Thrombotic disorders include myocardial infarction (MI), acute ischemic stroke (AIS) and venous thromboembolism (VTE), which encompasses pulmonary embolism (PE), and deep vein thrombosis (DVT). To prevent further complications or mortality in patients with MI and AIS, rapid restoration of blood flow is needed to minimize organ damage. Such treatment also is needed in patients with massive PE. Blood flow can be restored mechanically via percutaneous coronary intervention with stent implantation for MI and by thrombectomy in patients with AIS or PE. Alternatively, pharmacological reperfusion can be achieved by systemic administration of plasminogen activators (PAs). PAs convert plasminogen to the fibrinolytic enzyme, plasmin. Plasmin then degrades the clot into soluble fragments. Streptokinase (SK) and urokinase (UK) were the first therapeutic clot dissolving drugs but both lead to excessive bleeding complications because of non-specific effects. Current therapy focuses on clot specific agents such as recombinant tissue-PA (rt-PA) or tenecteplase (TNK), a rt-PA variant. However, there is a risk of intracranial bleeding in at least 1% of patients, which can be fatal or disabling. Thus, a need exists for new strategies to enable safer reperfusion that are not associated with potentially fatal side effects. This study focuses on the therapeutic role of alpha2-antiplasmin (α2AP). α2AP is the primary inhibitor of plasmin. One approach to thrombolysis is to attenuate α2AP with an inhibitory antibody (A2AP IgG). Inhibition of α2AP would enable clot lysis with lower doses of PAs, thereby reducing the risk of bleeding and serving as a safer approach to thrombolytic therapy. We aimed to characterize A2AP IgG and evaluate its effect on fibrinolysis in vitro and in vivo. A2AP IgG1 was selected and developed using phage display and an antibody gene library with human and rabbit α2AP as the antigen. Affinity maturation was performed and the Fc portion of the A2AP IgG1 was subsequently changed to the IgG4 isotype which yielded A2AP IgG4. A2AP IgG4 binds α2AP with 63-fold higher affinity than A2AP IgG1 as determined using surface plasmon resonance (SPR). SDS-PAGE and western blot analysis reveals that both antibodies bind to the plasmin-α2AP (PAP) complex, fibrinogen, and fragment X but not to α2AP; results confirmed by ELISA. In functional studies, A2AP IgG1 significantly reduced plasmin inhibition by α2AP by 5.5-fold. Both A2AP IgG1 and A2AP IgG4 shortened tissue-PA (t-PA)-mediated clot lysis in a concentration dependent manner. A2AP IgG4 was 2.2-fold more potent than A2AP IgG1 in human plasma and 1.4-fold more potent in rabbit plasma. Compared with t-PA or TNK alone, addition of either antibody enhanced the lysis of preformed plasma clots. Combining A2AP IgG4 with 10% of the highest t-PA or TNK dose produced more clot lysis than the highest dose of t-PA or TNK alone. In a rabbit jugular vein thrombosis model, A2AP IgG4 alone produced 20% lysis. When combined with a low dose of TNK, 40% clot lysis resulted, which was significantly greater than the 30% clot lysis observed with a higher dose of TNK. A2AP IgG4 alone or in combination with a lower dose of TNK did not cause significantly more bleeding than the higher dose of TNK alone and did not degrade circulating fibrinogen. Thus, we have shown that by inactivating α2AP, A2AP IgG attenuates α2AP activity, and accelerates clot lysis in vitro and in vivo. This demonstrates that antibody-mediated inhibition of α2AP, enhances thrombolysis and enables use of lower doses of PAs. / Thesis / Master of Science in Medical Sciences (MSMS)

Duchenne muscular dystrophy (DMD) and congenital muscular dystrophy 1A (MDC1A) are the two most common forms of muscular dystrophy in humans, caused by mutations in dystrophin and laminin α2 genes respectively. Both are severe forms of the disease that lead to premature death due and are both now known to have a significant effect on the central nervous system. This project investigated the role of both proteins involved in each of these diseases in cerebellar Purkinje cells of two murine models of disease: the mdx mouse a dystrophin-deficient model of DMD and the dy2J a laminin α2-deficient murine model of MDC1A. In the case of dystrophin further studies were undertaken in order to determine if increasing age had any effects on cerebellar function. It was found that there is no difference in electrophysiological characteristics (RMP, IR, eEPSP) of the cells when compared to appropriate control groups, nor was there any difference when young and aged dystrophin-deficient mdx groups were compared. Evoked IPSP characteristics were examined in young mdx cerebellar Purkinje cells and again no difference was found when compared to wildtype. There was a significant difference in response to the GABAA antagonist bicuculline, with wildtype increasing eEPSP amplitude by almost double that found in mdx. There was no difference in short term plasticity as measured by paired pulse facilitation in any of these groups. There was no difference in paired pulse depression at the inhibitory interneuron- Purkinje cell synapse of young wildtype and mdx cerebellar Purkinje cells. There a significant blunting of long term depression (LTD, (a form of long term synaptic plasticity) between young wildtype and mdx. When young wildtype animals were compared to aged wildtype animals LTD was found to be similar, when young mdx was compared to aged mdx, there was a recovery of LTD seen in the aged population. There was also significant differences in LTD found when littermate controls were compared to dy2J (laminin α2 mutants). A third of the phenotypic animals (dy2J) potentiated. Finally when rebound potentiation (a GABA-ergic form of long term synaptic plasticity in the cerebellum) was compared in young wildtype and mdx mice, mdx mice displayed depression, rather than the expected potentiation in contrast to potentiation (or no change) as seen in all wildtype cells.

Duchenne muscular dystrophy (DMD) and congenital muscular dystrophy 1A (MDC1A) are the two most common forms of muscular dystrophy in humans, caused by mutations in dystrophin and laminin α2 genes respectively. Both are severe forms of the disease that lead to premature death due and are both now known to have a significant effect on the central nervous system. This project investigated the role of both proteins involved in each of these diseases in cerebellar Purkinje cells of two murine models of disease: the mdx mouse a dystrophin-deficient model of DMD and the dy2J a laminin α2-deficient murine model of MDC1A. In the case of dystrophin further studies were undertaken in order to determine if increasing age had any effects on cerebellar function. It was found that there is no difference in electrophysiological characteristics (RMP, IR, eEPSP) of the cells when compared to appropriate control groups, nor was there any difference when young and aged dystrophin-deficient mdx groups were compared. Evoked IPSP characteristics were examined in young mdx cerebellar Purkinje cells and again no difference was found when compared to wildtype. There was a significant difference in response to the GABAA antagonist bicuculline, with wildtype increasing eEPSP amplitude by almost double that found in mdx. There was no difference in short term plasticity as measured by paired pulse facilitation in any of these groups. There was no difference in paired pulse depression at the inhibitory interneuron- Purkinje cell synapse of young wildtype and mdx cerebellar Purkinje cells. There a significant blunting of long term depression (LTD, (a form of long term synaptic plasticity) between young wildtype and mdx. When young wildtype animals were compared to aged wildtype animals LTD was found to be similar, when young mdx was compared to aged mdx, there was a recovery of LTD seen in the aged population. There was also significant differences in LTD found when littermate controls were compared to dy2J (laminin α2 mutants). A third of the phenotypic animals (dy2J) potentiated. Finally when rebound potentiation (a GABA-ergic form of long term synaptic plasticity in the cerebellum) was compared in young wildtype and mdx mice, mdx mice displayed depression, rather than the expected potentiation in contrast to potentiation (or no change) as seen in all wildtype cells.

No description available.

Reproduction de : Thèse doctorat : Médecine. Biochimie et biologie moléculaire : Reims : 2008. / Titre provenant de l'écran-titre. Bibliogr. f.175-194.

L'étude systématique de l'hémostase post-thrombolytique a été peu étudiée. Chez 80 malades thrombolysés consécutifs, une étude prospective a comporté l'étude – aux heures 0, 2 et 24 – des facteurs de l'hémostase suivants: fibrinogène, plasminogène, PDF (produits de dégradation de la fibrine et du fibrinogène), D-dimères, alpha2-antiplasmine et facteur XIII, ainsi que l'hématocrite et la numération plaquettaire. Des calculs statistiques approfondis ont exploré les corrélations des variations des facteurs hémostatiques entre eux et avec 37 paramètres cliniques et étiologiques. Processus moléculaires post-thrombolytiques. Le rt-PA induit deux processus, indépendants statistiquement à la 2ème heure: d'une part une élévation des PDF et des D-dimères; d'autre part, une baisse du fibrinogène, corrélée à une baisse du plasminogène (r=0,48, p=0.01), de l'alpha2-antiplasmine (r=0.48, p =0.004) et du facteur XIII (r=0.44, p=0.01). La baisse du plasminogène est corrélée significativement avec celle de l'alpha2-antiplasmine (r=0.77, p<0.001), et du facteur XIII (r=0.47, p=0.02). La mise en jeu de facteurs anti-fibrinolytiques, qui n'avait jamais été décrite précédemment, peut jouer un rôle dans une limitation de la fibrinolyse et dans la rethrombose. Des corrélations sont notées entre la baisse précoce du plasminogène et l'étiologie cardioembolique (p=0.04), et un mauvais pronostic final (p=0.03), possiblement en rapport la thrombolyse intense de gros caillots. Les hématomes intra-cérébraux parenchymateux (HP) sont liés significativement à la baisse du fibrinogène (p=0.01) et à l'augmentation des PDF (p=0.01). Une baisse du fibrinogène au-dessous de 2g/L multiplie la probabilité de HP précoce par un facteur 12,82. Ainsi est confirmé le modèle d'une “coagulopathie précoce avec dégradation du fibrinogène”», prédictive de l'hématome, proposé par l'équipe lyonnaise de thrombolyse en 2004 / A systematic study of post-thrombolytic haemostasis has rarely been performed. In 80 consecutive patients, we have prospectively studied at hours 0, 2 and 24 the following parameters: fibrinogen, plasminogen, alpha2-antiplasmin, factor XIII, fibrin(ogen) Degradation Products (FDP), D-dimers, haematocrit and platelet count. Comprehensive statistical studies calculated correlations of the haemostatic values betwen themselves and with 38 etiological and clinical parameters. Molecular dynamics. Two changes between h0 and h2 were statistically independent: an increase in FDP and D-Dimers; a decrease in fibrinogen, plasminogen, alpha2-antiplasmin and factor XIII. At h2, the decrease in fibrinogen was significantly correlated with that of plasminogen (0.48, p = 0.01), alpha2-antiplasmin (0.48, p = 0.004), and factor XIII (0.44, p = 0.01). The decrease in plasminogen was significantly correlated with those of antifibrinolytic components, alpha2-antiplasmin (r=0.77, p<0.001) and factor XIII (0.47, p=0.02). To our knowledge, such an activation of antifibrinolytic components had not hitherto been mentioned. The h2 decrease of plasminogen was correlated with cardioembolic etiology (p=0.04) and final poor oucome (p=0.03), a fact possibly due to intense thrombolysis of large clots. Patients having early parenchymal hematomas (PH) showed h2 haemostasis disturbances: high FDP (p=0.01), and low fibrinogen (p=0.01). The decrease in fibrinogen less than 2g/L multiplies the odds of early PH by a factor 12.82. Thus, we confirm the model of an “early fibrinogen degradation coagulopathy” predictive of hematomas, which had been coined by the Lyon thrombolysis team in 2004

Müller cells are major glial cells in the retina and have a broad range of functions that are vital for the retinal neurons. During retinal injury gliotic response either leads to Müller cell dedifferentiation and formation of a retinal progenitor or to maintenance of mature Müller cell functions. The overall aim of this thesis was to investigate the intra- and extracellular signaling of Müller cells, to understand how Müller cells communicate during an injury and how their properties can be regulated after injury. Focus has been on the α2-adrenergic receptor (α2-ADR) and endothelin receptor (EDNR)-induced modulation of Müller cell-properties after injury. The results show that α2-ADR stimulation by brimonidine (BMD) triggers Src-kinase mediated ligand-dependent and ligand-independent transactivation of epidermal growth factor receptor (EGFR) in both chicken and human Müller cells. The effects of this transactivation in injured retina attenuate injury-induced activation and dedifferentiation of Müller cells by attenuating injury-induced ERK signaling. The attenuation was concomitant with a synergistic up-regulation of negative ERK- and RTK-feedback regulators during injury. The data suggest that adrenergic stress-signals modulate glial responses during retinal injury and that α2-ADR pharmacology can be used to modulate glial injury-response. We studied the effects of this attenuation of Müller cell dedifferentiation on injured retina from the perspective of neuroprotection. We analyzed retinal ganglion cell (RGC) survival after α2-ADR stimulation of excitotoxically injured chicken retina and our results show that α2-ADR stimulation protects RGCs against the excitotoxic injury. We propose that α2-ADR-induced protection of RGCs in injured retina is due to enhancing the attenuation of the glial injury response and to sustaining mature glial functions. Moreover, we studied endothelin-induced intracellular signaling in Müller cells and our results show that stimulation of EDNRB transactivates EGFR in Müller cells in a similar way as seen after α2-ADR stimulation. These results outline a mechanism of how injury-induced endothelins may modulate the gliotic responses of Müller cells. The results obtained in this thesis are pivotal and provide new insights into glial functions, thereby uncovering possibilities to target Müller cells by designing neuroprotective treatments of retinal degenerative diseases or acute retinal injury.

No description available.

O cachorro-do-mato (Cerdocyon thous) é um canídeo neotropical que necessita ser contido por meios farmacológicos para a realização de certos procedimentos médicos e de manejo, em função de características comportamentais de defesa e grande susceptibilidade ao estresse. A combinação de seis protocolos (tiletamina-zolazepam-xilazina-atropina e azaperone; dextrocetamina-diazepam-xilazina-atropina e azaperone; cetamina racêmica-diazepam-xilazina-atropina e azaperone; tiletamina-zolazepam-detomidina-atropina e azaperone; dextrocetamina-diazepam-detomidina-atropina e azaperone; cetamina racêmica-diazepam-detomidina-atropina e azaperone) foram administradas, por via intramuscular, a dez cachorros-do-mato (nove machos e uma fêmea) com pesos médio 5,85 ± 0,83 kg, para possibilitar a realização de procedimentos de que incluíam marcação, exame físico, colheita de amostras de sangue, colheita de medula óssea e outros procedimentos pouco invasivos de moderada duração em Cerdocyon thous de cativeiro. Após a verificação dos pesos de cada cachorro-do-mato, a dose individual de cada uma das drogas foi calculada por meio de extrapolação alométrica interespecífica. O método proposto mostrou-se plenamente adequado à contenção farmacológica de exemplares de Cerdocyon thous que necessitem ser submetidos a procedimentos medianamente dolorosos ou incômodos, como exame físico e colheita de sangue e medula óssea. Não é indicado, porém, para procedimentos cirúrgicos. / The Crab-eating Fox (Cerdocyon thous) is a neotropical carnivorous that requires chemical restraint for handling due to its susceptibility to stress and characteristics of defensive behavior. Ten Crab-eating Fox (9 males and 1 female) weighing 5.85 ± 0.83 kg were given the combination of six protocols (tiletamine-zolazepam-xylazine-atropine and azaperone; dextroketamine-diazepam-xylazine-atropine and azaperone; racemic ketamine-diazepam-xylazine-atropine and azaperone; tiletamine-zolazepam-detomidine-atropine and azaperone; dextroketamina-diazepam-detomidine-atropine and azaperone; racemic ketamine-diazepam-detomidine-atropine and azaperone) by i.m. injection during field procedures that included identification, physical examination, blood sampling, bone marrow harvesting and other mildly invasive procedures of moderate duration in Cerdocyon thous of captivity. After checking the weights of each Crab-eating Fox, the individual dose of each drug was calculated by means of interspecific allometric extrapolation. The proposed method was safe for both the animal and the human personnel and it is recommended for routine management and stressful but not painful medical procedures like physical examination, measuring, sexing, and bone marrow and blood collection in Cerdocyon thous. / Tese (Doutorado)

[pt] A noradrenalina desempenha um papel central em diversos transtornos relacionados ao medo, como o transtorno de ansiedade generalizada (TAG). Estudos farmacológicos em humanos e animais mostraram que os comportamentos relacionados ao medo podem ser regulados pela aplicação sistêmica de drogas noradrenérgicas. No entanto, as diferenças individuais na ansiedade-traço são frequentemente negligenciadas ao estudar os efeitos não apenas de drogas noradrenérgicas, mas de outros compostos. No presente estudo, examinamos os efeitos da ioimbina, um antagonista do receptor alfa2-adrenérgico, em duas linhagens de ratos criados para respostas de alto e baixo congelamento à pistas contextuais previamente associadas a choques nos pés (ratos Carioca de Alto e Baixo Congelamento - CAC e CBC, respectivamente). Descobrimos que a administração sistêmica de ioimbina no segundo dia de condicionamento do medo contextual (sessão de teste) diminuiu significativamente as respostas de congelamento de fêmeas CAC, mas não de machos. No entanto, o tratamento com ioimbina induziu um aumento significativo no comportamento de congelamento de ratos CBC machos e fêmeas. Resultados semelhantes foram observados quando os grupos foram novamente expostos à mesma câmara de condicionamento 6 dias depois. Nossos resultados indicam que, embora a ioimbina leve a efeitos ansiolíticos em ratos CAC, ela tem um efeito ansiogênico nos ratos CBC. Entretanto, esse efeito foi mais evidente nas fêmeas do que nos machos. Nossas descobertas apontam para o papel da noradrenalina na regulação e mediação das respostas de medo em diferentes traços de ansiedade. Além disso, nossos resultados também ressaltam a relevância do uso de ambos os sexos em estudos comportamentais e farmacológicos usando modelos animais de transtornos de ansiedade. / [en] Norepinephrine plays a central role in several fear-related disorders, such as generalized anxiety disorder (GAD). Pharmacological studies in humans and animals have shown that fear-related behaviors can be regulated by the systemic application of noradrenergic drugs. However, individual differences in trait anxiety are often overlooked when studying the effects of not only noradrenergic drugs, but other compounds. In the present study we examined the effects of yohimbine, an alpha2-adrenergic receptor antagonist, in two lines of rats bred for high and low freezing responses to contextual cues previously associated with footshocks (Carioca high- and low-conditioned freezing rats - CHF and CLF, respectively). We found that systemic administration of yohimbine on the second day of contextual fear conditioning (test session) significantly decreased the freezing responses of CHF females, but not CHF males. Yet, yohimbine treatment induced a significant increase in freezing behavior of both male and female CLF rats. Similar results were observed when groups were re-exposed to the same conditioning chamber 6 days later. Our findings indicate that while yohimbine leads to anxiolytic effects on CHF rats, it has an anxiogenic effect on CLF ones. However, such effect was more evident in females than in males. Our findings point to the role of norepinephrine in regulating and mediating fear responses in different anxiety traits. Furthermore, our findings also underscore the relevance of using both sexes in behavioral and pharmacological studies using animal models of anxiety disorders.