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In this paper, we provide a mathematical model of the effect of aspirin on blood clotting. The model tracks the enzyme prostaglandin H synthase and an important blood clotting factor, thromboxane A2, in the form of thromboxane B2. Through model analysis, we determine conditions under which the reactions of prostaglandin H synthase are self-sustaining. Lastly, through numerical simulations, we demonstrate that the model accurately captures the steady-state chemical concentrations of interest in blood, both with and without aspirin treatment.

A utilização de células humanas para a produção do fator VIII de coagulação recombinante (rFVIII) visa obter padrões de glicosilação equivalentes aos encontrados na proteína normal. O objetivo do trabalho foi obter um processo de produção do rFVIII em biorreator em perfusão, devido à sua labilidade térmica. Foram realizados estudos preliminares em Spinner e biorreator utilizando uma linhagem de rHeLa, cujos resultados embasaram os estudos com a linhagem produtora rSkHep. Foram utilizados microcarregadores nos cultivos com esta linhagem devido à dificuldade de adaptação da mesma à suspensão. Ensaios preliminares identificaram a melhor condição de cultivo com 3 g/L Mic e 1 cel/mic e, a partir destes valores, realizou-se um ensaio em perfusão, com tempo de residência de 24 h, no qual as variáveis controladas foram mantidas constantes durante três tempos de residência. A concentração de rFVIII obtida foi semelhante 2 UI/ mL. / The interest in using human cells for the recombinant coagulation factor VIII (rFVIII) lies in obtaining glycosylation patterns similar to the ones found in the normal protein. The objective of this work was to obtain a process for rFVIII production in bioreactor, in perfusion mode, due to the thermal lability of the protein. Using a recombinant HeLa cell line adapted to suspension growth a group of studies in a bioreactor in batch mode were performed. These results were the basis for the studies performed with the producing cell line rSkHep. Microcarriers (micc) were used due to the harshness to adapt the cell line to suspension and to serum-free medium. Preliminary tests identified the best culture condition with 3 g micc/L and 3 cell/micc and, from its values, it was performed a bioreactor study in perfusion mode, with a residence time of 24 hours. The controlled variables were kept constant for three residence times. The maximum rFVIII concentration obtained was 2 UI/mL.

O fator VIII da coagulação é utilizado para o tratamento da hemofilia A e pode ser obtido a partir de concentrados do plasma humano ou na sua forma recombinante (rFVIII). Nosso laboratório tem explorado uma alternativa mais eficiente para a produção do rFVIII em células de mamíferos, utilizando um variante artificial do rFVIII humano (rFVIII-lab). O objetivo principal deste trabalho foi avaliar a imunogenicidade do rFVIII-lab utilizando camundongos modelo da hemofilia A, tendo como objetivos experimentais a purificação, caracterização de atividade funcional in vivo e caracterização de imunogenicidade do rFVIII-lab comparada a produtos de referência, um derivado de plasma e outro recombinante. Os resultados indicam que o perfil de imunogenicidade observado para o rFVIII-lab foi menos intenso e a atividade funcional observada foi similar quando comparado aos produtos de referência. A expectativa é que o presente estudo contribua para o estabelecimento de uma plataforma de produção do rFVIII no país visando o tratamento dos pacientes hemofílicos brasileiros. / Factor VIII (FVIII) replacement therapy employing either FVIII concentrates from blood plasma or recombinant FVIII is the standard of care for management of hemophilia A. Our group has been exploring a more efficient alternative for recombinant FVIII production in mammalian cells employing an engineered artificial variant of the protein (rFVIII-lab). The main objective of this study was to evaluate the immunogenicity of the rFVIII-lab using a murine model of hemophilia A and the specific experimental objectives were to purify, evaluate the in vivo functional activity and the immunogenicity of rFVIII-lab compared to plasma derived and recombinant reference products. Data revealed reduced immunogenicity of rFVIII-lab whereas functional activity was similar when compared to the reference products. The presented study is expected to contribute to the establishment of a locally production platform for the rFVIII aiming at the treatment of Brazilian hemophilic patients.

Von Willebrand factor (VWF) promotes platelet adhesion and aggregation at sites of vascular damage. This function is directly related to the multimer size of VWF. The VWF-specific metalloprotease ADAMTS13 decreases VWF multimer size by cleaving at Y1605-M1606 in the VWF A2 domain. This thesis examined the sensitivity of ADAMTS13 cleavage to mutagenesis of the full-length multimerized VWF substrate, and a small VWF A2 domain fragment, VWF115. The ADAMTS13 cleavage site at Y1605-M1606 was mutated with the most severe loss of cleavage observed in Y1605A/M1606A. In addition, 4 single nucleotide polymorphisms were examined, with D1472H, Q1571H, P1601T proteins all showing increased resistance to cleavage. In contrast, G1643S has enhanced cleavage in the full-length VWF substrate but shows cleavage resistance in VWF115. Three von Willebrand disease mutations were also examined. In patients, R1597W has enhanced ADAMTS13 cleavage and a loss of high molecular weight multimers, while R1205H has enhanced protein clearance resulting in very low VWF levels and Y1584C patients have moderately low VWF levels. R1597W has enhanced cleavage of full-length VWF, while a slight cleavage increase is observed in VWF115 for Y1584C, and no change is seen with R1205H. The VWF mutations R1597W, Y1605A/M1606A, R1205H and Y1584C were further examined in the VWF knockout mouse using recombinant VWF protein infusion and hydrodynamic delivery of VWF cDNA to determine the effects these mutations produce on VWF antigen levels, multimer structure, secretion, clearance and function in a thrombotic injury model. All four mutations had different pathogenic mechanisms. R1597W showed accelerated clearance with loss of multimer structure, and greatly increased time to thrombotic occlusion. Y1605A/M1606A showed accelerated clearance with normal or supranormal multimer structure, a loss of thrombotic occlusion but increased platelet accumulation. Y1584C showed no change in protein clearance, with decreased VWF antigen level, reduced multimer structure, and reduced thrombotic potential. R1205H demonstrated a synthetic defect in vitro and in vivo increased clearance with a decrease in VWF antigen levels and normal multimer structure and a variable thrombotic potential. These results validate the use of the genetically-modified VWF knockout mouse model for evaluating the pathogenic mechanisms of putative VWF mutations. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2010-07-28 10:24:40.654

Factor C is a multi-domain serine protease which recognizes Gram-negative bacteria via binding to lipopolysaccharides and triggers hemolymph clotting cascade in the horseshoe crab. A closely related molecule was also found to be present in the genome of the tick Ixodes scapularis. In this work, the full sequence of Factor C ortholog from Ixodes ricinus (IrFC) was determined. IrFC is mainly expressed in tick hemocytes and the heavy chain of the activated molecules is present in tick hemolymph as confirmed by Western blotting with antibodies raised against recombinant fragments of IrFC. The function of the IrFC in tick innate immunity was assessed using its silencing by RNA interference.

The freshwater crayfish, Pacifastacus leniusculus, has been used as a model for studying hematopoiesis or blood cell production or hematopoiesis and immunity. The work of this thesis aims to investigate the impact of factors such as ROS signaling, Ast1, and the PVF/PVR signaling pathway in controlling stem cell behavior during hematopoiesis and specifically the role of the crosslinking enzyme transglutaminase (TGase) in regulation of hematopoiesis. The role of ROS in crayfish hematopoiesis was characterized by using the antioxidant named NAC to inhibit ROS production. Low ROS level resulted in a prolonged decrease in hemocyte numbers and a combined injection of LPS and NAC caused a slower rate of new hemocyte production. A low ROS level in cell cultures supplemented with crude Ast1 was found to inhibit cell spreading and a high extracellular TGase activity was detected on the surfaces of APC and HPT cells. We suggest that ROS serves as a prime signal to control proliferation and differentiation of progenitor cells by affecting extracellular TGase activity. We reported an inhibitory effect of Ast1 on TGase enzyme activity and on its crosslinking activity and consequently Ast1 affects the clot formation and thus coagulation by inhibiting the crosslinking activity of the TGase enzyme. Secretion of the clot protein (CP) and the production of CP filament network between spreading cells were observed in HPT cell cultures in vitro. In the presence of CP together with Ast1 in 3D-collagen-I cultures, HPT cells were found to be more elongated and they formed chains of cells throughout the surrounding matrix. In the HPT tissue, CP was located around the HPT cells or around the lobules of HPT, and thus, CP was demonstrated to be a part of ECM and to possibly function together with collagen in generating a suitable environment for HPT progenitor cells. The inhibition of PVF/PVR downstream signaling pathway by Sunitinib malate resulted in a dramatic change of cell morphology and induction of an increase cell surface area during cell culture. The addition of crude Ast1 into the cell cultures in vitro enhanced this effect. Consequently, cell migration was stimulated and a high extracellular TGase activity on HPT cell surface was found after this inhibition. In conclusion, the work in this thesis provides new insight in understanding the role of the extracellular matrix (ECM) and extracellular TGase activity in controlling stem cell activity.

Orientadores: Marcelo Cristianini, Alline Artigiani Lima Tribst / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-24T06:58:37Z (GMT). No. of bitstreams: 1 LeiteJunior_BunoRicardodeCastro_M.pdf: 4205771 bytes, checksum: 2ccdbecbd8ae2dc92dd1978fbf86b48b (MD5) Previous issue date: 2014 / Resumo: A homogeneização à alta pressão (HAP) é um processo capaz de alterar a conformação e funcionalidade de enzimas. Os objetivos deste trabalho foram: (i) avaliar a influência da HAP até 190 MPa nas atividades proteolítica e de coagulação do leite bem como na estabilidade de quatro enzimas coagulantes do leite, (ii) acompanhar o processo de coagulação por ensaios reológicos e (iii) avaliar o desenvolvimento dos géis por 24 horas por meio das análises de proteólise, sinérese, reologia e microscopia. As avaliações foram feitas comparando-se os resultados obtidos com as enzimas processadas e não processadas. O coalho de vitelo processado a 190 MPa apresentou redução de 52% na atividade proteolítica, aumento da taxa de coagulação do leite e gel formado mais consistente. A avaliação deste gel por 24h indicou a formação de uma rede proteica com menor proteólise, maior sinérese, maior consistência e menor porosidade. Após processamento a 150 MPa, o coalho de bovino adulto apresentou redução da atividade proteolítica, aumento da atividade e estabilidade de coagulação do leite, maior taxa de coagulação do leite e formação de gel com maior consistência. O gel se mostrou mais compacto, firme e com maior expulsão do soro da matriz proteica nas 24h em que foi avaliado. A protease fúngica do Rhizomucor miehei foi a enzima mais resistente ao processo de HAP, sofrendo mínima ou nenhuma alteração na atividade proteolítica e de coagulação do leite quando processada até 190 MPa em diferentes concentrações e em múltiplos processos consecutivos. Entretanto, na avaliação reológica da coagulação do leite utilizando-se a protease fúngica homogeneizada a 190 MPa por até 3 ciclos ou quando homogeneizada a 190 MPa em soluções com concetração de 20 % foi observado aumento da consistência do gel. Para pepsina suína, as alterações na atividade proteolítica e de coagulação do leite só foram observadas durante a estocagem, com redução na atividade proteolítica e um aumento na atividade de coagulação do leite para enzima processada a 150 MPa. No entanto, esta enzima processada promoveu uma coagulação do leite mais rápida formando um gel mais consistente, mesmo imediatamente após o processamento por HAP. Durante a observação deste gel por 24h, este se mostrou mais compacto, firme, menos poroso e com maior liberação de soro da matriz proteica. De uma forma geral foi possível concluir que as maiores pressões aplicadas (150 MPa e 190 MPa) afetaram positivamente as enzimas avaliadas, com redução da atividade proteolítica inespecífica e aumento da atividade de coagulação de leite, com consequente formação de géis com menores níveis de proteólise, o que favorece a manutenção da rede proteica rígida, firme e coesa. Desta forma, conclui-se que a HAP é um processo promissor que pode ser aplicado como uma tecnologia para melhorar as características hidrolíticas das enzimas coagulantes do leite, especialmente quando se deseja diminuir atividade proteolítica e aumentar sua atividade de coagulação do leite. Além disso, a menor proteólise no gel pode resultar numa extensão da vida de prateleira de queijos frescos, por, possivelmente, reduzir a formação de compostos de sabor indesejável / Abstract: High pressure homogenization (HPH) is a process that can alter the conformation and functionality of enzymes. The objectives of this study were: (i) evaluate the influence of HPH up to 190 MPa on the proteolytic and milk-clotting activities and stability of four milk-clotting enzymes, (ii) monitor the coagulation process by rheological assays and (iii) evaluate the gel development for 24 hours analyzing proteolysis, syneresis, rheological and microstructural behavior. The evaluations were performed by comparing the results between the processed and non-processed enzymes. The calf rennet processed at 190 MPa decreased 52 % its proteolytic activity, increased the rate of milk-clotting and a more consistent gel was formed. The evaluation of the gel for 24 hours indicated the formation of a protein network with lower proteolysis, higher syneresis, higher consistency and lower porosity. After processing at 150 MPa adult bovine rennet showed a reduction proteolytic activity, increase activity and stability of milk-clotting, higher milk-clotting rate and formed more consistent gels. This gel was more compact, firm and higher whey separation of protein matrix during the 24 hours of evaluation. The fungal protease from Rhizomucor miehei was the most resistant enzyme to the HPH process, showing minimal or no change in proteolytic activity and milk coagulation when processed up to 190 MPa at different concentrations and multiple consecutive processes. However, in the rheological evaluation of milk coagulation using fungal protease homogenized to 190 MPa for up 3 cycles or when homogenized in a solution with a concentration of 20% observed increase in the consistency of the gel. For porcine pepsin, changes on proteolytic activity and milk coagulation were only observed during storage, with reduction of proteolytic activity and an increase on the milk-clotting activity for the enzyme processed at 150 MPa. However, this enzyme promoted a faster coagulation of milk forming more consistent gel immediately after the processing by HPH. During the observation of this gel for 24 hours, this was more compact, firm, less porous and more release of whey of the protein matrix. Overall it was concluded that the highest applied pressures (150 MPa and 190 MPa) positively affected the enzymes with reduced nonspecific proteolytic activity and increased milk-clotting activity, with consequent formation of gels with lower levels of proteolysis, which favors the maintenance of a network of protein rigid, firm and cohesive. Thus, it is concluded that HPH is a promising process that can be applied as a technology to improve the hydrolytic characteristics of milk coagulating enzymes, especially to reduce proteolytic activity and increase the milk-clotting activity. Furthermore, the lower proteolysis in the gel may result in an extension of the shelf life of fresh cheese, by possibly reducing the formation of bitterness flavor / Mestrado / Tecnologia de Alimentos / Mestre em Tecnologia de Alimentos

A utilização de células humanas para a produção do fator VIII de coagulação recombinante (rFVIII) visa obter padrões de glicosilação equivalentes aos encontrados na proteína normal. O objetivo do trabalho foi obter um processo de produção do rFVIII em biorreator em perfusão, devido à sua labilidade térmica. Foram realizados estudos preliminares em Spinner e biorreator utilizando uma linhagem de rHeLa, cujos resultados embasaram os estudos com a linhagem produtora rSkHep. Foram utilizados microcarregadores nos cultivos com esta linhagem devido à dificuldade de adaptação da mesma à suspensão. Ensaios preliminares identificaram a melhor condição de cultivo com 3 g/L Mic e 1 cel/mic e, a partir destes valores, realizou-se um ensaio em perfusão, com tempo de residência de 24 h, no qual as variáveis controladas foram mantidas constantes durante três tempos de residência. A concentração de rFVIII obtida foi semelhante 2 UI/ mL. / The interest in using human cells for the recombinant coagulation factor VIII (rFVIII) lies in obtaining glycosylation patterns similar to the ones found in the normal protein. The objective of this work was to obtain a process for rFVIII production in bioreactor, in perfusion mode, due to the thermal lability of the protein. Using a recombinant HeLa cell line adapted to suspension growth a group of studies in a bioreactor in batch mode were performed. These results were the basis for the studies performed with the producing cell line rSkHep. Microcarriers (micc) were used due to the harshness to adapt the cell line to suspension and to serum-free medium. Preliminary tests identified the best culture condition with 3 g micc/L and 3 cell/micc and, from its values, it was performed a bioreactor study in perfusion mode, with a residence time of 24 hours. The controlled variables were kept constant for three residence times. The maximum rFVIII concentration obtained was 2 UI/mL.

O fator VIII da coagulação é utilizado para o tratamento da hemofilia A e pode ser obtido a partir de concentrados do plasma humano ou na sua forma recombinante (rFVIII). Nosso laboratório tem explorado uma alternativa mais eficiente para a produção do rFVIII em células de mamíferos, utilizando um variante artificial do rFVIII humano (rFVIII-lab). O objetivo principal deste trabalho foi avaliar a imunogenicidade do rFVIII-lab utilizando camundongos modelo da hemofilia A, tendo como objetivos experimentais a purificação, caracterização de atividade funcional in vivo e caracterização de imunogenicidade do rFVIII-lab comparada a produtos de referência, um derivado de plasma e outro recombinante. Os resultados indicam que o perfil de imunogenicidade observado para o rFVIII-lab foi menos intenso e a atividade funcional observada foi similar quando comparado aos produtos de referência. A expectativa é que o presente estudo contribua para o estabelecimento de uma plataforma de produção do rFVIII no país visando o tratamento dos pacientes hemofílicos brasileiros. / Factor VIII (FVIII) replacement therapy employing either FVIII concentrates from blood plasma or recombinant FVIII is the standard of care for management of hemophilia A. Our group has been exploring a more efficient alternative for recombinant FVIII production in mammalian cells employing an engineered artificial variant of the protein (rFVIII-lab). The main objective of this study was to evaluate the immunogenicity of the rFVIII-lab using a murine model of hemophilia A and the specific experimental objectives were to purify, evaluate the in vivo functional activity and the immunogenicity of rFVIII-lab compared to plasma derived and recombinant reference products. Data revealed reduced immunogenicity of rFVIII-lab whereas functional activity was similar when compared to the reference products. The presented study is expected to contribute to the establishment of a locally production platform for the rFVIII aiming at the treatment of Brazilian hemophilic patients.