Programa de Pós-graduação em Genética e Biologia Molecular
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Navegando Programa de Pós-graduação em Genética e Biologia Molecular por Por Orientador "Paccez, Juliano Domiraci"
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Item Triagem in silico de candidatos vacinais contra Toxoplasma gondii(Universidade Federal de Goiás, 2018-03-06) Inácio, Moisés Morais; Cravo, Pedro Vitor Lemos; http://lattes.cnpq.br/1059199347781390; Paccez, Juliano Domiraci; http://lattes.cnpq.br/2350706025601982; Castro, Ana Maria de; Rocha, Thiago LopesToxoplasma gondii is the causative agent of congenital toxoplasmosis, which manifests as mild chorioretinitis, miscarriage, mental retardation, microcephaly, hydrocephalus, and seizures. Treatment of this disease is limited and a new vaccine represents the best strategy for prevention of the infection. In the present study, the reverse vaccinology combined with immunomics was applied for the development of a vaccine against T. gondii. Using an in silico approach, we identified T. gondii’s proteins that contain signal peptide and transmembrane domain using the ToxoDB® database. We evaluated the homology of these proteins with the human proteome and predicted their epitopes using Blastp, NetMHCpan 3.0 and NetMHCIIpan 3.1 tools. Class I and II HLA alleles with frequency greater than 1% in the population of South America, North America and Europe were obtained using the dbMHC database. Processing of the MHC class I epitopes were evaluated by MHC I Processing on the IEDB® database and the B lymphocyte epitopes were obtained through the Bcpred and BCTOPE servers. Finally, the antigenicity of the potential targets was analyzed by the VAxiJen server. A total of 1228 proteins were obtained, from which 349 showed no homology with human proteins. For the South American population, among the proteins identified with promiscuous epitopes, we observed proteins that are part of the virulence arsenal of the pathogen such as ROP8, ROP7, ROM4, Cathepsin C / B, rhoptry neck protein and LMBR1 family region protein. In the North American and European populations, we identified common proteins to both populations, such as MIC15, ROP7, HECT-domain (ubiquitin-transferase) domain-containing protein and rhoptry neck protein. ROP31 and subtilisin SUB2 are exclusive to the North American population. These proteins are involved in the invasion process and were shown to be positive in all the parameters adopted in this study. Regarding B lymphocyte epitopes, proteins such as ROP7, ROP8, ROM4, MIC15, HECT were identified. These proteins also presented promiscuous epitopes to class I and II HLAs from the analyzed populations. In addition, MIC2, ROM5, ROP9, MIC8, and MIC9 also showed B lymphocyte epitopes, but MIC9 was noteworthy with the highest score, high expression in the bradyzoite stage, and lack of vaccine test. ROP7, ROP8, ROM4, MIC8 and MIC9 were selected for in vivo and in vitro testing. Thus, our results demonstrate that immunochemical reverse vaccination has been shown not only to identify potential vaccine candidates against pathogens with complex life cycles.Item Caracterização molecular da proteína histidina quinase de Paracoccidioides brasiliensis(Universidade Federal de Goiás, 2019-03-08) Santos, Dener Lucas Araújo dos; Paccez, Juliano Domiraci; http://lattes.cnpq.br/2350706025601982; Paccez, Juliano Domiraci; Bailão, Alexandre Melo; Tomazett, Mariana VieiraProkaryotic and eukaryotic organisms (except those belonging to the phylum metazoa) have developed a system named phosphorelay, which is responsible for adaptation to different environmental conditions such as oxidative stress, osmotic stress and thermal shock. Studies have demonstrated that inhibition of hybrid histidine kinase, the stimulus sensory protein in the phosphorelay pathway, affects morphological transition from mycelial fungi to yeast. Among the thermodymorphic fungi, Paracoccidioides spp. are among the most prevalent in Latin America, mainly in Brazil, as the cause of the systemic mycosis paracoccidioidomycosis (PCM). The exposure of fungi from this complex to the host body temperature (37 ° C), leads to the transition from the mycelial to the parasitic yeast form. It is notable that the morphological transition is necessary for the pathogenicity of Paracoccidioides spp.. Thus, the objective of this study is to characterize the hybrid histidine kinase (HHK) protein and to evaluate its level of expression during the dimorphism of Paracoccidioides brasiliensis. We carried out similarity analyzes that indicated that HHK is highly conserved in other dimorphic fungi such as H. capsulatum and B. dermatitidis. In addition, a region of the HHK gene was cloned into pET32a expression vector and the recombinant protein was generated. After confirming the identity of HHK in UPLC-MSE, we produced polyclonal anti-histidine kinase antibodies. RT-qPCR revealed that PbHHK is expressed in greater amounts transition when compared to the other morphological phases. In conclusion, the high conservation of histidine kinase in dimorphic fungi combined with their increased expression in the transition phase demonstrates the importance of PbHHK for the morphological transition of P. brasiliensis.