Fetal-Derived MyD88 Signaling Contributes to Poor Pregnancy Outcomes During Gestational Malaria

Placental malaria (PM) remains a severe public health problem in areas of high malaria transmission. Despite the efforts to prevent infection poor outcomes in Plasmodium endemic areas, there is still a considerable number of preterm births and newborns with low birth weight resulting from PM. Although local inflammation triggered in response to malaria is considered crucial in inducing placental damage, little is known about the differential influence of maternal and fetal immune responses to the disease progression. Therefore, using a PM mouse model, we sought to determine the contribution of maternal and fetal innate immune responses to PM development. For this, we conducted a series of cross-breeding experiments between mice that had differential expression of the MyD88 adaptor protein to obtain mother and correspondent fetuses with distinct genetic backgrounds. By evaluating fetal weight and placental vascular spaces, we have shown that the expression of MyD88 in fetal tissue has a significant impact on PM outcomes. Our results highlighted the existence of a distinct contribution of maternal and fetal immune responses to PM onset. Thus, contributing to the understanding of how inflammatory processes lead to the dysregulation of placental homeostasis ultimately impairing fetal development

Characterization of oncogenic pathways in pancreatic cancer: microRNAs and IKKβ

O adenocarcinoma ductal pancreático (PDAC) é o tumor sólido mais letal em humanos, sendo que menos de 10% dos pacientes sobrevivem 5 anos ou mais. Essa baixa sobrevida é atribuída ao fenótipo altamente metastático, à recidiva e à ineficácia das terapias disponíveis. Assim, a presente tese buscou caracterizar vias oncogênicas que contribuem para a malignidade de PDAC, visando assim aumentar o conhecimento necessário para desenvolvimento de novas terapias, sendo esta tese dividida em dois capítulos. No Capítulo 1, avaliamos a via de microRNAs (miRNAs) regulados pela KRAS oncogênica, que está presente em mais de 90% dos tumores de PDAC e ainda permanece sem tratamento disponível para este tipo de tumor. Dado que miRNAs efetores de KRAS ainda são pouco explorados, nosso objetivo foi identificar miRNAs induzidos pela KRAS oncogênica, que contribuem para o fenótipo maligno do PDAC<. Usando um ensaio de microarranjo de DNA, identificamos 17 miRNAs regulados positivamente por KRAS. A análise global de alvos destacou o supressor tumoral PTEN como o alvo regulado pelo maior número destes miRNAs (7 dos 17). Por meio da superexpressão e redução de expressão de KRAS, confirmamos que KRAS regula negativamente PTEN e regula positivamente 4 destes 7 miRNAs que têm PTEN como alvo. Para avaliar se a regulação de PTEN por KRAS poderia ser mediada por estes miRNAs, usamos ensaios de luciferase contendo trechos do 3\'UTR de PTEN. Tanto a KRAS, quanto o mir-19b-3p são capazes de regular a atividade de luciferase em PDAC. Como conclusão deste capítulo, nós identificamos uma assinatura de miRNAs induzidos por KRAS em PDAC que tem PTEN como alvo e validamos o miR-19b-3p como um dos miRNAs efetores na regulação dos níveis de PTEN por KRAS, contribuindo para o fenótipo maligno de PDAC. No Capítulo 2, investigamos o papel da quinase IKK para o fenótipo tronco-tumoral das células iniciadoras de tumor (CITs) pancreáticas, que constituem uma subpopulação de células tumorais consideradas responsáveis pela metástase e recidiva tumoral. A IKK desempenha um papel chave na ativação da via do NF-&#954;B, constitutivamente ativa em cerca de 70% dos casos de PDAC e que promove o fenótipo tronco-tumoral em diversos tipos de tumores. Assim, nosso objetivo foi determinar como a IKK&#946; afeta o fenótipo tronco-tumoral e metastático de células pancreáticas visando o desenvolvimento desta quinase como alvo terapêutico. Para avaliar CITs em linhagens de PDAC, utilizamos a cultura de tumoresferas e estabelecemos a linhagem AsPC-1-SORE6 expressando o gene repórter GFP sob o controle dos fatores de pluripotência SOX2 e OCT4. Embora a inibição da expressão de IKK&#946; por interferência de RNA tenha reduzido a formação de tumoresferas, a inibição farmacológica de IKK&#946; foi ineficaz em inibir o fenótipo tronco-tumoral das CITs pancreáticas, não alterando a formação de tumoresferas ou alterando transitoriamente a frequência da população SORE6- GFP+. Por outro lado, a inibição de IKK&#946; reduziu a migração e invasão celulares. Em conclusão, a IKK&#946; afeta o fenótipo tronco-tumoral de maneira quinase-independente e interfere na migração e invasão celulares, características importantes para metástase, podendo representar um potencial alvo terapêutico anti-metastático em PDAC.Pancreatic ductal adenocarcinoma (PDAC) is the deadliest solid tumor in humans, with a 5-year survival rate of less than 10%. This low survival rate is attributed to its highly metastatic phenotype, tumor relapse and to the inefficacy of available therapies. Thus, this thesis sought to characterize oncogenic pathways that contribute to PDAC malignancy, aiming to increase the knowledge needed for the development of new therapies, with the thesis being divided into two chapters. In Chapter 1, we evaluated the microRNA (miRNA) pathway regulated by oncogenic KRAS, which is mutated in more than 90% of PDAC tumors and lack effective therapies for this type of tumor. Given that miRNAs that act as KRAS effectors are still poorly explored, our objective was to identify miRNAs induced by oncogenic KRAS that contribute to the malignant phenotype of PDAC. Using DNA microarray, we identified 17 KRAS-upregulated miRNAs. miRNA target analysis highlighted the tumor suppressor PTEN as the miRNA target most often regulated by these miRNAs (7 out of 17). Through overexpression and downregulation of KRAS, we confirmed that KRAS negatively regulates PTEN levels and upregulates 4 of the 7 miRNAs that are predicted to target PTEN. To investigate whether the negative regulation of PTEN by KRAS could be mediated by these miRNAs, we used luciferase reporters containing PTEN 3\'UTR inserts. Both KRAS and mir-19b-3p were able to regulate luciferase reporter activity in PDAC. As a conclusion of this chapter, we identified a signature of KRAS-induced miRNAs in PDAC that target PTEN and have validated miR-19b- 3p as one of the effector miRNAs in the regulation of PTEN levels by KRAS, contributing to the malignant phenotype of PDAC. In Chapter 2, we investigated the role of IKK kinase for the cancer stem phenotype of pancreatic tumor initiating cells (TICs), which constitute a subpopulation of tumor cells considered responsible for metastasis and tumor recurrence. IKK&#946; plays a key role in activating the NF-&#954;B pathway, which is constitutively active in about 70% of PDAC cases and promotes the cancer stem phenotype in several types of tumors. Thus, our objective was to determine how IKK&#946; affects the cancer stem and metastatic phenotype of pancreatic cells aiming at the development of this kinase as a therapeutic target. To evaluate TICs in PDAC cell lines, we used the tumor sphere assays and established the AsPC-1-SORE6 cell line expressing the GFP reporter gene under control of pluripotency factors SOX2 and OCT4. Although inhibition of IKK expression by RNA interference reduced tumor sphere formation, its pharmacological inhibition did not impair the cancer stem phenotype of pancreatic CITs, not affecting tumor sphere formation or only transiently altering the SORE6-GFP+ population frequency. On the other hand, IKK&#946; inhibition reduced cell migration and invasion. In conclusion, IKK&#946; seems to affect the cancer stem phenotype in a kinase-independent manner and interferes with cell migration and invasion, important hallmarks of metastasis, and may represent a potential anti-metastatic therapeutic target for PDAC

MyD88 signaling is directly involved in the development of murine placental Malaria

Malaria is a widespread infectious disease caused by the parasite Plasmodium. During pregnancy, malaria infection leads to a range of complications that can affect both the mother and fetus, including stillbirth, infant mortality, and low birth weight. In this study, we utilized a mouse model of placental malaria (PM) infection to determine the importance of the protein MyD88 in the host immune response to Plasmodium during pregnancy. Initially, we demonstrated that Plasmodium berghei NK65GFP adhered to placental tissue via chondroitin sulfate A and induced PM in mice with a C57BL/6 genetic background. To evaluate the involvement of MyD88 in the pathology of PM, we performed a histopathological analysis of placentas obtained from MyD88(-/-) and wild-type (WT) mice following infection on the 19th gestational day. Our data demonstrated that the detrimental placental alterations observed in the infected mice were correlated with the expression of MyD88. Moreover, in the absence of this protein, production of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) was significantly reduced in the infected mice. More importantly, in contrast to fetuses from infected WT mice, which exhibited a reduction in body weight, the fetuses from infected MyD88(-/-) mice did not display significant weight loss compared to their noninfected littermates. In addition, we observed a decrement of maternal care associated with malaria infection, which was attenuated in the MyD88-deficient mice. Collectively, the results of this study illustrate the pivotal importance of the MyD88 signaling pathway in the pathogenesis of placental malaria, thus presenting new possibilities for targeting MyD88 in therapeutic interventions.Programa Estratégico de Ciência, Tecnologia & Inovação nas Fundações Estaduais de Saúde (PECTI/AM Saúde), FAPEAMSão Paulo Research Foundation (FAPESP), 2009/53889-0São Paulo Research Foundation (FAPESP), 2009/53256-7São Paulo Research Foundation (FAPESP), 2011/17880-8São Paulo Research Foundation (FAPESP), 2012/02270-2Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES, AUX-PE-PNPD 2751/2010Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES, AUX-PE-PNPD 258/2010Conselho Nacional do Desenvolvimento Científico e Tecnológico - CNPq, 475771/2009-5Conselho Nacional do Desenvolvimento Científico e Tecnológico - CNPq, 404213/201