Neste trabalho estudou-se a purificação de fator VIII de coagulação (FVIII) e da Proteína C (PC) por cromatografia. Em coluna ANX Sepharose FF como primeira etapa de purificação do plasma permite a eluição do FVIII e da PC com bom fator de purificação, mas não a separação destas. Propomos a separação de FVIII e PC empregando cromatografia de afinidade a metal (IMAC) empregando Cu2+, Ni2+, Zn2+, Co2+ e Fe3+ e dessorção empregando imidazol, cloreto de amônio e variação de pH. Em colunas de Fe3+ e Ni2+ as proteínas praticamente não se ligaram à resina. Em IMAC-Co2+, a PC não é adsorvida pela resina enquanto o FVIII pode ser eluído com imidazol 100 mM. Em IMAC-Cu2+ a PC eluiu com imidazol 10mM e o FVIII com 200mM. Não foi possível eluir as proteínas nem com NH4Cl 1M nem quando o pH foi abaixado até 4,0. Em IMAC-Zn2+ a PC não é adsorvida e o FVIII eluiu com imidazol 200mM ou NH4Cl 1M. Diminuindo-se o pH, a recuperação da atividade do FVIII foi baixa. Concluímos que IMAC-Co2+ apresentou os melhores rendimentos e melhores fatores de purificação para as 2 protéinas. / In this work purification methods for the coagulation factor VIII (FVIII) and Protein C (PC) by chromatography was studied. The use of ANX Sepharose FF column as the first purification step allows the elution of FVIII and PC with good purification factors, but not its separation. We propose the separation of FVIII and PC using immobilized metal ion affinity chromatography (IMAC) using Cu2+, Ni2+, Zn2+, Co2+ and Fe3+ and desorption employing imidazole, ammonium chloride and pH variation. In the columns containing Fe3+ and Ni2+ metal ions, proteins did not adsorb to the resin. In IMAC-Co2+, PC was not adsorbed to the resin, while FVIII could be eluted with 100 mM imidazole. In IMAC-Cu2+ PC could be eluted with 10 mM imidazole and FVIII with 200 mM. It was not possible to elute the proteins from IMAC-Cu2+ column with either 1M NH4Cl or when pH was descreased to 4,0. In IMAC-Zn2+, PC was not adsorbed and FVIII could be eluted with 200 mM imidazole or 1 M NH4Cl. We conclude that IMAC-Co2+ presented the best yield and purification factors for the 2 proteins.

A mathematical model of blood coagulation under defined flow conditions, initiated and modulated by spatially discrete regions of surface bound tissue factor (TF) and thrombomodulin (TM), respectively, is presented. The model incorporates fluid phase and surface-associated reactions of the extrinsic, intrinsic, and common pathways, as well as three inhibitory pathways. The spatially heterogeneous model is formulated by finite element method, and an effective prothrombotic zone, which quantifies the spatial propagation of thrombin generation is defined. Characteristic features of coagulation are simulated under physiologic conditions, and the behavior of the system in response to perturbations in TF and TM surface densities, TF site dimensions, and wall shear rate is explored. The major findings of these studies include: (i) The model system responds in an 'all-or-none', threshold-like manner to changes in model parameters. (ii) It was found that prothrombotic effects may extend significantly beyond the dimensions of the spatially discrete site of TF expression in both axial and radial directions. (iii) The relationship between the length of the effective prothrombotic zone and the interval distance between tandem sites of TF expression dictate the net response of the system. Additive prothrombotic effects of sub-clinical lesions as well as suppressive antithrombotic effects of intervening TM-containing regions were observed. Secondly, the computational model is applied to calculate an individualized, systems-based metric of clotting potential for 210 pre-menopausal women in the Leiden Thrombophilia Study (LETS). The simulated variable was found to be a highly predictive parameter for deep venous thrombosis risk.

M.Sc. (Zoology) / A number of chemical substances in mining, industrial, agricultural and domestic effluents are likely to contaminate watercourses. These toxicants have a definite effect on all aquatic life, even at sublethal concentrations. Due to the extensive copper mining activities in the Phalaborwa region, there is concern that the sublethal effect of copper might affect the physiology of fish in the Olifants River, Kruger National Park, Transvaal. Copper can be found as an essential micronutrient in nearly all waters, although anthropogenic sources such as metals from mining and planting industries might produce environmental concentrations causing toxic effects to fish. Several effects of exposure to sublethal concentrations of copper have already been reported in fish, In the present study, the blood coagulation, general haematology, osmoregulation and differential white blood cell counts of the Mozambique tilapia, Oreochromis mossambicus were investigated after the exposures to sublethal concentrations of copper, for a short-term (96 hours) exposure as well as a long-term (four weeks) exposure in experimental flow through systems, at both 29 ± I°C and 19 ± I°C. After the respective sublethal exposures, changes in the values of blood coagulation, general haematology, osmoregulation and differential white blood cell counts were obtained, to determine the effects of the chosen copper concentrations on the blood physiology of O. mossambicus, at 29 + 10 and 19 ± I°C, respectively. After a photographic observation of blood, during all the phases of coagulation was made, it was evident that the exposure to copper, at both temperatures, resulted in delays of the blood coagulation times as well as decreases in the elasticity of the clots that formed. Copper was found to induce haemophilia at 29 ± I°C and 19 ± l°C. whilst at 19 ± I°C it also induced thrombocytopenia. Thus exposure to copper lead to coagulation defects which caused haemorrhage which can eventually cause the death of these fish...

Orientador: Joyce Maria Annichinno-Bizzacchi / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-08T02:29:39Z (GMT). No. of bitstreams: 1 Mello_TayanaBezerraTeixeira_D.pdf: 2343820 bytes, checksum: be3d8e4b057cd84ec6771c67b8b1407d (MD5) Previous issue date: 2006 / Resumo: Nos últimos anos tem sido demonstrada uma associação entre níveis elevados de certos fatores da coagulação e risco de tromboembolismo venoso (TEV). Entretanto, o número de estudos é pequeno e a maioria estão restritos a populações caucasóides. Neste estudo caso-controle emparelhado por idade, sexo e etnia investigamos os níveis plasmáticos do fibrinogênio, FVII, FVIII, FIX, FX, FXI, FvW e grupo sanguíneo (GS) ABO no risco trombótico. Foram analisados 175 pacientes com TEV (122 mulheres e 53 homens), com idade mediana de 36 anos (13-63), acompanhados no Ambulatório de Hemostasia da Unicamp no período de julho de 2002 a julho de 2005. Na análise univariada, níveis elevados de FVIII (OR: 5,3 IC95% 2,9-9,6), FvW (OR: 4,9 IC95% 2,7-8,9), FIX (OR: 2,4 IC95% 1,3-4,4), FXI (OR: 2,1 IC95% 1,1-4,0) e GS não O (OR: 3,0 IC95% 1,9-4,6) foram fatores de risco para TEV. O FVIII, FvW e GS não O foram associados ao risco tanto em membro inferior como em sítio incomum da doença. A análise de todas estas variáveis, segundo critério de seleção ¿stepwise¿, mostrou o FVIII (OR: 3,1 IC95% 1,6-6,0), FvW (OR:2,8 IC95% 1,4-5,4) e o GS não-O (OR:2,2 IC95% 1,3-3,5) como fatores de risco independentes para TEV e que o FIX e FXI agiram sinergicamente a estas variáveis no acréscimo do risco trombótico. Risco de recorrência foi conferido por elevações do FIX (OR: 4,7 IC95% 1,8-11,9) e FXI (OR: 3,4 IC95% 1,8-8,7). Os determinantes dos níveis plasmáticos do FVIII não estão totalmente esclarecidos. Como a LRP (Low density lipoprotein receptor related protein) tem um papel no catabolismo do FVIII, foram pesquisados os polimorfimos C200T, o A775P e o D2080N no gene codificador dessa proteína, como fator de risco para TEV e a influência dos mesmos sobre os níveis do FVIII e FvW. Não houve diferença nas prevalências destes polimorfismos entre pacientes e controles. No entanto, no grupo-controle, o genótipo DN, do polimorfismo D2080N, foi associado a menores concentrações do FVIII (77,4 UI/dl) e FvW (70,2 UI/dl), quando comparado ao genótipo DD (127 UI/dl e 108,4 UI/dl, respectivamente p<0,05). Em conclusão, nesta população brasileira miscigenada, o FVIII, FvW , FIX, FXI e GS não O foram associados ao risco de TEV e o polimorfismo D2080N, no gene da LRP, interferiu com os níveis plasmáticos de FVIII e FvW / Abstract: During the last years an association between high levels of certain coagulation factors and the risk for venous thromboembolism (VTE) has been demonstrated. The number of studies, however, is small, and most of them are restricted to Caucasian populations. In this case control study, matched for age, sex and ethnicity, we investigated the plasma levels of fibrinogen, FVII, FVIII, FIX, FX, FXI, vWF and the ABO blood group (BG) in the thrombotic risk. It was analyzed 175 patients with VTE (122 women and 53 men), median age 36 years (13-63), followed at the hemostasis outpatient clinic at the State University of Campinas ¿ UNICAMP, between July 2002 and July 2005. In univariate analysis, elevated levels of FVIII (OR: 5.3 95%CI 2.9-9.6), FvW (OR: 4.9 95%CI 2.7-8.9), FIX (OR: 2.4 95%CI 1.3-4.4), FXI (OR: 2.1 95%CI 1.1-4.0) and non O BG (OR: 3.0 95%CI 1.9-4.6) were risk factors for VTE. FVIII, FvW and non O BG were associated with the risk both in lower limbs and unusual sites of disease. Analysis of all these variables by stepwise selection criteria showed FVIII (OR:3.1 95%CI 1.6-6.0), FvW (OR:2.8 95%CI 1.4-5.4) and non O BG (OR:2.2 95%CI 1.3-3.5) as independent risk factors for VTE, and FIX and FXI increased synergistically the risk of thrombosis of these variables. Risk of recurrence was conferred by FIX (OR:4.7 IC95% 1.8-11.9) and FXI (OR:3.4 IC95% 1.8-8.7). The FVIII plasma levels determinants are not well established. Since LRP (Low density lipoprotein receptor related protein) has a role in the catabolism of FVIII, we evaluated the C200T, A775P and, D2080N polymorphisms in the gene coding of this protein, as risk factor for VTE and their influence upon the levels of FVIII and vWF. There was no difference regarding the prevalence of these polymorphisms between patients and controls. However, in the control group, the DN genotype, of the D2080N polymorphism, was associated with lower concentrations of FVIII (77.4 UI/dl) and vWF (70.2 UI/dl), when compared to DD genotype (127 UI/dl e 108.4 UI/dl, respectively p<0.05). In conclusion, in these Brazilian miscigenous population, increased levels of FVIII, FvW, FIX, FXI and non O BG were associated with VTE risk and the D2080N polymorphism, in the LRP gene, influenced plasma levels of FVIII and vWF / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Fisiopatologia Medica