como ligante de plasminogênio e possui participação na adesão de Paracoccidioides às células do hospedeiro. Selecionamos esta proteína para ensaios de validação e foi observada sua presença na superfície e em vesículas secretoras do fungo. Confirmamos a capacidade da FBA de converter plasminogênio em plasmina na presença do ativador tecidual de plasminogênio (tPA) e essa interação promoveu a degradação de fibrina. Em ensaios de infecção, a adição de anticorpos que bloqueiam o sítio de ligação da FBA reduziu a interação do fungo com macrófagos e a interação de FBA com Plg aumentou o índice de invasão celular. Esses dados sugerem que a FBA pode contribuir no processo de adesão, invasão e disseminação do fungo.

La neuroinflammation est un dénominateur commun de plusieurs troubles du système nerveux central. Les réactions inflammatoires sont souvent médiées par plusieurs voies de signalisation qui conduisent à l'ouverture de la barrière hémato-encéphalique. L'activateur tissulaire du plasminogène (tPA) est une serine protéase qui induit l'ouverture de la barrière hémato-encéphalique. Au cours des dernières années, il a également été montré que les récepteurs NMDA situés dans les cellules endothéliales peuvent jouer un rôle crucial dans la propagation de la réaction inflammatoire.Mon travail au cours de ma thèse a mis l'accent sur la découverte des mécanismes par lesquels le récepteur NMDA effectue une médiation de l'ouverture de la barrière hémato-encéphalique induite par le TPA. Dans notre première étude, nous montrons que les récepteurs NMDA endothéliaux sont des cibles thérapeutiques potentielles pour prévenir l'infiltration et l'inflammation des cellules immunitaires médiées par l'EAE. Nous montrons que l'anticorps monoclonal du récepteur NMDA spécifique à la souris, le Glunomab, pourrait protéger la barrière de la moelle épinière de dommages inflammatoires. Nous montrons également que les récepteurs NMDA sont exprimés en étroite association avec les protéines de jonction serrées dans les cellules endothéliales cérébrales. Dans notre deuxième étude, nous montrons pour la première fois que les récepteurs NMDA neuroendothéliaux peuvent présenter une action métabotropique lors de l'inflammation. Nous soulignons également que ces récepteurs sont en effet des récepteurs NMDA non conventionnels exprimant la sous unité GluN3A. En outre, nous rapportons que le tPA accélère l'ouverture de la barrière hémato-encéphalique en présence d'une agoniste rare de la glycine par un mécanisme dépendant de l'activation de RhoA. Les résultats de mon projet apportent une nouvelle vision du rôle des récepteurs NMDA métabotropiques dans les cellules endothéliales cérébrales. En outre, il fournit également des détails plus précis sur l'ouverture de la barrière hémato-encéphalique via l’activateur tissulaire du plasminogène. / Neuroinflammation is a common denominator of several central nervous system disorders. Inflammatory reactions are often mediated by several signaling pathways which lead to the opening of the blood brain barrier. Tissue plasminogen activator (tPA) is a serine protease induces opening of the blood brain barrier. In recent years, it has also been shown that NMDA receptors located in endothelial cells can play a crucial role in propagation of inflammatory reaction. My doctoral study focused on the finding the underlying mechanisms of action(s) by which NMDA receptor mediates tPA induced opening of the blood brain barrier. In our first study we show that endothelial NMDA receptors are potential therapeutic targets to prevent EAE mediated immune cell infiltration and inflammation. We show that NMDA receptor specific mouse monoclonal antibody Glunomab could prevent the brain spinal cord barrier from inflammatory damage. We also show that NMDA receptors are expressed in close association of tight junction proteins in cerebral endothelial cells. In our second study, we show for the first time that, neuroendothelial NMDA receptors can exhibit metabotropic mode of action during inflammation. We also highlight that these receptors are indeed GluN3A expressing non-conventional NMDA receptors. In addition, we report that tPA accelerates the opening of blood brain barrier in presence of an uncommon agonist glycine by RhoA activation dependent mechanism.My project results provide a nouvelle insight for the role of metabotropic NMDA receptors in cerebral endothelial cells. In addition it also provides more precise details of blood brain barrier opening mediated by tissue plasminogen activator.

Le système d’activation du plasminogène (SAP), initialement décrit dans la circulation sanguine, intervient dans la dégradation des caillots de fibrine (fibrinolyse). Ce système permet de convertir un zymogène inactif (le plasminogène) en enzyme active (la plasmine) via l’action de l’activateur tissulaire du plasminogène (tPA), lui-même inhibé par la serpine inhibitrice de l’activateur du plasminogène de type 1 (PAI-1). Il a été suggéré qu’une dérégulation du SAP dans le système nerveux central (SNC) pouvait être un processus physiopathologique dans la sclérose en plaques (SEP). Cependant, l’origine et les conséquences de cette dérégulation restent peu caractérisées dans cette pathologie. Durant cette thèse, nous avons donc cherché à mieux caractériser la dérégulation du SAP dans la SEP et à déterminer son rôle dans la physiopathologie de cette maladie. Les travaux réalisés au cours de ce doctorat ont permis de montrer qu’une forte surexpression de PAI-1 survient dans le SNC lors des phases symptomatiques de modèles murins de SEP, entraînant une inhibition de la fibrinolyse intraparenchymateuse. Cette altération du SAP est une cause de la formation des zones de lésion, et coïncide temporellement avec les phases symptomatiques des modèles murins de SEP. Les astrocytes réactifs pro-inflammatoires sont les responsables de cette surexpression parenchymateuse délétère de PAI-1. Il apparaît qu’une intervention à visée thérapeutique permettant la restauration de l’activité fibrinolytique intraparenchymateuse, via l’inhibition de PAI-1, s’avère bénéfique dans des modèles animaux de SEP. En conclusion, nos travaux révèlent que la présence d’astrocytes réactifs est à l’origine de la surexpression de PAI-1 engendrant l’inhibition de la fibrinolyse intraparenchymateuse constatée dans la SEP. Ce processus physiopathologique délétère est une des causes menant à la formation de zones lésionnelles dans la SEP. / Degradation of fibrin blood clots (fibrinolysis) is mediated by the plasminogen activation system (PAS), initially discovered in the blood. This system allows the inactive plasminogen conversion into active plasmin by tissue-type plasminogen activator (tPA). The plasminogen activator inhibitor type 1 (PAI-1) is able to inhibit tPA. It has been suggested that in the central nervous system (CNS), a PAS dysregulation could be a physiopathological mechanism of multiple sclerosis (MS). However, this dysregulation cause and consequences are poorly described in this disease. This work aimed to characterise the PAS dysregulation occurring in MS, and to better define its role in MS physiopathology. Our results describe a strong PAI-1 overexpression in the CNS during symptomatic periods in animal models of MS, leading to an intraparenchymal fibrinolysis inhibition. The PAS dysregulation is a cause of lesion formation, and temporally coincides with symptomatic periods in these models. Pro-inflammatory reactive astrocytes overexpress PAI-1. It appears that an increase of parenchymal fibrinolysis by inhibiting PAI-1 reduces EAE severity. To conclude, our results highlight a role of reactive astrocytes in MS, leading to an over-expression of PAI-1 and an impairment of parenchymal fibrinolysis. This physiopathological mechanism is implied in lesion formation in MS.