CONSIDERAÇÕES FINAIS Este trabalho investigou o papel de

genes da imunidade inata em macrófagos de cães frente à leishmaniose visceral canina, com objetivo de identificar componentes que contribuam para a resistência natural do cão à doença. O estudo solidificou e desenvolveu metodologias. Além disso, demonstrou o perfil de expressão basal de importantes genes associados à imunidade inata em macrófagos caninos. Revelou que macrófagos de cães resistentes e susceptíveis a leishmaniose visceral apresentam expressão de citocinas distinta. Adicionalmente, foi traçada uma correlação entre a expressão de NOD2

e a sobrevivência do parasito intracelular. Resultados, estes, que abrem portas para futuros estudos. Devido à importância para saúde pública da leishmaniose visceral, esta importante linha de pesquisa visa contribuir para um melhor entendimento da resposta imune inata do cão frente à infecção, com consequente ampliação do conhecimento sobre a doença e impacto significativo em seu controle. Finalmente, o potencial de estudos com TLR, NLR e outros genes da imunidade inata em cães é extremamente amplo e pode auxiliar a desvendar a patogênese de inúmeras doenças que acometem essa espécie.

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48 9. _ANEXOS 9.1. Soluções • Acetato de amônio 7 M Acetato de amônio 57,75 g Água Milli-Q q.s.p. 100 mL • Clorofórmio 2-pentanol Clorofórmio 96 mL Álcool isopropílico 4 mL • EDTA 0,5 M EDTA 18,61 g Água Milli-Q q.s.p. 100 mL pH 8 • Formaldeído 12,3 M Formaldeído 41 mL

Água Milli Q tratada com DEPC q.s.p. 50 mL

• Isotono

Ácido cítrico 2,1 g

Cloreto de sódio (NaCl) 1,4 g

Formol PA 1 mL Água destilada q.s.p 200 mL • PBS Na2HPO4 bibásico 1,09 g NaHPO4 monobásico 0,32 g NaCl 9 g Água destilada q.s.p. 1000 mL pH entre 7,2 e 7,4

• PBS para Elisa R & D Systems

Na2HPO4 bibásico 2,1708 g KH2PO4 monobásico 0,204 g NaCl 7,946 g KCl 0,2025 g Água destilada q.s.p. 1000 mL pH entre 7,2 e 7,4

49 • Reagente diluente BSA 1 g PBS 100 mL pH entre 7,2 e 7,4 • RPMI completo RPMI 500 mL Glutamina 5,5 mL

Aminoácidos não essenciais 5,5 mL

Piruvato 5,5 mL

Bicarbonato de sódio 12 mL Penicilina/estreptomicina 5,5 mL Soro fetal bovino inativado 50 mL

• Sarcosyl 10 %

N-Lauroylsarcosine sodium salt 10 g Água Milli-Q q.s.p. 100 mL • Solução de bloqueio (PBSC) Caseína 20 g PBS 1000 mL • Solução de extração Tiocionato de guanidina 30 g EDTA 0,5 M pH 8 10 mL Sarcosyl 10% 2,5 mL Água Milli-Q q.s.p. 50 mL

• Solução de lavagem (PBS-tween)

PBS 1000 mL Tween 20 0,5 mL pH entre 7,2 e 7,4 • Solução de revelação OPD 0,02 g Tampão citrato 30 mL Peróxido de hidrogênio 12 µL • Solução substrato Tetrametilbenzidina 10 mL Água oxigenada 10 mL

50 • Tampão carbonato (tampão de ligação)

Na2CO3 (carbonato de sódio) 1,59 g

NaHCO3 (bicarbonato de sódio) 2,93 g

Água destilada q.s.p. 1000 mL pH 9,6, conservar a 4ºC

• Tampão citrato (tampão substrato)

Na2HPO4 (fosfato de sódio bibásico) 7,19 g

Ácido cítrico 5,19 g Água destilada q.s.p. 1000 mL pH 5, conservar a 4ºC • Tampão de corrida EDTA 0,5 M pH 8 20 µL Azul de bromofenol 0,025 g Xileno cianol 0,025 g Glicerol 5 mL

Água Milli Q tratada com DEPC q.s.p. 10 mL

• Tampão de lise de hemácias

Tris base 0,20587 g Cloreto de amônio 0,77 g Água Milli Q q.s.p. 100 mL • Tampão MOPS (10x) Acetato de sódio 6,804 g EDTA 1,8612 g MOPS 57,8 g

Água Milli Q tratada com DEPC q.s.p. 500 mL pH 7 • Tampão TBE Tris base 10,8 g Ácido bórico 5,5 g EDTA 0,744 g Água destilada q.s.p. 1000mL • Tampão TE Tris HCl 1 M pH 8 1 mL EDTA 0,5 M pH 8 0,2 mL Água Milli-Q q.s.p. 100 mL

51 • Tris HCl 1 M

Tris base 6,055 g

Água Milli-Q q.s.p. 50 mL pH 8

52 9.2. Certificado do CETEA