Dentre os países da América Latina, o Brasil é o país que mais recebe doações de sangue em volume, alcançando mais de 3,5 milhões de bolsas anualmente só na hemorrede pública. Para que possam ocorrer as transfusões e processamento de hemoderivados é mandatória a realização de testes para detecção de agentes patológicos. Neste cenário está inserido o Instituto de Biologia Molecular do Paraná (IBMP) que, em parceria com a FIOCRUZ, é responsável pelo fornecimento do kit NAT destinado a detecção dos vírus da imunodeficiência humana (HIV), hepatite C (HCV) e hepatite B (HVB) à hemorrede pública brasileira. Visando implementar melhorias e novas tecnologias aos processos existentes, o Instituto adquiriu a licença para utilização da técnica de gelificação na produção de kits para diagnóstico. Esta técnica propõe a estabilização de biomoléculas sem afetar sua funcionalidade, proporcionando a redução de custos de produção, além de possibilitar que os kits sejam armazenados e transportados à temperatura ambiente, aspecto fundamental para simplificar sua cadeia logística e viabilizar a distribuição destes produtos em áreas remotas do Brasil e do mundo. No entanto, ainda há escassez de trabalhos que avaliem aspectos financeiros da implementação desta técnica na indústria. Cabe salientar ainda que a realização de testes em campo seria complexa e demandaria um alto investimento. Uma alternativa para realizar este tipo de avaliação é pelo uso de simulação computadorizada do processo. As ferramentas de simulação possibilitam avaliar diferentes cenários, obtendo dessa forma informações imprescindíveis para melhoria operacional e para tomada de decisões. Neste trabalho, são avaliados os impactos da implementação da técnica de gelificação no custo direto de produção de insumos para diagnóstico por meio de simulação discreta usando o software FlexSim®. Dados obtidos a partir de dezesseis cenários simulados usando a nova tecnologia foram comparados com registros históricos do IBMP na produção de insumos para diagnóstico pelo processo convencional. Dentre os cenários avaliados, seis atenderam às restrições impostas pelo processo e em todos foi possível verificar a redução nos custos diretos, que variou de 3,73% até 5,41% em relação às condições atuais. As reduções de custo ocorreram principalmente devido a reduções nos custos com matéria-prima, que alcançaram economia de 4,1% e nos custos com mão de obra, dada redução do número de operadores no processo. Ademais, os resultados indicam que a implementação da tecnologia propicia melhor aproveitamento da estrutura física da planta sem que ocorra saturação de linhas de processamento. Portanto, baseado em modelos de simulação, este trabalho demonstra a viabilidade do emprego da tecnologia de gelificação no IBMP em termos de custo direto de produção de insumos para diagnóstico. / Among Latin American countries, Brazil is the one that receives more blood donations in volume, reaching up to 3.5 million blood bags in the public blood donation network annually. In order to enable transfusions and processing of blood-derived products, it is mandatory to test the blood against pathologic agents. In association with the Oswaldo Cruz Foundation (FIOCRUZ), the Molecular Biology Institute of Paraná (IBMP) supplies the kit NAT for the detection of the human immunodeficiency, hepatitis B and C viruses (HIV, HBV and HCV) to the Brazilian public blood donation network. Aiming at the implementation of improvements and the introduction of new technologies to its processes, the Institute acquired a license to use the gelification technique in the production of diagnostic kits. The gelification technique proposes the stabilization of biomolecules without harming their functionality, while reducing production costs and allowing the products to be stored and transported at room temperature. It is a key aspect to simplify the logistics chain used to distribute the kit, allowing it to reach World`s remote areas. However, there is still a lack of published studies evaluating financial aspects of the implementation of this technique in the industry. It is worth emphasizing the cost to conduct field experiments of this kind of implementation. One alternative for such an evaluation is by the use of process computer simulation. Simulation tools could be used to analyze different scenarios, providing valuable information for process improvement and decision-making. In this work, the impacts of the gelification technique implementation in the direct production cost of reagents for diagnostic were evaluated using discrete simulation with FlexSim®. Data obtained from sixteen simulated scenarios deploying gelification were compared with historic records of the production of reagents for diagnosis using the conventional process at IBMP. Six of the sixteen evaluated scenarios satisfied the restrictions imposed by the process and all of them indicated a reduction of direct production costs, varying from 3.73% to 5.41% when compared to current conditions. The cost reduction is mainly associated to lower expenses with raw materials, representing savings of up to 4.1% and to the reduction in the number of operators needed. Additionally, results indicate that the implementation of the new technology leads to a better use of the physical structure of the production facility without saturating processing lines. Therefore, based on simulation models, this work shows that the employment of the gelification technology in the manufacturing process of diagnostic reagents at IBMP is viable in terms of direct production costs.

Indiana University-Purdue University Indianapolis (IUPUI) / Juvenile Myelomonocytic Leukemia (JMML) is rare, fatal myeloproliferative disease (MPD) affecting young children, and is characterized by expansion of monocyte lineage cells and hypersensitivity to Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) stimulation. JMML is frequently associated with gain-of-function mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase, Shp2. Activating Shp2 mutations are known to promote hyperactivation of the Ras-Erk signaling pathway, but Akt is also observed to have enhanced phosphorylation, suggesting a potential role for Phosphatidylinositol-3-Kinase (PI3K)-Akt signaling in mutant Shp2-induced GM-CSF hypersensitivity and leukemogenesis. Having demonstrated that Class IA PI3K is hyperactivated in the presence of mutant Shp2 and contributes to GM-CSF hypersensitivity, I hypothesized the hematopoietic-specific Class IA PI3K catalytic subunit p110δ is a crucial mediator of mutant Shp2-induced PI3K hyperactivation and GM-CSF hypersensitivity in vitro and MPD development in vivo. I crossed gain-of-function mutant Shp2 D61Y inducible knockin mice, which develop fatal MPD, with mice expressing kinase-dead mutant p110δ D910A to evaluate p110δ’s role in mutant Shp2-induced GM-CSF hypersensitivity in vitro and MPD development in vivo. As a comparison, I also crossed Shp2 D61Y inducible knockin mice with mice bearing inducible knockout of the ubiquitously expressed Class IA PI3K catalytic subunit, p110α. I found that genetic interruption of p110δ, but not p110α, significantly reduced GM-CSF-stimulated hyperactivation of both the Ras-Erk and PI3K-Akt signaling pathways, and as a consequence, resulted in reduced GM-CSF-stimulated hyper-proliferation in vitro. Furthermore, I found that mice bearing genetic disruption of p110δ, but not p110α, in the presence of gain-of-function mutant Shp2 D61Y, had on average, smaller spleen sizes, suggesting that loss of p110δ activity reduced MPD severity in vivo. I also investigated the effects of three PI3K inhibitors with high specificity for p110δ, IC87114, GDC-0941, and GS-9820 (formerly known as CAL-120), on mutant Shp2-induced GM-CSF hypersensitivity. These inhibitors with high specificity for p110δ significantly reduced GM-CSF-stimulated hyperactivation of PI3K-Akt and Ras-Erk signaling and reduced GM-CSF-stimulated hyperproliferation in cells expressing gain-of-function Shp2 mutants. Collectively, these findings show that p110δ-dependent PI3K hyperactivation contributes to mutant Shp2-induced GM-CSF hypersensitivity and MPD development, and that p110δ represents a potential novel therapeutic target for JMML.