Os piretróides sintéticos, em especial os do tipo II, como a cialotrina, são extensivamente utilizados para o controle de uma ampla variedade de ectoparasitas que acometem os animais de produção. Entretanto, no Brasil e em outros países, sua utilização vai além da saúde animal, sendo utilizados também em saúde pública, no controle de diversos vetores, como é o caso do vetor da dengue, dentre outros. Visto que a cialotrina modifica a atividade de macrófagos peritoneais, o objetivo deste trabalho foi investigar os prováveis mecanismos através dos quais este piretróide modifica a atividade destas células. Os presentes resultados, analisados em seu conjunto, mostram de maneira inequívoca que a cialotrina tem um efeito direto e/ou indireto sobre a atividade de macrófagos peritoneais. Especificamente, observou-se neste trabalho que o praguicida causou em ratos: 1 ? marcação fos positiva em neurônios do núcleo paraventricular do hipotálamo (NPH), após a dose de 3,0 mg/kg/dia; 2 - diminuição do percentual e intensidade de fagocitose de macrófagos peritoneais ativados e avaliados por citometria de fluxo; 3 - diminuição dose-dependente da produção de nitrito (NO2); 4 ? diminuição do percentual e intensidade de fagocitose de macrófagos peritoneais ativados, em ratos adrenalectomizados e/ou tratados com metirapona (inibidor da síntese de corticosterona) e RU 486 (antagonista de receptores glicocorticóides) com a finalidade de modular os níveis de glicocorticóides, e tratados com 3,0 mg/kg/dia de cialotrina; 5 ? aumento dos níveis de noradrenalina hipotalâmica em animais tratados com a dose de 3,0mg/kg/dia de cialotrina; 6 - diminuição do percentual e intensidade de fagocitose, bem como diminuição da produção de nitrito de macrófagos peritoneais ativados, em ratos simpatectomizados químicamente com 6-OHDA; 7 - diminuição dose dependente do percentual e intensidade de fagocitose, bem como da produção de nitrito de macrófagos peritoneais ativados e tratados in vitro com 10 e 100 nM de cialotrina. No entanto, não observamos: 1 ? alterações na produção de nitrito realizada por macrófagos peritoneais ativados, em ratos adrenalectomizados e/ou tratados com metirapona e RU 486; 2 - alterações na viabilidade celular induzida pelo tratamento in vitro com a cialotrina na concentração de 10 e 100 nM e 3 ? alterações nos efeitos da cialotrina sobre a atividade de macrófagos tratados in vitro com os ligantes de receptores benzodiazepínicos periféricos. Em conjunto, os presentes dados mostram que a cialotrina interfere com a atividade de macrófagos por atuar indiretamente, através da ativação do eixo Hipotálamo-Hipófise-Adrenal (HHA), e/ou diretamente sobre os mesmos modulando sua atividade. É muito provável que o efeito resultante do tratamento in vivo com este praguicida esteja ligado à somatória destas ações / Synthetic pyrethroids, particularly those of type II, such as cyhalothrin, are extensively used in agriculture for the control of a broad range of ectoparasites in farm animals. However, in Brazil and some other countries, these pyrethroids have also been used in public health, for the control of insects that are known to be vectors of diseases such as dengue. Since it has been suggested that cyhalothrin alters activity of peritoneal macrophages, the objective of our study was to investigate the putative mechanisms for the changes induced by pyrethroid in these cells. The results presented here show, in an unequivocal manner, that cyhalothrin has a direct or indirect (or both) effect on the activity of peritoneal macrophages. We specifically observed in this work that this pesticide induced in rats: 1- Fos-positive immunostaining in neurons of the paraventricular nucleus of the hypothalamus (NPH), after 3.0 mg/kg/day; 2 ? a reduction in the percentage and intensity of phagocytosis by activated peritoneal macrophages, evaluated by flow cytometry; 3 ? a dose-dependent reduction in nitrite production (NO2); 4 ? a reduction in the percentage and intensity of phagocytosis by activated peritoneal macrophage from adrenalectomized rats treated or not with metirapone (inhibitor of corticosterone synthesis) or RU 486 (antagonist of glicocorticoids receptors) with the propose of modulating the levels of glicocorticoids, and treated with 3.0 mg/kg/day of cyhalothrin; 5 ? an increase in the hypothalamic levels of noradrenaline in rats treated with 3.0 mg/kg/day of cyhalothrin; 6 ? a reduction in the percentage and intensity of phagocytosis and also a decrease in the production of nitrite by activated peritoneal macrophages, after chemical sympatectomy with 6-OHDA; 7 ? a dose-dependent reduction of the percentage and intensity of phagocytosis, and also a decrement in nitrite production by activated peritoneal macrophages treated in vitro with 10 and 100 nM of cyhalothrin. However, we found no differences on: 1 ? nitrite production by activated peritoneal macrophages after adrenalectomy, treated or not with metirapone or RU 486; 2 ?cell viability of peritoneal macrophages treated in vitro with 10 and 100 nM of cyhalothrin, and 3 ? the effects of cyhalothrin on macrophage activity after in vitro treatment with peripheral benzodiazepine receptor ligands. Altogether, the present results show that cyhalothrin interferes with the activity of peritoneal macrophages by acting indirectly, via activation of the hypothalamus-pituitary-adrenal axis, or directly on these cells, altering their activity. As a matter of fact, it is quite possible that the results of in vivo cyhalothrin treatment on macrophage activity would be related to the combined effect of these direct and indirect influences

O estresse é uma realidade na produção avícola mundial. Sabe-se que ambientes estressores prejudicam o bem-estar, os parâmetros produtivos e a imunidade de frangos de corte. Sabe-se, também, que o estresse por calor diminui a atividade de macrófagos em frangos de corte e, que existem, inúmeros estressores ambientais que insidem sobre a produção animal e podem aumentar a susceptibilidade às doenças. A Salmonella spp. é uma das maiores zoonoses do mundo, causando mais de 1 bilhão de casos de infecção. Nesse sentido, o presente trabalho analisa os efeitos do estresse por calor (31±1°C) sobre os índices zootécnicos, a imunidade, a invasão bacteriana e a integridade intestinal em frangos de corte infectados com Salmonella enteritidis; os dados obtidos foram discutidos dentro de uma perspectiva neuroimune. Os frangos foram divididos em quatro grupos: 1) Controle (C); 2) Estresse por Calor a 31±1 °C (HS31°C); 3) Controle infectados com Salmonella enteritidis (Controle Positivo [PC]) e; 4) Estresse por calor a 31±1 °C e infectados com Salmonella (PHS31°C). Nossos resultados mostraram que o estresse por calor em uma situação de infecção experimental por Salmonella enteritidis (grupo PHS31°C) 1) diminuiu os índices zootécnicos; especificamente, diminuiu o ganho de peso, consumo de alimento e a conversão alimentar; 2) diminuiu os níveis plasmáticos de INF-γ e IgA; 3) diminuiu a expressão de, IL-6 e IL-12 em baço e diminui IL1- β, IL-10 e TGF-β em tonsila cecal; 4) diminuiu a expressão de AvBD-4 e AvBD-6 em tonsila cecal e; 5) diminuiu a expressão de TLR-2 em baço e tonsila cecal. Observamos, também, 6) aumento dos níveis séricos de corticosterona nos animais dos grupos HS31°C e PHS31°C e; 7) piora no quadro de enterite produzida pela Salmonella enteritidis, quando os animais foram estressados por calor, caracterizando-se uma enterite moderada ao longo de todo o intestino delgado. Finalmente, 8) observamos que o estresse por calor aumentou a migração de Salmonella enteritidis para baço das aves do grupo PHS31°C, porém esse aumento não foi observado no fígado; observamos, também, presença de Salmonella na medula osssea dos animais estressados e infectado com essa bactéria. Os dados obtidos sugerem que a somatória dos fatores estresse por calor e infecção por Samonella prejudicou os parâmetros produtivos, a integridade intestinal, a imunidade e, em especial a ativação e atividade de macrófagos, possibilitando um aumento da migração de Salmonella enteritidis para o baço e medula óssea dos frangos de corte. Neste sentido, o estresse por calor teria prejudicado a qualidade da barreira imune intestinal, via ativação do eixo HPA e aumento dos níveis de corticosterona, diminuindo a imunidade inata proporcionando a migração das bactérias patogênicas através da mucosa intestinal para o baço e a medula óssea das aves estressadas. / Stress is a reality in the world poultry production. Environmental stressors impair both welfare, performance parameters and immunity in broiler chickens. Heat stress decreases macrophage activity in broiler chickens and many environmental stressors that impact animal production increases animal\'s susceptibility to diseases. Samonella spp is one of the most endemic zoonotic diseases of the world, inducing more than 1 billion infection cases per year. In this way, we studied the effects of 31±1°C heat stress on performance parameters, immunity, bacteria invasion and intestinal integrity in broiler chickens experimentally infected with Salmonella enteritidis; the data were discussed under a neuroimmune perspective. The broiler chickens were divided into four different groups: 1) Control group (C); 2) 31±1 °C heat stressed group (HS31°C); 3) Control group infected with Salmonella enteritidis Positive control (PC) and; 4) 31±1 °C heat stressed and Salmonella enteritidis infected group (PHS31°C). We showed the heat stress applied in the course of Salmonella enteritidis infection (PHS31°C group) decreased poultry performance parameters; specifically, it decreased the body weight gain, the feed intake and the food conversion; 2) decresead INF-γ and IgA plasmatic levels; 3) decreased the mRNA expression of IL-6 and IL- 12 in spleen and the mRNA expression of IL1-β, IL-10 and TGF-β in cecal tonsil; 4) decreased the mRNA expression of AvBD-4 and AvBD-6 in cecal tonsil and; 5) decreased the mRNA expression of TLR-2 in spleen and cecal tonsil. We also observed 6) an increase in corticosterone serum levels in the animals of the HS31°C and the PHS31°C groups and, 7) more severe intestinal inflamation produced by Salmonella enteritidis in heat stressed chickens, characterized by a moderate enteritis throughout all the small intestine mucosa (PHS31°C group). Finally, 8) we showed that the heat stress increased splenic Salmonella enteritides invasion in PHS31°C broiler chickens; we also observed the presence of Salmonella in the bone marrow of stressed and infected broiler chickens. These data suggest that heat stress and Salmonella infection working together impair chicken\'s performance parameters, intestinal integrity and immunity (specially the macrophage activity), increasing ate the same time splenic and bone marrow Salmonella enteritidis invasion. Thus, heat stress could have impared the intestinal immunity barrier quality, via HPA axis activation and corticosterone serum levels release, decreasing the inate immunity and, providing pathogenic bacteria migration through the intestinal mucosa for spleen and bone marrow of the heat stressed chickens.

Submitted by PPG Pediatria e Sa?de da Crian?a (pediatria-pg@pucrs.br) on 2018-08-30T19:28:55Z No. of bitstreams: 1 Disserta??o Final Gabriela Fabiano de Souza.pdf: 1295606 bytes, checksum: 81a673082dab9c871a133af1f8517135 (MD5) / Approved for entry into archive by Sheila Dias (sheila.dias@pucrs.br) on 2018-09-03T12:11:04Z (GMT) No. of bitstreams: 1 Disserta??o Final Gabriela Fabiano de Souza.pdf: 1295606 bytes, checksum: 81a673082dab9c871a133af1f8517135 (MD5) / Made available in DSpace on 2018-09-03T12:19:46Z (GMT). No. of bitstreams: 1 Disserta??o Final Gabriela Fabiano de Souza.pdf: 1295606 bytes, checksum: 81a673082dab9c871a133af1f8517135 (MD5) Previous issue date: 2018-03-19 / Conselho Nacional de Pesquisa e Desenvolvimento Cient?fico e Tecnol?gico - CNPq / Respiratory syncytial virus (RSV) is the major cause of infection in children up to five years of age. Reinfection is very common among patients, causing symptoms such as cold or allergy. However, in children, immunosuppressed patients and elderly infection is exacerbated leading to hospitalization and in some case, even death. The number of hospitalizations each year is alarming, even more so because up to now there is still no vaccine for RSV. Tissue damage in the lung caused by RSV leads to an immune response, where infected cells signal activation of signaling pathways, production of reactive oxygen species (ROS), and massive production of inflammatory mediators. Among this production is the macrophage migration inhibitory factor (MIF), which is a pro-inflammatory cytokine, which has been shown to play an important role in the immune response. Knowing this importance, we evaluated MIF expression macrophages from BALB/c mice. The cells were infected with different concentrations of RSV and analyzed by western blot, real-time PCR and Cytometric Bead Array (CBA). After confirmation of MIF expression by the infection, different inhibitors of signaling pathways and ROS were used to evaluate its importance for the expression of MIF. From the results obtained, we showed the dependence of ROS, 5-lipoxygenase (5-LOX), COX, PI3K and partially of P38 MAPK, for MIF expression, besides the need for viral activity. MIF was shown to be important for the release of cytokines such as TNF?, MCP-1 and IL-10. Based on this information MIF may play an important role in the exacerbation of infection, so it was extremely important to explore mechanisms involved in the expression of MIF in relation to RSV. / O V?rus sincicial respirat?rio (VSR) ? a maior causa de infec??o em crian?as at? os cinco anos de idade. A reinfec??o ? muito comum entre os pacientes, causando sintomas como de uma gripe ou alergia, no entanto, em crian?as, pacientes imunossuprimidos e idosos a infec??o ? muito mais exacerbada, o que acaba levando a necessidade de interna??o, podendo levar o paciente a ?bito. O n?mero de interna??es a cada ano ? alarmante, ainda mais que at? os dias atuais ainda n?o se tem uma vacina para o VSR. O dano tecidual no pulm?o, causado por VSR leva a uma resposta imune, onde c?lulas infectadas sinalizam para que ocorra a ativa??o de vias de sinaliza??o, produ??o de esp?cies reativas de oxig?nio (EROs) e tamb?m uma produ??o massiva de mediadores inflamat?rios. Dentre essa produ??o, est? o fator inibidor de migra??o de macr?fagos (MIF), que ? uma citocina pr?-inflamat?ria, que tem demonstrado um importante papel na resposta imune. Sabendo dessa import?ncia, avaliamos a express?o de MIF em macr?fagos de camundongos BALB/c. As c?lulas foram infectadas com diferentes concentra??es de VSR e analisadas por western blot, PCR em tempo real e Cytometric Bead Array (CBA). Ap?s a confirma??o da express?o de MIF pela infec??o, foram utilizados diferentes inibidores de vias de sinaliza??o e de EROs, para que fosse poss?vel avaliar sua import?ncia para a express?o de MIF. A partir dos resultados obtidos mostramos a depend?ncia de EROS, 5-lipoxigenase (5-LOX), COX, PI3K e parcialmente de P38 MAPK, para a express?o de MIF, al?m da necessidade de atividade viral. MIF se mostrou importante para a libera??o de citocinas como TNF ?, MCP-1 e IL-10. Baseado nessas informa??es MIF pode desempenhar um papel importante na exacerba??o da infec??o, sendo assim, foi de extrema import?ncia explorar mecanismos envolvidos na express?o de MIF em rela??o ao VSR.

Benzodiazepínicos (BDZ) são fármacos amplamente utilizados devido às suas propriedades ansiolíticas e sedativas, mediadas pelo complexo GABAA no Sistema Nervoso Central (SNC). Além destes receptores centrais, os BDZ possuem afinidade por receptores do tipo periféricos (PBR) os quais estão presentes em células do sistema imune, como linfócitos e macrófagos, em células tumorais e em glândulas adrenais. O presente trabalho avaliou os efeitos do diazepam, um BDZ freqüentemente utilizado, sobre o crescimento tumoral e a imunidade de animais portadores do Tumor Ascítico de Ehrlich (TAE). Mais especificamente, este trabalho avaliou os seguintes parâmetros da resposta imune: atividade de macrófagos, populações de linfócitos B, T helper e citotóxicos esplênicos, citotoxicidade de células Natural Killer (NK). Além disso, a marcação para PBR em células do TAE e avaliação de possíveis efeitos do diazepam ou Ro5-4864 in vitro sobre o ciclo celular do TAE foram avaliadas. Os resultados mostraram os seguintes efeitos do tratamento com diazepam in vivo (3,0 mg/kg): (1) aumento do crescimento do TAE; sem modificação das fases do ciclo celular do tumor, após 7 dias, (2) diminuição do número de leucócitos da cavidade peritoneal, da produção de NO e do índice de espraiamento; mas sem interferência com a produção de peróxido de hidrogênio e o índice de fagocitose, após 2 dias (3) não modificou o peso relativo do baço e a porcentagem de linfócitos esplênicos, após 2 dias ou 7 dias (4) aumento da citotoxicidade de células NK, após 3 dias (5) diminuição da porcentagem de células do TAE marcadas para PBR, após 7 dias. O tratamento in vitro com diazepam ou Ro5-4864 mostrou um aumento da proliferação de células do TAE. Já o tratamento in vivo com diazepam em doses menores (0,3 mg/kg e 1mg/kg) não modificou o crescimento do TAE, após 7 dias. Desta forma, sugere-se que o diazepam na dose de 3,0 mg/kg tenha aumentado o crescimento do TAE e diminuído a resposta imune inata, observada por meio da diminuição da atividade dos macrófagos peritoneais. Parece-nos plausível excluir de nossos resultados a participação de linfócitos B, T helper e citotóxicos. Por outro lado, não foi possível precisar a relevância das células NK para o desenvolvimento do tumor. Além disso, pode-se afirmar que há expressão de PBRs em células do TAE e que o tratamento in vitro com diazepam ou Ro5-4684 aumentou a proliferação destas células. Desta forma, os resultados dos dois últimos experimentos sugerem que a existência de efeitos do diazepam sobre crescimento tumoral in vivo pode também ser atribuída, ao menos em parte, a uma ação direta deste fármaco sobre células do TAE / Benzodiazepines (BDZ) are drugs widely used due to their anxiolytic and sedative properties, acting on specific sites coupled to GABAA complex in the Central Nervous System (CNS). Besides these central receptors, BDZ have affinity for peripheral-type receptors (PBR), which have been found in immune cells, such as lymphocytes and macrophages, in tumor cells and in the adrenal glands. The present study evaluated the effects of diazepam, a commonly used BDZ, on tumor growth and immunity of mice bearing Ehrlich Ascitic Tumor (EAT). Specifically, this study evaluated the following parameters of the immune system: macrophage activity, populations of B, helper and cytotoxic T lymphocytes, and Natural Killer (NK) cells cytotoxicity. Furthermore, the evaluation of PBR expression in EAT cells and possible in vitro effects of diazepam or Ro5-4864 on EAT cell cycle were performed. Results showed the following diazepam effects in vivo (3.0 mg/kg per day): (1) increased tumor growth without changes in cell cycle, after 7 days; (2) decreased the number of leucocytes in the peritoneal cavity, the production of NO and the spreading index, but did not modify the production of hydrogen peroxide and the phagocytosis index, after 2 days; (3) did not modify the relative spleen weight and the population of lymphocytes after 2 or 7 days, (4) increased NK cytotoxicity after 3 days; (5) reduced the percentage of EAT cells expressing PBR after 7 days. Experiments performed in vitro showed that diazepam or Ro5-4864 increased the proliferation of EAT cells. Diazepam treatment in vivo using lower doses (0.3 mg/kg and 1mg/kg) did not modify tumor growth. Therefore, diazepam in the dose of 3.0 mg/kg increased the growth of EAT and reduced innate immunity, probably through the decrease in the activity of peritoneal macrophages. A role of B and helper or cytotoxic T lymphocytes in our experiments seems unlikely since the population of these cells types remained unchanged. On the other hand, it was not possible to determine the relevance of NK cells cytotoxicity on tumor development. The expression of PBR in EAT cells and the increase of their proliferation induced by in vitro treatment with diazepam or Ro5-4684 were observed. The results of these two last experiments suggest that the increase on tumor growth following diazepam treatment in vivo can be attributed, at least in part, to a direct action of this drug on EAT cells

This dissertation represents efforts to determine the functional consequences acquired by vascular smooth muscle cells (SMC) in response to CD40 ligation by activated CD154+ T cells, and to elucidate components of the signaling pathway(s) activated in response to CD40 signaling in both monocytes and SMC. To study the consequences of CD40 stimulation, primary human monocytes and aortic SMC were treated with plasma membranes purified from CD154 + , CD4+ T cells. The results presented in this dissertation demonstrate that SMC, like monocytes/macrophages, are capable of interacting with T cells in a manner that results in reciprocal activation events. SMC were shown to present antigen to, and activate T cells. In turn T cell stimulus resulted in the activation of proinflammatory function in SMC initiated through the CD154:CD40 interaction. CD40 stimulation of SMC resulted in the production of the chemokines interleukin 8 (IL-8) and macrophage chemotactic protein-1 (MCP-1), and the upregulation of intercellular adhesion molecule (ICAM). Examination of the intracellular signaling pathways activated through CD40 signaling revealed the involvement of MAPKs in the pathway leading to induction of proinflammatory activity. Evaluation of CD40 signaling in monocytes demonstrated the activation of the MAPK family members ERK1/2, but not the MAPK family members p38 or c-jun-N-terminal kinase (JNK). In contrast, CD40 signaling in SMC was shown to result in ERK1/2 and p38 activation, and both of these kinases were shown to play a critical role in the induction of chemokine synthesis. An examination of the ability of anti-inflammatory cytokines to modulate CD40 signaling in monocytes and SMC demonstrated that the anti-inflammatory cytokines IL-4 and IL-10 abrogate CD40-mediated induction of inflammatory cytokine production by monocytes. This inhibition was shown to be a result of a negative influence of IL-4 and IL-10 on CD40 mediated ERK1/2, activation in monocytes. However, IL-4 and IL-10 did not inhibit SMC proinflammatory responses indicating a difference in the intracellular responses to these cytokines by the two cell types. (Abstract shortened by UMI.)

H2O2 generated by alveolar macrophages has been linked to the development pulmonary fibrosis, but little is known about its source, mechanism of production and exact role upon alveolar macrophage activation. In this study, we found that alveolar macrophages from asbestosis patients spontaneously produce high levels of H2O2 and have high expression of Cu,Zn-SOD. Cu,Zn-SOD localized to the mitochondrial intermembrane space (IMS) in asbestosis patients and asbestos induced translocation of Cu,Zn-SOD to the IMS. This process was unique to macrophages and dependent on functional mitochondrial respiration. The presence of at least one of the conserved cysteines was required for disulfide bond formation and mitochondrial translocation. These conserved cysteine residues were also necessary for enzyme activation and H2O2 generation. Cu,Zn-SOD-mediated H2O2 generation was inhibited by knockdown of the iron-sulfur protein, Rieske, in complex III. The role of Cu,Zn-SOD was biologically relevant as Cu,Zn-SOD-/- mice generated significantly less H2O2, had less oxidative stress, and were protected from developing pulmonary fibrosis. This protective mechanism is closely related to the alveolar macrophage activation and polarization in Cu,Zn-SOD-/- mice, as they had a dominant pro-inflammatory phenotype. Macrophages not only initiate and accentuate inflammation after tissue injury, but they are also involved in resolution and repair. The pro-inflammatory M1 macrophages have microbicidal and tumoricidal activity, whereas the M2 macrophages are involved in tumor progression and tissue remodeling, and can be pro-fibrotic in certain settings. We demonstrate that overexpression of Cu,Zn-SOD promoted macrophages polarization into an M2 phenotype. Furthermore, overexpression of Cu,Zn-SOD in mice resulted in a pro-fibrotic environment and accelerated the development of pulmonary fibrosis. The mechanism which Cu,Zn-SOD-mediated H2O2 utilizes to modulate macrophage M2 polarization is through redox regulation of a critical cysteine in STAT6. The polarization process, at least partially, was regulated by epigenetic modulation. We show that STAT6 was indispensable for Cu,Zn-SOD-mediated M2 polarization. STAT6 upregulated Jmjd3, a histone H3 lysine 27 demethylase, and initiated M2 gene transcriptional activation. Targeting STAT6 with leflunomide, which can reduce cellular ROS production and inhibit STAT6 phosphorylation, abolished M2 polarization and ameliorated the fibrotic development. Taken together, these observations provide a novel mechanism for the pathogenesis of pulmonary fibrosis whereby the antioxidant enzyme Cu,Zn-SOD plays a paradoxical role. The study highlights the importance of mitochondrial Cu,Zn-SOD and redox signals in macrophage polarization and fibrosis development. These observations demonstrate that the Cu,Zn-SOD-STAT6-Jmjd3 pathway is a novel regulatory mechanism for M2 polarization and that leflunomide is a potential therapeutic agent in the treatment of pulmonary fibrosis.

Aedes aegypti é importante vetor de patógeno causador de doença como dengue, febre amarela, febre Chikungunya e Zika. A fêmea realiza repasto sanguíneo a fim de adquirir nutriente para o desenvolvimento dos ovos. Neste contexto, a saliva possui papel fundamental, representando o elo entre o artrópode hematófago, seu hospedeiro vertebrado e o potencial patógeno a ser transmitido. Nessa saliva encontra-se um coquetel farmacológico com diversas atividades biológicas, como a presença de peptídeos antimicrobianos e moléculas com funções imunomoduladoras sobre células do hospedeiro vertebrado, com especial atenção aos macrófagos. Como os macrófagos também estão envolvidos nos mecanismos efetores da resposta contra protozoários do gênero Leishmania e a leishmaniose apresenta-se como uma doença de caráter zoonótico de grande relevância em saúde pública, o objetivo deste trabalho foi de avaliar o efeito do extrato de glândula salivar (EGS) de A. aegypti em infecções por Leishmania. Nossos resultados mostraram aumento da infecção in vivo e in vitro na presença do EGS, sugerindo fortemente que o EGS de A. aegypti é capaz de aumentar a infecção por Leishmania. / Aedes aegypti is an important vector of disease-causing pathogens such as dengue, yellow fever, Chikungunya and Zika fever. The mosquito female takes a blood meal in order to develop the eggs. In this context, a saliva plays a key role, representing the link between the hematophagous arthropod, its vertebrate host and the potential pathogen to be transmitted. During the evolutionary process, these insects developed a salivary cocktail with an arsenal of molecules presenting several immunomodulatory properties in host cells, such as macrophages. As macrophages are also involved in the mechanisms of response against protozoa of the genus Leishmania and leishmaniasis is a zoonotic disease of great relevance in public health, the aim of this work is to evaluate the effect of salivary gland extract (SGE) of A. aegypti in Leishmania infections. Our results showed increased infection, in vivo and in vitro, in the presence of SGE, strongly suggesting that A. aegypti EGS is able to increase infection by Leishmania.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotent cytokine involved in the production and function of hematopoietic cells, and GM-CSF plays in particular a major role in responses to infection and physiological and pathological inflammatory processes. GM-CSF is produced in many cell types, and increases in the intracellular Ca2+ concentration are, like in many other systems, of major importance in the intracellular signaling that determines GM-CSF expression after receptor stimulation of the cells. Previous studies have shown that the Ca2+/calmodulin-dependent phosphatase calcineurin (CN) mediates stimulation of GM-CSF transcription in response to Ca2+. This thesis shows that Ca2+ signaling also regulates GM-CSF transcription negatively through Ca2+/calmodulin-dependent kinase II (CaMK II) phosphorylation of serines in the autoinhibitory domain for DNA binding of the transcription factor Ets1. Mutation of the CaMK II target serines increased transactivation of the GM-CSF promoter/enhancer and decreased the sensitivity to inhibition by increased Ca2+ or constitutively active CaMK II. The Ca2+-dependent phosphorylation of Ets1 was also shown to reduce the binding of Ets1 to the GM-CSF promoter in vivo. RUNX1, also known as acute myeloid leukemia 1 (AML1), is one of three mammalian RUNX transcription factors and has many essential functions in hematopoiesis. RUNX1 has also many important roles in the immune system, and RUNX1 is the most frequent target for chromosomal translocation of genes in acute human leukemias. This thesis shows that RUNX1 directly interacts with both subunits of CN and that the strongest interaction is localised to the regulatory CN subunit and the DNA binding domain of the RUNX protein. Constitutively active CN was shown to activate the promoter/enhancer of GM-CSF synergistically with RUNX1, RUNX2 or RUNX3, and the Ets1 binding site of the promoter was shown to be essential for the synergy between RUNX1 and CN in Jurkat T cells. The analysis suggests that Ets1 phosphorylated by the protein kinase glycogen synthase kinase-3β is the target of RUNX1-recruited CN phosphatase at the GM-CSF promoter. Transforming growth factor-β (TGF-β) is another multipotent cytokine that often has a role opposite to that of GM-CSF in inflammatory responses since it is a potent suppressor of immune cells and therefore is anti-inflammatory. This thesis shows that TGF-β can decrease transcription from a GM-CSF promoter/enhancer. Certain constitutively active TGF-β receptors and the TGF-β activated transcription factor Smad3 could also repress GM-CSF transcription, whereas several other Smad proteins did not have this inhibitory effect. The inhibition required intact DNA binding ability of Smad3, and the 125 bp upstream of the transcription initiation site, which was sufficient for the inhibition, contains several weak Smad binding sites near the TATA box next to an Ets1 site of the promoter. Smad3 was able to bind to the promoter DNA together with Ets1 and could also be in complex with Ets1 in the absence of DNA. Surface plasmon resonance analysis revealed that Ets1 interacted with the DNA binding domain of Smad3, and the binding constant of this interaction was about 1 µM. The results identify a negative regulation of the GM-CSF promoter by TGF-β signaling through direct Smad3 binding and indicate that the mechanism is by Smad3 interaction with Ets1 and perhaps other proteins around the TATA box of the promoter. This thesis also identifies a novel transactivation domain in the N-terminal of RUNX1 including the N-terminal α-helix in the DNA binding domain. The domain was also required for RUNX2 and RUNX3 transactivation. Despite this, the N-terminal domain of RUNX1 was not essential for RUNX1 function in megakaryocytopoiesis in vitro from mouse embryonic stem cells.

Heat shock protein (HSP)27 is traditionally described as an intracellular chaperone and signaling molecule, but growing evidence suggests it is released from immune cells where it plays an anti-inflammatory role during atherogenesis. Previously, the O’Brien lab found that overexpression of HSP27 led to augmented HSP27 serum levels in female apolipoprotein E knockout (ApoE-/-) mice, attenuated atherogenesis, and inhibited macrophage foam cell formation via physical binding with scavenger receptor (SR)-A. However, the precise mechanism of atheroprotection remained elusive. This thesis sought to ascertain the mechanism(s) by which HSP27 prevents foam cell formation, and determine if SR-A, a key receptor involved in the uptake of lipid into macrophages, plays an important role in HSP27-mediated atheroprotection. Pre-treatment of human macrophages with recombinant HSP27 (rHSP27) inhibited acytelated low density lipoprotein (acLDL) binding and uptake independent from receptor competition effect. Reduction in uptake was associated with attenuation of expression of SR-A mRNA, total protein, and cell surface expression. To explore the signaling mechanism by which HSP27 modulated SR-A expression it was hypothesized that nuclear factor-kappa B (NF-kB), a major regulator of many atherosclerosis gene programs, is altered by extracellular HSP27. Indeed, rHSP27 markedly activated NF-kB signaling in macrophages. Using an inhibitor of NF-kBsignaling there was an attenuation of rHSP27-induced inhibition of SR-A gene and protein expression, as well as lipid uptake, suggesting that SR-A expression is regulated by NF-kB activation. Lastly, to investigate if SR-A is required for HSP27-mediated atheroprotection in vivo, ApoE-/- and ApoE-/-SR-A-/- mice fed a high fat diet were treated with rHSP25, the mouse orthologue of HSP27, or PBS for 3 weeks. While rHSP25 therapy equally reduced serum cholesterol levels in the mouse cohorts, aortic atherogenesis, assessed using en face and sinus cross-sectional analyses, was attenuated in ApoE-/- mice but not ApoE-/-SR-A-/- mice. In conclusion, rHSP27 inhibits foam cell formation by downregulating SR-A expression. This effect may be associated with NF-kB activation. Reductions in atherosclerotic burden by rHSP27 require SR-A, and are independent of changes in serum cholesterol levels, highlighting the importance of macrophage lipid uptake in atherogenesis. Results presented in this thesis demonstrate that SR-A is a major target for HSP27 atheroprotection in the vessel wall, and provide an impetus for further studies that investigate the potential therapeutic value of HSP27.

IL-12 family cytokines IL-23 and IL-27 play an important role linking innate and adaptive immunity, and regulating T-cell responses. The production of IL-12, a structurally similar cytokine, is decreased in chronic HIV infection; therefore IL-23 and IL-27 may also be influenced by HIV infection. I hypothesized that HIV inhibits LPS-induced IL-23 and IL-27 production in human MDMs by suppressing the activation of signalling pathways regulating their expression. In vitro HIV-infection of MDMs did not have any effect on basal secretion of IL-23 or IL-27; however, HIV inhibited LPS-induced production of IL-12/23 p40 and IL-23 p19, and IL-27 EBI3 and IL-27 p28 mRNA expression, and IL-23, IL-12/23 p40 and IL-27 secretion. In order to evaluate the molecular mechanisms by which HIV inhibits IL-23 and IL-27 in LPS-stimulated MDMs, the signalling pathways regulating their expression were evaluated. The PI3K, p38 MAPK, and JNK MAPK pathways were found to positively regulate LPS-induced IL-27 secretion. Interestingly, in vitro HIV infection inhibited LPS-induced p38 and JNK MAPK activation in MDMs. In summary, I have shown that HIV inhibits IL-23 and IL-27 production in LPS-stimulated MDMs and that HIV may inhibit LPS-induced IL-27 production through the inhibition of p38 and JNK MAPK activation. It is currently unknown whether PKCs regulate LPS-induced IL-23 or IL-27 in human monocytes/macrophages. I demonstrated that classical PKCs differentially regulate LPS-induced IL-23 and IL-27 secretion within THP-1 cells, primary monocytes, and MDMs. Classical PKCs were found to positively regulate LPS-induced IL-12/23 p40 and IL-27 p28 mRNA expression and IL-12/23 p40, IL-23, and IL-27 secretion in primary human monocytes. Similarly, the classical PKCs were found to positively regulate IL-27 p28 mRNA expression and IL-27 secretion in THP-1 cells. However, classical PKCs did not regulate LPS-induced IL-27 production in MDMs, or LPS-induced IL-23 production in THP-1 cells. Overall, this demonstrates that classical PKCs differentially regulate LPS-induced IL-23 and IL-27 production in different myeloid cells.