Made available in DSpace on 2015-04-20T12:31:24Z (GMT). No. of bitstreams: 1 Raimundo Felipe da Cruz Filho.pdf: 1412659 bytes, checksum: aba20cdd1aa35484f5932ead0d0bc3e7 (MD5) Previous issue date: 2013-04-18 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The fibrinolytic proteases degrade fibrin clots and therefore play an important role in the pharmaceutical industry as chemotherapeutic agents in the treatment of cardiovascular diseases. These biocatalysts were gradually discovered from plants, insects, earthworms, snakes and microorganisms (bacteria and fungi). Cardiovascular disease has been one of the leading causes of death in the world. A major cause of heart disease is the accumulation of fibrin in the arteries, causing thrombosis. According to the World Health Organization (WHO), about 17.5 million people will die of cardiovascular disease this year, and in 2030, this amount will be 23.6 million. Given the great potential of microbial biodiversity and the growing regional Amazon applicability of enzymes in the production of drugs, this research was conducted with the objective to (1) evaluate the growth of Bacillus stearothermophilus, (2) establish growth parameters associated with production of fibrinolytic proteases in submerged fermentation and (3) assess the effect of the extractive fermentation in the separation of these enzymes. In this study techniques of extractive fermentation and solid-state fermentation were employed. By submerged fermentation the following parameters were determined: Profile of growth of Bacillus stearothermophilus and the production of protease using 100 mL of the liquid medium [(g / L) 2 g KH2PO4, (NH4)2SO4 1 g, MgSO4 7H2O 0,1 g Na2HPO4 2H2O 0,9 g, yeast extract 1 g, distilled water 1000 ml] pH 7.2 supplemented with 0.5% gelatin in an 500 mL Erlenmeyer flask. The growth of the bacteria was determined at 610 nm, once every 2 hours for 36 hours. To determine the best conditions for the production of proteases were evaluated the influence of pH, stirring and temperature, age of inoculum and substrate concentration, the influence of natural sources of carbon (tapioca, arraruta and crueira), nitrogen sources and aeration. In the recovered extract was also performed a toxicity bioassay in Artemia salina and degradation tests in vitro of the blood clot by the fibrin plate method and the artificial clot degradation in tube. In addition, the partition coefficient (K), the purification factor (PF) and recovering the enzyme were determined. The solid-state fermentation was performed using as substrate 10g of manteiguinha bean [Vigna unguiculata (L.) Walp] with 60% humidity, pH 5.0 in a 250 ml Erlenmeyer flask. In extractive fermentation the best conditions were pH 5.0, 180 rpm and 25 °C in systems using PEG 1000 (g/mol-1) to 20% (w/w) and phosphate salts 15% (w/w) with K 1.05; FP 1.00; 152.54 Y. 34 mm halo in fibrin plate and partial degradation of the clot in tube. In the solid-state fermentation, the production of protease was 8.87 (U/mL), 23 mm of translucent halo in fibrin plate with total degradation of the blood clot in 24 hours. In this study, protease produced from Bacillus stearothermophilus by extractive fermentation and semi-solid fermentation was evaluated, showing in the optimum cultivation conditions that this microorganism presents physiology for industrial application in the production of the fibrinolytic protease / As proteases fibrinolíticas degradam coágulos de fibrina, por isso têm um importante papel na indústria farmacêutica como agentes quimioterapêuticos no tratamento de doenças cardiovasculares. Estes biocatalisadores foram descobertos gradualmente a partir de plantas, insetos, anelídeos, serpentes e micro-organismos (bactérias e fungos). Doenças cardiovasculares tem sido a principal causa de morte no mundo. Uma das principais causas de doenças cardíacas é o acúmulo de fibrina nas artérias, acarretando trombose. De acordo com Organização Mundial da Saúde (OMS), cerca de 17,5 milhões de pessoas morrerão este ano de doenças cardiovasculares, e em 2030, esse montante será de 23,6 milhões. Tendo em vista o grande potencial da biodiversidade microbiana regional Amazônica e a crescente aplicabilidade de enzimas na produção de medicamentos, esta pesquisa foi realizada com o objetivo de (1) avaliar o crescimento de Bacillus stearothermophilus, (2) estabelecer os parâmetros de crescimento associado a produção de proteases fibrinolíticas por fermentação submersa e (3) verifica o efeito da fermentação extrativa na separação dessas enzimas. Neste estudo foram empregados técnicas de fermentação extrativa e fermentação semi-sólida. Por fermentação submersa foram determinados os seguintes parâmetros: Perfil do crescimento de Bacillus stearothermophilus e a produção de protease utilizando 100mL do meio líquido [(g/L) KH2PO4 2g; (NH4)2SO4 1g; MgSO4 7H2O 0,1 g; Na2HPO4 2H2O 0,9 g; Extrato de Levedura 1 g; água destilada 1000mL] pH 7,2 suplementado com gelatina 0,5%, em frasco de Erlenmeyer de 500mL. O crescimento da bactéria foi determinado a 610nm, de 2 em 2 horas, durante 36 horas. Na Determinação das melhores condições para produção de proteases foram avaliados a influência do pH; agitação, temperatura, a idade do inóculo, da concentração do substrato, a influência das fontes de naturais de carbono (tapiocas, araruta e crueira), fontes de nitrogênio e aeração. No extrato recuperado foi realizado também bioensaio de toxicidade em Artemia salina e testes de degradação in vitro do coágulo sanguíneo pelos métodos da placa de fibrina e degradação do coagulo artificial em tubo. Foi determinado também o coeficiente de partição (K), o fator de purificação (FP) e a recuperação da enzima. A fermentação semi-sólida foi realizada utilizando como substrato 10g feijão manteiginha [Vigna unguiculata (L.) Walp], com 60% umidade, pH 5,0 em frasco Erlenmeyers de 250 mL. Na fermentação extrativa as melhores condições foram: pH 5,0; 180 rpm e 25 ºC, no sistemas utilizaram PEG 1000 (g/mol-1) a 20% (p/p) e sais fosfato a 15% (p/p) com K de 1,05; FP de 1,00; Y de 152,54. Halo de 34 mm na placa de fibrina e degradação parcial do coagulo em tubo. Na fermentação semi-sólida a produção de protease foi de 8,87 (U/mL), halo translucido de 23 mm em placa de fibrina com degradação total do coagulo de sangue em 24h. No presente estudo, protease de Bacillus stearothermophilus produzido em fermentação extrativa e fermentação semi-sólida foi avaliada, demonstrando nas condições ótimas de cultivo que este micro-organismo apresenta fisiologia para aplicações industriais na produção de protease fibrinolítica

O uso intravenoso do ativador tecidual do plasminogênio está aprovado para o tratamento do acidente vascular cerebral isquêmico há alguns anos e estão publicadas diretrizes e recomendações para o seu uso. O atendimento hospitalar precisa ser organizado a fim de tornar esse tratamento exeqüível e seguro, alcançando os resultados esperados. O objetivo deste estudo foi verificar a exeqüibilidade e a segurança do tratamento trombolítico intravenoso nos pacientes tratados pela Clínica Neurológica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, assim como avaliar as características desses pacientes, sua evolução clínica, as complicações do tratamento e os preditores prognósticos. Também foi avaliado o desempenho do atendimento hospitalar, através da análise do tempo das diversas etapas do atendimento, em quatro diferentes grupos de pacientes com base no local do primeiro atendimento médico. Foram tratados 51 pacientes entre Junho de 1998 e Agosto de 2005, primeiramente atendidos no Pronto Socorro de Neurologia (22 pacientes), Instituto do Coração (22 pacientes) e enfermarias do Instituto Central do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (3 pacientes), assim como no Hospital Universitário (4 pacientes). Os tempos do atendimento, representados por suas respectivas medianas, foram: entre o ictus e a admissão (55 minutos); entre a admissão e a análise da tomografia computadorizada de crânio (35 minutos); entre a admissão e o início do tratamento trombolítico (90 minutos) e entre o ictus e o início do tratamento trombolítico (160 minutos). De uma maneira geral, o desempenho do atendimento melhorou durante o período do estudo. Entretanto, a análise comparativa dos grupos revelou que os tempos entre a admissão e a análise da tomografia computadorizada de crânio e entre a admissão e o início do tratamento trombolítico foram maiores no Instituto do Coração (p = 0,002 e p = 0,01, respectivamente) do que no Pronto Socorro de Neurologia e Hospital Universitário. O principal mecanismo causador do acidente vascular cerebral isquêmico foi embolia de origem cardíaca (54%). A maioria dos pacientes tratados chegou ao hospital com déficits neurológicos graves (mediana 17 na Escala de AVC do NIH). Resultado funcional excelente em 3 meses, definido como pontuações 0 ou 1 na Escala de Rankin modificada, foi observado em 29% dos casos e hemorragia cerebral sintomática em 6% dos casos. Em conclusão, o tratamento trombolítico intravenoso com ativador tecidual do plasminogênio no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo foi exeqüível e seguro. A intensidade do déficit neurológico na admissão, mensurada pela Escala de AVC do NIH e a redução igual ou maior que quatro pontos nesta escala em 24 horas foram preditores prognósticos independentes. / Intravenous use of tissue plasminogen activator has been approved for acute ischemic stroke treatment for some years and guidelines and recommendations about its use have been published. Hospital attendance needs to be organized in order to become this treatment feasible and safe, reaching the expected results. The objective of this study was verify feasibility and safety of intravenous thrombolytic therapy in patients who were treated at Neurology Department of the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo as well evaluating characteristics of these patients, their clinical outcome, complications of therapy and outcome predictors. Hospital attendance performance was also evaluated through time analysis of several steps of attendance in four different patient groups based on the place of the first medical attendance. Fifty one patients were treated between June 1998 and August 2005, primarily attended at Neurology Emergency Department (22 patients), Heart Institute (22 patients) and wards of Central Institute of Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (3 patients) as well at University Hospital (4 patients). Attendance times, represented by its median were: between symptoms onset and hospital admission (55 minutes); between hospital admission and computed cranial tomography analysis (35 minutes); between hospital admission and thrombolytic therapy onset (90 minutes) and between symptoms onset and thrombolytic therapy onset (160 minutes). As a general rule, hospital attendance performance improved during study period. However, comparative analysis of groups disclosed that time between hospital admission and computed cranial tomography analysis as well time between hospital admission and thrombolytic therapy onset were longer at Heart Institute (p = 0,002 and p = 0,01, respectively) than at Neurology Emergency Department and University Hospital. The main acute ischemic stroke mechanism was cardiac emboli (54%). Most of treated patients arrived at hospital with serious neurologic impairment (NIH Stroke Scale median 17). Excellent functional outcome in 3 months, defined as scoring 0 or 1 by modified Rankin Scale was observed in 29% and symptomatic cerebral hemorrhage in 6% of the cases. In conclusion, intravenous thrombolytic therapy with tissue plasminogen activator at Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo was feasible and safe. The intensity of the neurological impairment at hospital admission, as measured by NIH Stroke Scale, and four-point or more score reduction on this scale at 24 hours were independent outcome predictors.

O uso intravenoso do ativador tecidual do plasminogênio está aprovado para o tratamento do acidente vascular cerebral isquêmico há alguns anos e estão publicadas diretrizes e recomendações para o seu uso. O atendimento hospitalar precisa ser organizado a fim de tornar esse tratamento exeqüível e seguro, alcançando os resultados esperados. O objetivo deste estudo foi verificar a exeqüibilidade e a segurança do tratamento trombolítico intravenoso nos pacientes tratados pela Clínica Neurológica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, assim como avaliar as características desses pacientes, sua evolução clínica, as complicações do tratamento e os preditores prognósticos. Também foi avaliado o desempenho do atendimento hospitalar, através da análise do tempo das diversas etapas do atendimento, em quatro diferentes grupos de pacientes com base no local do primeiro atendimento médico. Foram tratados 51 pacientes entre Junho de 1998 e Agosto de 2005, primeiramente atendidos no Pronto Socorro de Neurologia (22 pacientes), Instituto do Coração (22 pacientes) e enfermarias do Instituto Central do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (3 pacientes), assim como no Hospital Universitário (4 pacientes). Os tempos do atendimento, representados por suas respectivas medianas, foram: entre o ictus e a admissão (55 minutos); entre a admissão e a análise da tomografia computadorizada de crânio (35 minutos); entre a admissão e o início do tratamento trombolítico (90 minutos) e entre o ictus e o início do tratamento trombolítico (160 minutos). De uma maneira geral, o desempenho do atendimento melhorou durante o período do estudo. Entretanto, a análise comparativa dos grupos revelou que os tempos entre a admissão e a análise da tomografia computadorizada de crânio e entre a admissão e o início do tratamento trombolítico foram maiores no Instituto do Coração (p = 0,002 e p = 0,01, respectivamente) do que no Pronto Socorro de Neurologia e Hospital Universitário. O principal mecanismo causador do acidente vascular cerebral isquêmico foi embolia de origem cardíaca (54%). A maioria dos pacientes tratados chegou ao hospital com déficits neurológicos graves (mediana 17 na Escala de AVC do NIH). Resultado funcional excelente em 3 meses, definido como pontuações 0 ou 1 na Escala de Rankin modificada, foi observado em 29% dos casos e hemorragia cerebral sintomática em 6% dos casos. Em conclusão, o tratamento trombolítico intravenoso com ativador tecidual do plasminogênio no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo foi exeqüível e seguro. A intensidade do déficit neurológico na admissão, mensurada pela Escala de AVC do NIH e a redução igual ou maior que quatro pontos nesta escala em 24 horas foram preditores prognósticos independentes. / Intravenous use of tissue plasminogen activator has been approved for acute ischemic stroke treatment for some years and guidelines and recommendations about its use have been published. Hospital attendance needs to be organized in order to become this treatment feasible and safe, reaching the expected results. The objective of this study was verify feasibility and safety of intravenous thrombolytic therapy in patients who were treated at Neurology Department of the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo as well evaluating characteristics of these patients, their clinical outcome, complications of therapy and outcome predictors. Hospital attendance performance was also evaluated through time analysis of several steps of attendance in four different patient groups based on the place of the first medical attendance. Fifty one patients were treated between June 1998 and August 2005, primarily attended at Neurology Emergency Department (22 patients), Heart Institute (22 patients) and wards of Central Institute of Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (3 patients) as well at University Hospital (4 patients). Attendance times, represented by its median were: between symptoms onset and hospital admission (55 minutes); between hospital admission and computed cranial tomography analysis (35 minutes); between hospital admission and thrombolytic therapy onset (90 minutes) and between symptoms onset and thrombolytic therapy onset (160 minutes). As a general rule, hospital attendance performance improved during study period. However, comparative analysis of groups disclosed that time between hospital admission and computed cranial tomography analysis as well time between hospital admission and thrombolytic therapy onset were longer at Heart Institute (p = 0,002 and p = 0,01, respectively) than at Neurology Emergency Department and University Hospital. The main acute ischemic stroke mechanism was cardiac emboli (54%). Most of treated patients arrived at hospital with serious neurologic impairment (NIH Stroke Scale median 17). Excellent functional outcome in 3 months, defined as scoring 0 or 1 by modified Rankin Scale was observed in 29% and symptomatic cerebral hemorrhage in 6% of the cases. In conclusion, intravenous thrombolytic therapy with tissue plasminogen activator at Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo was feasible and safe. The intensity of the neurological impairment at hospital admission, as measured by NIH Stroke Scale, and four-point or more score reduction on this scale at 24 hours were independent outcome predictors.

Contexte L’occlusion d’une artère du cœur cause un syndrome coronarien aigu (SCA) soit avec une élévation du segment ST (IAMEST) ou sans élévation du segment ST (1). Le traitement des patients avec un IAMEST requiert soit une intervention coronarienne d’urgence (ICP primaire) ou une thérapie fibrinolytique (FL). La thérapie FL peut être administrée soit dans un contexte pré-hospitalier (PHL) ou à l’hôpital. Une prise en charge précoce des patients avec SCA peut être améliorée par un simple indice de risque. Objectifs Les objectifs de cette thèse étaient de : 1) comparer l’ICP primaire et la thérapie FL (2); décrire plusieurs systèmes internationaux de PHL; (3) développer et valider un indice de risque simplifié pour une stratification précoce des patients avec SCA. Méthodes Nous complétons des méta-analyses, de type hiérarchique Bayésiennes portant sur l’effet de la randomisation, d’études randomisées et observationnelles; complétons également un sondage sur des systèmes internationaux de PHL; développons et validons un nouvel indice de risque pour ACS (le C-ACS). Résultats Dans les études observationnelles, l’ICP primaire, comparée à la thérapie FL, est associée à une plus grande réduction de la mortalité à court-terme; mais ce sans bénéfices concluants à long terme. La FL pré-hospitalière peut être administrée par des professionnels de la santé possédant diverses expertises. Le C-ACS a des bonnes propriétés discriminatoires et pourrait être utilisé dans la stratification des patients avec SCA. Conclusion Nous avons comblé plusieurs lacunes importantes au niveau de la connaissance actuelle. Cette thèse de doctorat contribuera à améliorer l’accès à des soins de qualité élevée pour les patients ayant un SCA. / Background Acute occlusion of an artery of the heart results in acute coronary syndromes (ACS), either with ST-segment elevation (STEMI) or without ST-segment elevation (1). STEMI requires urgent treatment to restore coronary artery flow either by primary percutaneous coronary intervention (PCI) or fibrinolytic therapy (FL) (2). Although several randomized controlled trials (RCTs) demonstrate the superiority of primary PCI in reducing mortality compared to FL (2), the benefit of primary PCI over FL remains uncertain in unselected “real-life” patients (3,4). FL can be administered either in the pre-hospital setting (i.e., pre-hospital FL (PHL)) or at the hospital. PHL is rarely available outside Europe (5,6). Insights into the organization of PHL systems of care may promote more widespread use of PHL. Risk stratification of ACS patients should be prompt to ensure timely PCI for high-risk patients and to avoid unnecessary intervention in low-risk patients (7). Despite the availability of numerous ACS risk scores, there is still no simple risk score that can be easily applied in the initial management of ACS patients (8). Objectives The objectives of this doctoral dissertation were to address these current knowledge gaps in the optimal management of ACS. The objectives were to: 1) evaluate the efficacy, effectiveness, and safety of primary PCI and FL, (2) describe the infrastructure, processes and outcomes of several international PHL systems; and (3) develop and validate a novel clinical risk score for early risk stratification of ACS patients. Methods To address these objectives, I completed Bayesian hierarchical random-effects meta-analyses of published RCTs and observational studies which compare primary PCI and FL in patients with STEMI. I undertook a survey of the infrastructure, processes and outcomes of PHL in several European and North American pre-hospital emergency systems. Finally, I developed and validated an ACS risk score called the Canadian ACS (C-ACS). Results Primary PCI was superior to FL in reducing short-term mortality in RCTs and observational studies. However, the long-term survival benefit of primary PCI was noted only in RCTs, and not in the observational studies. PHL can be effectively delivered by health care professionals with variable levels of expertise. The new risk score, C-ACS, has good discriminant properties for short- and long-term mortality in patients with ACS. Conclusions The first manuscript of this dissertation has been recognized as one of the most valuable recent publications in STEMI management and has contributed to reorganization of STEMI care in Ontario. The other two manuscripts in this dissertation provide practical information and tools for health professionals caring for patients with ACS. In summary, this doctoral dissertation has and will continue to contribute to improve access to high quality care for patients with ACS.

Contexte L’occlusion d’une artère du cœur cause un syndrome coronarien aigu (SCA) soit avec une élévation du segment ST (IAMEST) ou sans élévation du segment ST (1). Le traitement des patients avec un IAMEST requiert soit une intervention coronarienne d’urgence (ICP primaire) ou une thérapie fibrinolytique (FL). La thérapie FL peut être administrée soit dans un contexte pré-hospitalier (PHL) ou à l’hôpital. Une prise en charge précoce des patients avec SCA peut être améliorée par un simple indice de risque. Objectifs Les objectifs de cette thèse étaient de : 1) comparer l’ICP primaire et la thérapie FL (2); décrire plusieurs systèmes internationaux de PHL; (3) développer et valider un indice de risque simplifié pour une stratification précoce des patients avec SCA. Méthodes Nous complétons des méta-analyses, de type hiérarchique Bayésiennes portant sur l’effet de la randomisation, d’études randomisées et observationnelles; complétons également un sondage sur des systèmes internationaux de PHL; développons et validons un nouvel indice de risque pour ACS (le C-ACS). Résultats Dans les études observationnelles, l’ICP primaire, comparée à la thérapie FL, est associée à une plus grande réduction de la mortalité à court-terme; mais ce sans bénéfices concluants à long terme. La FL pré-hospitalière peut être administrée par des professionnels de la santé possédant diverses expertises. Le C-ACS a des bonnes propriétés discriminatoires et pourrait être utilisé dans la stratification des patients avec SCA. Conclusion Nous avons comblé plusieurs lacunes importantes au niveau de la connaissance actuelle. Cette thèse de doctorat contribuera à améliorer l’accès à des soins de qualité élevée pour les patients ayant un SCA. / Background Acute occlusion of an artery of the heart results in acute coronary syndromes (ACS), either with ST-segment elevation (STEMI) or without ST-segment elevation (1). STEMI requires urgent treatment to restore coronary artery flow either by primary percutaneous coronary intervention (PCI) or fibrinolytic therapy (FL) (2). Although several randomized controlled trials (RCTs) demonstrate the superiority of primary PCI in reducing mortality compared to FL (2), the benefit of primary PCI over FL remains uncertain in unselected “real-life” patients (3,4). FL can be administered either in the pre-hospital setting (i.e., pre-hospital FL (PHL)) or at the hospital. PHL is rarely available outside Europe (5,6). Insights into the organization of PHL systems of care may promote more widespread use of PHL. Risk stratification of ACS patients should be prompt to ensure timely PCI for high-risk patients and to avoid unnecessary intervention in low-risk patients (7). Despite the availability of numerous ACS risk scores, there is still no simple risk score that can be easily applied in the initial management of ACS patients (8). Objectives The objectives of this doctoral dissertation were to address these current knowledge gaps in the optimal management of ACS. The objectives were to: 1) evaluate the efficacy, effectiveness, and safety of primary PCI and FL, (2) describe the infrastructure, processes and outcomes of several international PHL systems; and (3) develop and validate a novel clinical risk score for early risk stratification of ACS patients. Methods To address these objectives, I completed Bayesian hierarchical random-effects meta-analyses of published RCTs and observational studies which compare primary PCI and FL in patients with STEMI. I undertook a survey of the infrastructure, processes and outcomes of PHL in several European and North American pre-hospital emergency systems. Finally, I developed and validated an ACS risk score called the Canadian ACS (C-ACS). Results Primary PCI was superior to FL in reducing short-term mortality in RCTs and observational studies. However, the long-term survival benefit of primary PCI was noted only in RCTs, and not in the observational studies. PHL can be effectively delivered by health care professionals with variable levels of expertise. The new risk score, C-ACS, has good discriminant properties for short- and long-term mortality in patients with ACS. Conclusions The first manuscript of this dissertation has been recognized as one of the most valuable recent publications in STEMI management and has contributed to reorganization of STEMI care in Ontario. The other two manuscripts in this dissertation provide practical information and tools for health professionals caring for patients with ACS. In summary, this doctoral dissertation has and will continue to contribute to improve access to high quality care for patients with ACS.