Navegando por Autor "Gomes, Matheus de Souza"
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Item Accomplishing the genotype-specific serodiagnosis of single and dual Trypanosoma cruzi infections by flow cytometry Chagas- Flow ATE-IgG2a.(2018) Alessio, Glaucia Diniz; Araújo, Fernanda Fortes de; Sales Júnior, Policarpo Ademar; Gomes, Matheus de Souza; Amaral, Laurence Rodrigues do; Xavier, Marcelo Antônio Pascoal; Carvalho, Andréa Teixeira de; Lana, Marta de; Martins Filho, Olindo AssisThe methods currently available for genotype-specific diagnosis of T. cruzi infection still present relevant limitations, especially to identify mixed infection. In the present investigation, we have evaluated the performance of Chagas-Flow ATE-IgG2a test for early and late differential diagnosis of single and dual genotype-specific T. cruzi infections. Serum samples from Swiss mice at early and late stages of T. cruzi infection were assayed in parallel batches for genotype-specific diagnosis of single (TcI, TcVI or TcII) and dual (TcI+TcVI, TcVI+TcII or TcII+TcI) infections. The intrinsic reactivity to TcI, TcVI and TcII target antigens, including amastigote (AI/AVI/AII), trypomastigote-(TI/TVI/TII) and epimastigote (EI/ EVI/EII), at specific reverse of serum dilutions (500 to 64,000), was employed to provide reliable decision-trees for ªearlyº vs ªlateº, ªsingle vs ªdualº and ªgenotype-specificº serology. The results demonstrated that selective set of attributes ªEII 500/EI 2,000/AII 500º were able to provide high-quality accuracy (81%) to segregate early and late stages of T. cruzi infection. The sets ªTI 2,000/AI 1,000/EII 1,000º and ªTI 8,000/AII 32,000º presented expressive scores to discriminate single from dual T. cruzi infections at early (85%) and late stages (84%), respectively. Moreover, the attributes ªTI 4,000/TVI 500/TII 1,000º, ªTI 16,000/EI 2,000/EII 2,000/AI 500/TVI 500º showed good performance for genotype-specific diagnosis at early stage of single (72%) and dual (80%) T. cruzi infections, respectively. In addition, the attributes ªTI 4,000/AII 1,000/EVI 1,000º, ªTI 64,000/AVI 500/AI 2,000/AII 1,000/EII 4,000º showed moderate performance for genotype-specific diagnosis at late stage of single (69%) and dual (76%) T. cruzi infections, respectively. The sets of decision-trees were assembled to construct a sequential algorithm with expressive accuracy (81%) for serological diagnosis of T. cruzi infection. These findings engender new perspectives for the application of Chagas-Flow ATE-IgG2a method for genotype-specific diagnosis in humans, with relevant contributions for epidemiological surveys as well as clinical and post-therapeutic monitoring of Chagas disease.Item Caracterização do microbioma e do potencial multitolerante de bactérias associadas a brânquias e intestino de duas espécies de peixes detritívoros do Rio São Francisco.(2021) Damasceno, Maria Rosilene Alves; Garcia, Camila Carrião Machado; Moreira, Leandro Marcio; Garcia, Camila Carrião Machado; Moreira, Leandro Marcio; Zanette, Juliano; Gomes, Matheus de Souza; Cota, Renata Guerra de Sá; Silva, Silvana de QueirozComunidades microbianas altamente diversificadas integram a microbiota dos peixes, onde desempenham funções metabólicas essenciais à saúde dos hospedeiros. O perfil taxonômico e metabólico da microbiota dos teleósteos exibe associação com a filogenia, idade, dieta do hospedeiro e com as condições físico-químicas do seu habitat. A poluição e o estresse ambiental podem induzir alterações na estrutura das comunidades microbianas associadas aos peixes, podendo afetar negativamente as interações microbiota-hospedeiro. Estudos têm demonstrado que alterações do perfil da microbiota induzidas por contaminantes ambientais tem influenciado fortemente no declínio da ictiofauna. O objetivo deste estudo foi caracterizar e comparar a diversidade bacteriana associada à microbiota branquial e intestinal de Prochilodus argenteus e Rhinelepis aspera recrutados no médio rio São Francisco e em tanque de cultivo. As comunidades bacterianas associadas às espécies e tecidos (brânquias e intestino) nos diferentes ambientes foram caracterizadas por meio do sequenciamento de alto rendimento do gene rRNA 16S. Adicionalmente, foram realizados ensaios de tolerância com 277 isolados bacterianos da microbiota cultivável, obtidos dos mesmos tecidos dos animais do rio São Francisco e do tanque de cultivo. Estes microorganismos foram expostos a diferentes doses de metais (Cr, Cd, Ni, Cu, e Pb), NaCl e variações de pH, além da capacidade de formarem biofilme. O perfil taxonômico revelado pela análise metagenômica indicou grande diversidade bacteriana associada às vísceras dos peixes, composta por 33 filos, 61 classes, 133 ordens, 220 famílias e 295 gêneros. Em comum, a microbiota de P. argenteus e R. aspera foi majoritariamente composta pelos filos Proteobacteria e Fusobacteriota. As análises dos índices de diversidade α (Shanon e Simpson) e de dissimilaridade, estabelecida pela distância Unifrac ponderada, indicaram que a estrutura e diversidade microbiana foram fortemente influenciadas pelo ambiente, sem efeito significativo relacionado à espécie e tecidos viscerais. A diversidade bacteriana foi drasticamente reduzida na microbiota dos peixes mantidos em cativeiro comparado aos peixes selvagens. A ocorrência simpátrica das espécies aliada a convergência ecológica iliófaga pode ter exercido influência no aumento da convergência microbiana interespecífica na microbiota dos peixes selvagens, que ainda assim, mostrou-se distinta observada o expressivo número de táxons únicos nos diferentes tecidos. Um conjunto de filótipos bacterianos, entre eles, Cetobacterium_A, Aeromonas, Plesiomonas, Bacteroides, Pseudomonas_E, Shewanella e Edwardsiella prevaleceram em abundância variável e compartilhada em peixes selvagens e cultivados nos diferentes tecidos corporais, sugerindo serem constitutivos autóctones de um microbioma central. A microbiota cultivável isolada dos tecidos de animais de ambos os ambientes apresentou elevada tolerância ao chumbo. A identificação de bactérias multiressistentes a metais pesados realizada com base no gene 16S rRNA, indicou a presença de bactérias com potencial biotecnológico e biorremediador como Bacillus sp. e Anoxybacillus sp., e potenciais patógenos como Plesiomonas sp., Aeromonas sp., Klebisiella sp. e Serratia sp. Nossos dados de maneira conjunta, fornece informações inéditas sobre os microbiomas associados a brânquias e intestino de P. argenteus e R. aspera selvagens e aponta uma redução drástica das comunidades bacterianas induzida pelo estresse ambiental do confinamento, que poderá refletir adversamente na saúde animal e acarretar prejuízos à aquicultura. Futuramente, sugerimos caracterizar os microbiomas destas espécies procedentes de um ambiente aquático não impactado por pressões antrópicas, além de ensaios adicionais com a microbiota cultivável isolada neste estudo, para investigar o potencial biotecnológico destes microrganismos.Item Caracterização inicial dos constituintes da maquinaria de silenciamento gênico mediada por microRNAs em Schistosoma mansoni.(Programa de Pós-Graduação em Ciências Biológicas. Núcleo de Pesquisas em Ciências Biológicas, Pró-Reitoria de Pesquisa e Pós Graduação, Universidade Federal de Ouro Preto., 2008) Gomes, Matheus de Souza; Cota, Renata Guerra de SáO silenciamento de RNA refere-se a uma série de processos nucleares e citoplasmáticos envolvidos na regulação da expressão gênica pós-transcricional ou silenciamento pós-transcricional (PTGS), degradando ou silenciando seqüências específicas de mRNA. O mecanismo é conservado em animais incluindo alguns pontos distintos e vários pontos coincidentes, envolvidos tanto no silenciamento de pequenos RNAs por via endógena quanto por via exógena. O pequeno RNA melhor caracterizado é o microRNA (miRNAs), que predominantemente silencia genes através de uma regulação pós-transcricional. Neste trabalho, utilizamos bioinformática para identificar possíveis genes envolvidos na via de silenciamento gênico pós-transcricional mediado por miRNA em S. mansoni. Nós pesquisamos os bancos de dados do genoma e do transcriptoma de S. mansoni, com as seqüências de aminoácidos proteínas ortólogas relacionadas à via de miRNA. Identificamos o total de 13 prováveis proteínas envolvidas na via miRNA no parasito. Em seguida, o níveis de SmDicer1 e SmAgo 2, 3 e 4 foram identificados por qRT-PCR utilizando cercárias, vermes adultos, ovos e esquistossômulos com os seguintes períodos de cultivo in vitro 3,5; 8,5; 18,5; 24; 48 e 72 horas. Este resultado demonstrou que, os dois genes são diferencialmente expressos através do período de diferenciação cercária-esquistossômulo, tendo níveis similares em ovos e vermes adultos. Em cercárias SmDicer1 tem baixa expressão. Estes resultados sugerem que esta via é um importante mecanismo de regulação da expressão gênica neste parasita. Futuros experimentos são necessários para provar esta hipótese.Item Characterisation of major vault protein during the life cycle of the human parasite Schistosoma mansoni.(2014) Reis, Eneida Virgínia; Pereira, Roberta Verciano; Gomes, Matheus de Souza; Passos, Liana Konovaloff Jannotti; Babá, Élio Hideo; Coelho, Paulo Marcos Zech; Mattos, Ana Carolina Alves de; Couto, Flávia Fernanda Búbula; Borges, William de Castro; Cota, Renata Guerra de SáVaults are ribonucleoproteins (13MDa) highly conserved among lower and higher eukaryotes. Their association produces a complex composed of three proteins named Major Vault Protein (MVP), vault (PolyADP-ribose) polymerase (VPARP) and Telomerase-associated protein (TEP1), plus a small untranslated RNA. The exact function of this complex is unknown, although the biological role of vaults has been associated with multidrug resistance phenotypes and signal transduction pathways. Genomic analysis showed that model organisms, such as Caenorhabditis elegans and Drosophila melanogaster, do not possess genes encoding vaults. However, we have found that vault-related genes are present in the Schistosoma mansoni genome. These observations raised questions on the involvement of vaults in mechanisms of adaptation of the parasite in its mammalian host. Therefore, molecular characterisation of the putative Major Vault Protein performed using bioinformatics tools showed that this vault component is highly conserved in S. mansoni. The MVP expression level was quantified by qRT-PCR using total RNA from susceptible (LE) and resistant (LE-PZQ) adult worm lineages, cercariae and mechanically transformed schistosomula(MTS) cultured for 3.5, 24, 48 and 72h in vitro. Our results suggest a stage-specific expression in all developmental stages analysed. Western blotting has shown upregulation of SmMVP in the MTS-3.5, 72 h and resistant adult worms, and similar levels in all other stages. Furthermore, SmMVPwas found differentially expressed in adult males and females fromthe susceptible lineage. Further studies should clarify whether SmMVP is somehow linked to drug resistance in S. mansoni.Item Characterisation of the COP9 signalosome in Schistosoma mansoni parasites.(2013) Pereira, Roberta Verciano; Gomes, Matheus de Souza; Passos, Liana Konovaloff Jannotti; Borges, William de Castro; Cota, Renata Guerra de SáThe COP9 signalosome (CSN) is an eight-subunit complex found in all eukaryotes and shares structural features with both the 26S proteasome ‘lid’ and translation factor eIF3. Recent data have demonstrated that the CSN is a regulator of the ubiquitin (Ub) proteasome system (UPS). CSN controls substrate ubiquitination by cullin-RING Ub ligases, a step which determines substrate specificity of the UPS. Here, we reconstructed the CSN complex in Schistosoma mansoni and identified eight homologous components. Among these homologues, five subunits were predicted with their full-length sequences. Phylogenetic analysis confirmed the evolutionary conservation and the architecture of CSN, as well as the 26S proteasome ‘lid’. We performed quantitative reverse transcrip tion-polymerase chain reaction to detect the expression of the SmCSN transcripts. The Smcsn1, Smcsn2, Smcsn3, Smcsn4, Smcsn5, Smcsn6, Smcsn7 and Smcsn8 genes were upregulated in adult worms compared to cercariae, and the expression levels were similar to that of in vitro cultivated schistosomula. Taken together, these results suggest that the CSN complex may be important during cercariae, schistosome and adult worm development and might explain, at least in part, the differences among UPSs during the parasite life cycle.Item Characterization and mRNA expression analysis of PI31, an endogenous proteasome inhibitor from Schistosoma mansoni.(2010) Machado, Carla Botelho; Cabral, Fernanda Janku; Soares, Cláudia Sossai; Moreira, Érika Bueno de Carvalho; Morais, Enyara Rezende; Magalhães, Lizandra Guidi; Gomes, Matheus de Souza; Cota, Renata Guerra de Sá; Rosa, José César; Ruller, R.; Ward, R. J.; Rodrigues, VanderleiThe proline-rich inhibitor of 31 kDa (PI31) is highly conserved through metazoan evolution, and its activity in the proteasome inhibition is well-established although the precise mechanism of inhibition is unclear. The coding DNA sequence of Schistosoma mansoni PI31 (SmPI31) was cloned, and the recombinant protein was expressed in bacterial system. The correct amino acid sequence was confirmed by mass spectrometry and circular dichroism suggests that SmPI31 contains both α-helix and non-structured regions. Inhibition assays, using the SucLeu-Leu-Val-Tyr-4-MCA substrate for proteasome degradation, showed that the S. mansoni proteasome may be regulated by the inhibitory activity of SmPI31. A gene expression assay using qRT-PCR at various stages during the S. mansoni life cycle has shown that SmPI31 transcripts are expressed in all studied stages, suggesting that PI31 plays an important role during the developmental processes of the parasite. In this study first evidence is presented that PI31 has a conserved structure and plays a role as proteasome inhibitor in adult worms and it is expressed through life cycle.Item Characterization of export receptor exportins (XPOs) in the parasite Schistosoma mansoni.(2013) Abreu, Fabiano Carlos Pinto de; Pereira, Roberta Verciano; Oliveira, Victor Fernandes de; Gomes, Matheus de Souza; Passos, Liana Konovaloff Jannotti; Borges, William de Castro; Cota, Renata Guerra de SáSeveral proteins and different species of RNA that are produced in the nucleus are exported through the nuclear pore complexes, which require a family of conserved nuclear export receptors called exportins (XPOs). It has been reported that the XPOs (XPO1, XPO5, and XPOT) are directly involved in the transport processes of noncoding RNAs from the nucleus to the cytoplasm and/or from cytoplasm to the nucleus. All three genes are present in fungi, plants, and deuterostome metazoans. However, protostome metazoan species lack one of the three genes across evolution. In this report, we have demonstrated that all three XPO proteins are present in the parasite protostome Schistosoma mansoni . As this parasite has a complex life cycle presenting several stages in different hosts and environments, implying a differential gene regulation, we proposed a genomic analysis of XPOs to validate their annotation. The results showed the conservation of exportin family members and gene duplication events in S. mansoni .We performed quantitative RT-PCR, which revealed an upregulation of SmXPO1 in 24 h schistosomula (sixfold when compared with cercariae), and similar transcription levels were observed for SmXPO5 and SmXPOT in all the analyzed stages. These three XPO proteins have been identified for the first time in the protostome clade, which suggests a higher complexity in RNA transport in the parasite S. mansoni. Taken together, these results suggest that RNA transport by exportins might control cellular processes during cercariae, schistosomula, and adult worm development.Item Computational identification and evolutionary relationships of the MicroRNA gene cluster miR-71/2 in protostomes.(2013) Gomes, Matheus de Souza; Donoghue, Mark T. A.; Muniyappa, Mohan Kumar; Pereira, Roberta Verciano; Cota, Renata Guerra de Sá; Spillane, CharlesMicroRNAs (miRNAs) are small noncoding RNA molecules which are processed into *20–24 nt molecules that can regulate the gene expression posttranscriptionally. MiRNA gene clusters have been identified in a range of species, where in miRNAs are often processed from polycistronic transcripts. In this study, a computational approach is used to investigate the extent of evolutionary conservation of the miR-71/2 cluster in animals, and to identify novel miRNAs in the miRNA cluster miR-71/2. The miR-71/2 cluster, consisting of copies of the miR-71 and miR-2 (including miR-13) families, was found to be Protostome-specific. Although, this cluster is highly conserved across the Protostomia, the miR-2 family is completely absent from the Deuterostomia species, while miR-71 is absent from the Vertebrata and Urochordata. The evolutionary conservation and clustering propensity of the miR-71/2 family across the Protostomes could indicate the common functional roles across the member species of the Protostomia.Item Conservation and developmental expression of ubiquitin isopeptidases in Schistosoma mansoni.(2014) Pereira, Roberta Verciano; Vieira, Helaine Graziele Santos; Oliveira, Victor Fernandes de; Gomes, Matheus de Souza; Passos, Liana Konovaloff Jannotti; Borges, William de Castro; Cota, Renata Guerra de SáSeveral genes related to the ubiquitin (Ub)-proteasome pathway, including those coding for proteasome subunits and conjugation enzymes, are differentially expressed during the Schistosoma mansoni life cycle. Although deubiq¬uitinating enzymes have been reported to be negative regulators of protein ubiquitination and shown to play an im¬portant role in Ub-dependent processes, little is known about their role in S. mansoni. In this study, we analysed the Ub carboxyl-terminal hydrolase (UCHs) proteins found in the database of the parasite’s genome. An in silico ana-lysis (GeneDB and MEROPS) identified three different UCH family members in the genome, SmUCH-L3, SmUCH-L5 and SmBAP-1 and a phylogenetic analysis confirmed the evolutionary conservation of the proteins. We per¬formed quantitative reverse transcription-polymerase chain reaction and observed a differential expression profile for all of the investigated transcripts between the cercariae and adult worm stages. These results were corroborated by low rates of Z-Arg-Leu-Arg-Gly-Gly-AMC hydrolysis in a crude extract obtained from cercariae in parallel with high Ub conjugate levels in the same extracts. We suggest that the accumulation of ubiquitinated proteins in the cercaria and early schistosomulum stages is related to a decrease in 26S proteasome activity. Taken together, our data suggest that UCH family members contribute to regulating the activity of the Ub-proteasome system during the life cycle of this parasite.Item Deep sequencing of small RNAs reveals the repertoire of miRNAs and piRNAs in Biomphalaria glabrata.(2020) Queiroz, Fábio Ribeiro; Portilho, Laysa Gomes; Jeremias, Wander de Jesus; Babá, Élio Hideo; Amaral, Laurence Rodrigues do; Silva, Luciana Maria; Coelho, Paulo Marcos Zech; Caldeira, Roberta Lima; Gomes, Matheus de SouzaBACKGROUND Biomphalaria glabrata snails are widely distributed in schistosomiasis endemic areas like America and Caribe, displaying high susceptibility to infection by Schistosoma mansoni. After the availability of B. glabrata genome and transcriptome data, studies focusing on genetic markers and small non-coding RNAs have become more relevant. The small RNAs have been considered important through their ability to finely regulate the gene expression in several organisms, thus controlling the functions like cell growth, metabolism, and susceptibility/resistance to infection. OBJECTIVE The present study aims on identification and characterisation of the repertoire of small non-coding RNAs in B. glabrata (Bgl-small RNAs). METHODS By using small RNA sequencing, bioinformatics tools and quantitative reverse transcription polymerase chain reaction (RT-qPCR), we identified, characterised, and validated the presence of small RNAs in B. glabrata. FINDINGS 89 mature miRNAs were identified and five of them were classified as Mollusk-specific. When compared to model organisms, sequences of B. glabrata miRNAs showed a high degree of conservation. In addition, several target genes were predicted for all the mature miRNAs identified. Furthermore, piRNAs were identified in the genome of B. glabrata for the first time. The B. glabrata piRNAs showed strong conservation of uridine as first nucleotide at 5’ end, besides adenine at 10th position. Our results showed that B. glabrata has diverse repertoire of circulating ncRNAs, several which might be involved in mollusk susceptibility to infection, due to their potential roles in the regulation of S. mansoni development. MAIN CONCLUSIONS Further studies are necessary in order to confirm the role of the Bgl-small RNAs in the parasite/host relationship thus opening new perspectives on interference of small RNAs in the organism development and susceptibility to infection.Item Efeito do exercício físico e do consumo de sacarose na expressão de RNAs não codificadores longos e sirtuínas no tecido cardíaco e suas possíveis implicações biotecnológicas.(2020) Barroso, Yuri Windson Santos; Cota, Renata Guerra de Sá; Barboza, Natália Rocha; Oliveira, Lenice Kappes Becker; Gomes, Matheus de Souza; Cota, Renata Guerra de SáA Organização Mundial da Saúde relata que 01 em cada 04 indivíduos adultos são considerados sedentários, e no mundo totaliza aproximadamente 1,4 bilhões de pessoas nesta condição. Recentemente, vem sendo descrito que uma variedade de RNAs não codificadores longos (lncRNAs) tem um papel importante na gênese de várias doenças metabólicas, entretanto seu papel no desenvolvimento de doenças cardíacas, ainda é pouco compreendido. Os lncRNAs são moléculas de RNAs que possuem um tamanho superior a 200 nucleotídeos. Eles atuam na regulação de mecanismos epigenéticos, pós-transcricionais e pós-traducionais. As sirtuínas (SIRT) são uma família de sete enzimas (1-7) desacetilases dependentes de nicotinamida adenina dinucleotídeo (NAD+ ) reguladoras metabólicas, que executam papeis importantes na regulação da transcrição, na sobrevivência celular, no ritmo circadiano, inflamação e reparo celular. Junto eles são apontados como reguladores chaves na fisiologia e fisiopatologia do sistema cardiovascular. Desde 2008 que nosso grupo de pesquisa vem estudando os efeitos do uso crônico de uma dieta rica em carboidratos simples (DRC) em associação ou não com o treinamento físico de natação no desenvolvimento de doenças metabólicas. Continuando essa linha de pesquisa, a hipótese desse trabalho é que os lncRNAs e as sirtuinas são boas moléculas candidatas a biomarcadores associado ao metabolismo e patologias do tecido cardíaco. A seguinte metodologia foi adotada: utilizou de duas dietas isocalóricas, sendo uma DRCS (33% de leite condensado; 7% de açúcar cristal) e outra normocalórica. Os animais (N=12) foram submetidos ao treinamento aeróbico (65% Pmax) e eutanasiados após 18 semanas de treinamento/sedentário, conforme protocolo CEUA 2016/26. O trabalho teve os seguintes objetivos: (i) analisar o efeito da dieta e interação treinamento/sedentarismo sobre os parâmetros cardiovasculares; (ii) avaliar lesões nas fibras musculares utilizando abordagens morfológicas; (iii) analisar o efeito da dieta e interação treinamento/sedentarismo sobre a expressão dos lncRNAs FTX, HOTAIR, FENDERR,GAS5 e SIRT1 a 7. Os resultados demonstram efeito do treinamento na expressão dos lncRNAs FENDERR,FTX,HOTAIR e GAS5..Com relação as SIRTs, observamos efeito da DRC na expressão de SIRT3, do treinamento na expressão de SIRT4 e do treinamento e da DRC na expressão da SIRT6. Os animais do grupo sedentário, independente da dieta consumida apresentaram processos inflamatório significativo no tecido cardíaco. Os resultados permitem concluir que o treinamento aeróbico de natação reverteu, pelo menos em parte, os danos metabólicos associados ao uso crônico de uma DCS e que novos estudos serão necessários para confirmar o papel de biomarcador epigenético da SIRTs e dos lncRNAs.Item Epitope mapping of the HSP83.1 protein of Leishmania braziliensis discloses novel targets for immunodiagnosis of tegumentary and visceral clinical forms of leishmaniasis.(2014) Souza, Daniel Menezes; Mendes, Tiago Antônio de Oliveira; Gomes, Matheus de Souza; Cunha, João Luís Reis; Nagem, Ronaldo Alves Pinto; Carneiro, Cláudia Martins; Coelho, Eduardo Antônio Ferraz; Galvão, Lúcia Maria da Cunha; Fujiwara, Ricardo Toshio; Bartholomeu, Daniella CastanheiraGold standard serological diagnostic methods focus on antigens that elicit a strong humoral immune response that is specific to a certain pathogen. In this study, we used bioinformatics approaches to identify linear B-cell epitopes that are conserved among Leishmania species but are divergent from the host species Homo sapiens and Canis familiaris and from Trypanosoma cruzi, the parasite that causes Chagas disease, to select potential targets for the immunodiagnosis of leishmaniasis. Using these criteria, we selected heat shock protein 83.1 of Leishmania braziliensis for this study. We predicted three linear B-cell epitopes in its sequence. These peptides and the recombinant heat shock protein 83.1 (rHSP83.1) were tested in enzyme-linked immunosorbent assays (ELISAs) against serum samples from patients with tegumentary leishmaniasis (TL) and visceral leishmaniasis (VL) and from dogs infected with Leishmania infantum (canine VL [CVL]). Our data show that rHSP83.1 is a promising target in the diagnosis of TL. We also identified specific epitopes derived from HSP83.1 that can be used in the diagnosis of human TL (peptide 3), both human and canine VL (peptides 1 and 3), and all TL, VL, and CVL clinical manifestations (peptide 3). Receiver operating characteristic (ROC) curves confirmed the superior performance of rHSP83.1 and peptides 1 and 3 compared to that of the soluble L. braziliensis antigen and the reference test kit for the diagnosis of CVL in Brazil (EIE-LVC kit; Bio-Manguinhos, Fiocruz). Our study thus provides proof-of-principle evidence of the feasibility of using bioinformatics to identify novel targets for the immunodiagnosis of parasitic diseases using proteins that are highly conserved throughout evolution.Item Genome-wide identification of novel microRNAs and their target genes in the human parasite Schistosoma mansoni.(2011) Gomes, Matheus de Souza; Muniyappa, Mohan Kumar; Carvalho, Sávio Gonçalves; Cota, Renata Guerra de Sá; Spillane, CharlesMature microRNAs (miRNAs) are small, non-coding regulatory RNAs which can elicit post-transcriptional repression of mRNA levels of target genes. Here, we report the identification of 67 mature and 42 precursor miRNAs in the Schistosoma mansoni parasite. The evolutionarily conserved S. mansoni miRNAs consisted of 26 precursor miRNAs and 35 mature miRNAs, while we identified 16 precursor miRNAs and 32 mature miRNAs that displayed no conservation. These S. mansoni miRNAs are located on seven autosomal chromosomes and a sex (W) chromosome. miRNA expansion through gene duplication was suggested for at least two miRNA families miR-71 and mir-2. miRNA target finding analysis identified 389 predicted mRNA targets for the identified miRNAs and suggests that the sma-mir-71 may be involved in female sexual maturation. Given the important roles of miRNAs in animals, the identification and characterization of miRNAs in S. mansoni will facilitate novel approaches towards prevention and treatment of Schistosomiasis.Item Genome-wide identification, characterisation and expression profiling of the ubiquitin-proteasome genes in Biomphalaria glabrata.(2019) Portilho, Laysa Gomes; Duarte, Bruna Custódio Dias; Queiroz, Fábio Ribeiro; Ribeiro, Thales Henrique Cherubino; Jeremias, Wander de Jesus; Babá, Élio Hideo; Coelho, Paulo Marcos Zech; Morais, Enyara Rezende; Cabral, Fernanda Janku; Caldeira, Roberta Lima; Gomes, Matheus de SouzaBACKGROUND Biomphalaria glabrata is the major species used for the study of schistosomiasis-related parasite-host relationships, and understanding its gene regulation may aid in this endeavor. The ubiquitin-proteasome system (UPS) performs post-translational regulation in order to maintain cellular protein homeostasis and is related to several mechanisms, including immune responses. OBJECTIVE The aims of this work were to identify and characterise the putative genes and proteins involved in UPS using bioinformatic tools and also their expression on different tissues of B. glabrata. METHODS The putative genes and proteins of UPS in B. glabrata were predicted using BLASTp and as queries reference proteins from model organism. We characterised these putative proteins using PFAM and CDD software describing the conserved domains and active sites. The phylogenetic analysis was performed using ClustalX2 and MEGA5.2. Expression evaluation was performed from 12 snail tissues using RPKM. FINDINGS 119 sequences involved in the UPS in B. glabrata were identified, which 86 have been related to the ubiquitination pathway and 33 to proteasome. In addition, the conserved domains found were associated with the ubiquitin family, UQ_con, HECT, U-box and proteasome. The main active sites were lysine and cysteine residues. Lysines are responsible and the starting point for the formation of polyubiquitin chains, while the cysteine residues of the enzymes are responsible for binding to ubiquitin. The phylogenetic analysis showed an organised distribution between the organisms and the clades of the sequences, corresponding to the tree of life of the animals, for all groups of sequences analysed. The ubiquitin sequence was the only one with a high expression profile found in all libraries, inferring its wide range of performance. MAIN CONCLUSIONS Our results show the presence, conservation and expression profile of the UPS in this mollusk, providing a basis and new knowledge for other studies involving this system. Due to the importance of the UPS and B. glabrata, this work may influence the search for new methodologies for the control of schistosomiasis.Item miRNAs em Schistosoma mansoni : biogênese, predição, validação e alvos potenciais.(Programa de Pós-Graduação em Ciências Biológicas. Núcleo de Pesquisas em Ciências Biológicas, Pró-Reitoria de Pesquisa e Pós Graduação, Universidade Federal de Ouro Preto., 2012) Gomes, Matheus de Souza; Cota, Renata Guerra de SáOs miRNAs constituem uma classe extensa de pequenos RNAs não codificadores de proteínas possuindo, em sua forma madura, de 20 a 24 nucleotídeos, cuja função principal é regular a expressão gênica. Seus precursores, denominados pré-miRNAs, têm como característica principal a formação de estruturas secundárias estáveis em forma de grampos (hairpins) possuindo um tamanho variável de 70 a 120 nucleotídeos. Os resultados publicados na última década sugerem que o silenciamento gênico mediado pelos miRNAs é um processo evolutivamente conservado e envolve, em um primeiro momento, a interação entre o miRNA e o respectivo RNA mensageiro alvo, promovendo sua clivagem ou repressão de sua tradução. Apesar de o Schistosoma mansoni ser um organismo com genoma conhecido, pouco se conhece sobre o repertório de miRNAs presentes neste parasito. Resultados anteriores do nosso laboratório demonstraram que genes centrais da biogênese de miRNAs são mais expressos em cercárias e esquistossômulos jovens quando comparados a vermes adultos, levando a hipótese de que os mecanismos de adaptação do parasito ao hospedeiro mamífero, imediatamente pós-infecção, podem envolver a participação de miRNAs. Para investigar este hipótese, foram utilizados neste trabalho abordagens computacionais e experimentais para identificar miRNAs em S. mansoni, bem como determinar suas características estruturais e padrão de expressão, correlacionando em paralelo com o perfil de expressão de 13 genes envolvidos em sua biogênese. Os resultados obtidos sugerem que os genes envolvidos na biogênese de miRNAs são conservados e apresentam um perfil de expressão gênica diferencial durante a transição cercária-esquistossômulos jovens. Foram identificados neste trabalho 35 miRNAs conservados, 32 não conservados e mais de 300 prováveis RNA mensageiros alvos no genoma de S. mansoni. Dentre os miRNAs identificados foram caracterizados duplicações gênicas, clusteres (mir-71/2) e miRNAs sexo-específicos. As análises da expressão gênica dos miRNAs conservados e não conservados evidenciaram padrões de expressão gênero-específicos (sma-miR-61, sma-miR-125a e sma-miR-124-3p) e estágio-específicos (sma-mir-new10-5p, sma-miR-125a). Observou-se também uma correlação positiva entre a alta expressão de miRNAs no parasito e baixa expressão de alvos específicos. Estes dados sugerem que a regulação do proteoma do parasito, principalmente nas fases de cercária e esquistossômulos, provavelmente é mediada por regulação pós-transcricional mediada por miRNAs viabilizando a instalação efetiva do parasito no hospedeiro mamífero. Este conjunto de resultados abre perspectiva para avaliação do perfil de expressão em esporocistos demonstrando que transcritos produzidos nesta fase do ciclo biológico do S. mansoni podem ser silenciados e posteriormente traduzidos em cercárias ou esquistossômulos.Item Molecular characterization of SUMO E2 conjugation enzyme : differential expression profile in Schistosoma mansoni.(2011) Pereira, Roberta Verciano; Cabral, Fernanda Janku; Gomes, Matheus de Souza; Babá, Élio Hideo; Passos, Liana Konovaloff Jannotti; Carvalho, Omar; Rodrigues, Vanderlei; Afonso, Robson José de Cássia Franco; Borges, William de Castro; Cota, Renata Guerra de SáSUMO-dependent post-translational modification is implicated in a variety of cellular functions including gene expression regulation, nuclear sub-localization, and signal transduction. Conjugation of SUMO to other proteins occurs in a similar process to ubiquitination, which involves three classes of enzymes: an E1 activating, an E2 conjugating, and an E3 target-specific ligase. Ubc9 is the unique SUMO E2 enzyme known to conjugate SUMO to target substrates. Here, we present the molecular characterization of this enzyme and demonstrate its expression profile during the S. mansoni life cycle. We have used bioinformatic approaches to identify the SUMO-conjugating enzyme, the SmUbc9-like protein, in the Schistosoma mansoni databases. Quantitative RT-PCR was employed to measure the transcript levels of SUMO E2 in cercariae, adult worms, and in vitro cultivated schistosomula. Furthermore, recombinant SmUbc9 was expressed using the Gateway system, and antibodies raised in rats were used to measure SmUbc9 protein levels in S. mansoni stages by Western blotting. Our data revealed upregulation of the SmUbc9 transcript in early schistosomula followed by a marked differential gene expression in the other analyzed stages. The protein levels were maintained fairly constant suggesting a post-transcriptional regulation of the SmUbc9 mRNA. Our results show for the first time that S. mansoni employs a functional SUMO E2 enzyme, for the conjugation of the SUMO proteins to its target substrates.Item Molecular cloning, sequencing, and expression analysis of presenilin cDNA from Schistosoma mansoni.(2009) Magalhães, Lizandra Guidi; Borges, William de Castro; Gomes, Matheus de Souza; Cota, Renata Guerra de Sá; Rodrigues, VanderleiPresenilins (PS) are integral membrane proteins involved, among other functions, in regulated intramembrane proteolysis. In this study, we report the identification and characterization of a complementary DNA from Schistosoma mansoni exhibiting a significant homology to human and nonvertebrate presinilins. S. mansoni contained a 1,485 bp open reading frame encoding a predicted protein of 494 amino acids. Alignment of predicted amino acid sequence of S. mansoni with PS (SmPS) from other species revealed up to 40% similarity shared among the investigated organisms. In addition, phylogenetic analyses demonstrated SmPS being closely related to its orthologues found in Schistosoma japonicum and Caenorhabditis elegans. Expression analysis of SmPS using quantitative real-time PCR revealed that the transcript is up-regulated in the egg stage. We hypothesize that the high level of SmPS in the S. mansoni embryo correlates to an important role during cellular signaling associated to larval development. To our knowledge, this study represents the first attempt to investigate the existence and abundance of PS from a helminth parasite.Item NEDD8 conjugation in Schistosoma mansoni : genome analysis and expression profiles.(2013) Pereira, Roberta Verciano; Gomes, Matheus de Souza; Olmo, Roenick Proveti; Sousa, Daniel M.; Passos, Liana Konovaloff Jannotti; Babá, Élio Hideo; Borges, William de Castro; Cota, Renata Guerra de SáNEDD8 is an ubiquitin-like molecule that covalently binds to target proteins through an enzymatic cascade analogous to ubiquitylation. This modifier is known to bind to p53 and p73, as well as all Cullin family proteins, which are essential components of Skp1/Cul-1/F-box protein (SCF)-like Ub ligase complexes. Here, we focused on a genomic analysis of the genes involved in the NEDD8 conjugation pathway in Schistosoma mansoni. The results revealed seven genes related to NEDD8 conjugation that are conserved in Schistosoma japonicum, Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens. We performed quantitative RT-PCR (qRT-PCR), which showed differential profiles for Smnedd8, Smapp1, Smuba3, Smube2f, Smdcn1, Smrbx and Smsenp8 throughout the life cycle of S. mansoni. Upregulation was observed in 3-day-old schistosomula and adult worms for all analysed genes. We also analysed the transcription levels of Cullin family members Smp63 and Smp73, and observed upregulation in early schistosomula, while cercariae and adult worms showed expression levels similar to one another. Taken together, these results suggest that the NEDDylation/ DeNEDDylation pathway controls important cellular regulators during worm development from cercariae to schistosomula and, finally, to adult.Item Performance of TcI/TcVI/TcII Chagas-Flow ATE-IgG2a for universal and genotype-specific serodiagnosis of Trypanosoma cruzi infection.(2017) Alessio, Glaucia Diniz; Araújo, Fernanda Fortes de; Côrtes, Denise Fonseca; Sales Júnior, Policarpo Ademar; Lima, Daniela Cristina; Gomes, Matheus de Souza; Amaral, Laurence Rodrigues do; Xavier, Marcelo Antônio Pascoal; Carvalho, Andréa Teixeira de; Martins Filho, Olindo Assis; Lana, Marta deDistinct Trypanosoma cruzi genotypes have been considered relevant for patient management and therapeutic response of Chagas disease. However, typing strategies for genotype- specific serodiagnosis of Chagas disease are still unavailable and requires standardization for practical application. In this study, an innovative TcI/TcVI/TcII Chagas Flow ATE-IgG2a technique was developed with applicability for universal and genotypespecific diagnosis of T. cruzi infection. For this purpose, the reactivity of serum samples (percentage of positive fluorescent parasites-PPFP) obtained from mice chronically infected with TcI/Colombiana, TcVI/CL or TcII/Y strain as well as non-infected controls were determined using amastigote-AMA, trypomastigote-TRYPO and epimastigote-EPI in parallel batches of TcI, TcVI and TcII target antigens. Data demonstrated that ªα-TcII-TRYPO/ 1:500, cut-off/PPFP = 20%º presented an excellent performance for universal diagnosis of T. cruzi infection (AUC = 1.0, Se and Sp = 100%). The combined set of attributes ªα-TcITRYPO/ 1:4,000, cut-off/PPFP = 50%º, ªα-TcII-AMA/1:1,000, cut-off/PPFP = 40%º and ªα- TcVI-EPI/1:1,000, cut-off/PPFP = 45%º showed good performance to segregate infections with TcI/Colombiana, TcVI/CL or TcII/Y strain. Overall, hosts infected with TcI/Colombiana and TcII/Y strains displayed opposite patterns of reactivity with ªα-TcI TRYPOº and ªα-TcII AMAº. Hosts infected with TcVI/CL strain showed a typical interweaved distribution pattern. The method presented a good performance for genotype-specific diagnosis, with global accuracy of 69% when the population/prototype scenario include TcI, TcVI and TcII infections and 94% when comprise only TcI and TcII infections. This study also proposes a receiver operating reactivity panel, providing a feasible tool to classify serum samples from hosts infected with distinct T. cruzi genotypes, supporting the potential of this method for universal and genotype-specific diagnosis of T. cruzi infection.Item Physical localization of the retrotransposons Boudicca and Perere 03 in Schistosoma mansoni.(2008) Valentim, Cláudia Laignier Lage; Gomes, Matheus de Souza; Jeremias, Wander de Jesus; Cunha, Juliana Cecília; Oliveira, Guilherme Corrêa de; Botelho, Ana Cristina de Carvalho; Pimenta, Paulo Filemon Paolucci; Passos, Liana Konovaloff Jannotti; Cota, Renata Guerra de Sá; Babá, Élio HideoSchistosoma mansoni is 1 of the causative agents of schistosomiasis, an endemic disease in 76 countries of the world. The study of its genome, estimated to be 270 Mb, is very important to understanding schistosome biology, the mechanisms of drug resistance, and immune evasion. Repetitive elements constitute more than 40% of the S. mansoni genome and may play a role in the parasite evolution. The retrotransposons Boudicca, a long terminal repeat (LTR), and Perere 03, a non-LTR, are present in a high number in the S. mansoni genome and were localized with the use of fluorescence in situ hybridization (FISH) and primed in situ labeling (PRINS). Bacterial artificial chromosomes (BAC) clones containing the retrotransposons Boudicca and Perere 03 were selected by bioinformatic analysis and used as probes in FISH. Using metaphase chromosomes from sporocysts and the FISH and PRINS techniques, we were able to map these retrotransposons. Perere 03 was localized in the euchromatic regions of the short arm of chromosome 2 and Boudicca in