Estudo de associação entre variantes dos genes HLA-DRB e CTLA4 e pênfigo foliáceo endêmico (fogo selvagem)

Orientadora: Maria Luiza Petzl-ErlerDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências BiológicasResumo: O pênfigo foliáceo endêmico (fogo-selvagem) é uma doença caracterizada por bolhas intra-epidérmicas e por anticorpos anti-desmogleína 1, uma proteína dos desmossomos, estruturas de adesão celular. A área de endemicidade localiza-se na região central da América do Sul, principalmente no centro-oeste do Brasil. Os auto-anticorpos promovem a perda da adesão entre os queratinócitos, um processo chamado acantólise. As bolhas resultantes localizam-se nas camadas superficiais da pele. Proteínas HLA têm um papel fundamental no desenvolvimento da resposta imune, apresentando antígenos aos linfócitos T e formando o repertório de linfócitos que participará da resposta imune periférica. Associações entre alelos de genes HLA e doenças autoimunes são frequentes na literatura. Já foram observadas associações entre alelos dos genes HLA-DRB e DQB e pênfigo foliáceo endêmico. O gene CTLA4 tem um papel fundamental na homeostase do sistema imune. Várias associações já foram observadas entre variantes de CTLA4 e doenças autoimunes. O objetivo deste trabalho foi investigar possíveis associações entre variantes dos genes HLA-DRB (DRB1, DRB3 e DRB5) e CTLA4 e pênfigo foliáceo endêmico. A amostra de pacientes e de controles foi composta por 147 e 182 indivíduos, respectivamente. A maior parte dos pacientes foi coletada no Hospital Adventista do Pênfigo (Campo Grande/MS) e a amostra de controles foi coletada em Campo Grande e Curitiba. As variantes gênicas foram tipadas através da técnica PCR-SSO. A análise estatística foi realizada utilizando o programa computacional RxC (algoritmo metropolis) e a magnitude das associações foi expressa através da OR (odds ratio). Associações positivas com a doença foram observadas com os alelos DRB1*0102, *0404, e *1602 (OR = 6,25; 1,98 e 2,66, respectivamente). Também foram observadas associações positivas com os alelos DRB1*0406 e *1406 (OR = 14,09 e 16,77, respectivamente). Estas OR elevadas são devidas, provavelmente, ao pequeno número de indivíduos que possuem estes alelos, por isso devem ser examinadas com cautela. Associações negativas foram observadas com os alelos *0301, *0701, *1101, *1104, *1301 e *1303 (OR = 0,23; 0,07; 0,05; 0,12; 0,33 e 0,09, respectivamente). Observou-se uma associação positiva com o alelo DRB5*0202 e uma associação negativa com DRB3*0202. Não foi possível determinar se a associação primária é com os alelos DRB1*16 ou com DRB5*0202. A associação com DRB3*0202 é consequente do desequilíbrio de ligação com alelos de DRB1 associados negativamente. Os resultados sugerem que os genes HLA-DRB tem um importante papel no estabelecimento da patogênese do pênfigo foliáceo endêmico, e que o gene CTLA4 não está influenciando de forma expressiva o desenvolvimento desta doença. No entanto, parece haver uma interação entre determinados genótipos de CTLA4 e HLA-DRB 1 na determinação do pênfigo foliáceo endêmico

Leitura e formação no Ensino Superior : problematização sobre a formação de leitores no Brasil e em Portugal

Neste artigo discute-se o problema da leitura e da formação de leitores no ensino superior, no Brasil e em Portugal, sendo nosso objetivo perceber se estes estudantes são ainda leitores em construção. A metodologia para a discussão centrou-se nas investigações em torno da formação leitora desses estudantes, o que permitiu chegar a algumas conclusões afins: são fundamentais estudos de maior dimensão nessas áreas; é basilar que todos os docentes do ensino superior tomem consciência das dificuldades desses alunos na leitura/escrita/literacias académicas e que nas suas disciplinas possam dar um contributo para colmatar esses problemas; os alunos do ensino superior são ainda leitores em construção.info:eu-repo/semantics/publishedVersio

Predicting the Proteins of Angomonas deanei, Strigomonas culicis and Their Respective Endosymbionts Reveals New Aspects of the Trypanosomatidae Family

Endosymbiont-bearing trypanosomatids have been considered excellent models for the study of cell evolution because the host protozoan co-evolves with an intracellular bacterium in a mutualistic relationship. Such protozoa inhabit a single invertebrate host during their entire life cycle and exhibit special characteristics that group them in a particular phylogenetic cluster of the Trypanosomatidae family, thus classified as monoxenics. in an effort to better understand such symbiotic association, we used DNA pyrosequencing and a reference-guided assembly to generate reads that predicted 16,960 and 12,162 open reading frames (ORFs) in two symbiont-bearing trypanosomatids, Angomonas deanei (previously named as Crithidia deanei) and Strigomonas culicis (first known as Blastocrithidia culicis), respectively. Identification of each ORF was based primarily on TriTrypDB using tblastn, and each ORF was confirmed by employing getorf from EMBOSS and Newbler 2.6 when necessary. the monoxenic organisms revealed conserved housekeeping functions when compared to other trypanosomatids, especially compared with Leishmania major. However, major differences were found in ORFs corresponding to the cytoskeleton, the kinetoplast, and the paraflagellar structure. the monoxenic organisms also contain a large number of genes for cytosolic calpain-like and surface gp63 metalloproteases and a reduced number of compartmentalized cysteine proteases in comparison to other TriTryp organisms, reflecting adaptations to the presence of the symbiont. the assembled bacterial endosymbiont sequences exhibit a high A+T content with a total of 787 and 769 ORFs for the Angomonas deanei and Strigomonas culicis endosymbionts, respectively, and indicate that these organisms hold a common ancestor related to the Alcaligenaceae family. Importantly, both symbionts contain enzymes that complement essential host cell biosynthetic pathways, such as those for amino acid, lipid and purine/pyrimidine metabolism. These findings increase our understanding of the intricate symbiotic relationship between the bacterium and the trypanosomatid host and provide clues to better understand eukaryotic cell evolution.Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)ERC AdG SISYPHEUniv Fed Rio de Janeiro, Inst Biofis Carlos Chagas Filho, Lab Ultraestrutura Celular Hertha Meyer, BR-21941 Rio de Janeiro, BrazilUniv Fed Rio de Janeiro, Inst Biofis Carlos Chagas Filho, Lab Metab Macromol Firmino Torres de Castro, BR-21941 Rio de Janeiro, BrazilLab Bioinformat, Lab Nacl Computacao Cient, Rio de Janeiro, BrazilINRIA Grenoble Rhone Alpes, BAMBOO Team, Villeurbanne, FranceUniv Lyon 1, CNRS, UMR5558, Lab Biometrie & Biol Evolut, F-69622 Villeurbanne, FranceUniv Estadual Campinas, Inst Biol, Dept Genet Evolucao & Bioagentes, São Paulo, BrazilUniv São Paulo, Fac Ciencias Farmaceut Ribeirao Preto, Dept Ciencias Farmaceut, São Paulo, BrazilLab Nacl Ciencia & Tecnol Bioetano, São Paulo, BrazilUniv Fed Minas Gerais, Inst Ciencias Biol, Dept Bioquim & Imunol, Belo Horizonte, MG, BrazilUniv Fed Goias, Inst Ciencias Biol, Mol Biol Lab, Goiania, Go, BrazilFundacao Oswaldo Cruz, Inst Carlos Chagas, Lab Biol Mol Tripanossomatideos, Curitiba, Parana, BrazilFundacao Oswaldo Cruz, Inst Carlos Chagas, Lab Genom Func, Curitiba, Parana, BrazilUniv Estadual Campinas, Ctr Pluridisciplinar Pesquisas Quim Biol & Agr, São Paulo, BrazilUniv Fed Minas Gerais, Inst Ciencias Biol, Dept Parasitol, Belo Horizonte, MG, BrazilUniv Fed Santa Catarina, Dept Microbiol Imunol & Parasitol, Ctr Ciencias Biol, Lab Protozool & Bioinformat, Florianopolis, SC, BrazilUniv Fed Vicosa, Dept Bioquim & Biol Mol, Ctr Ciencias Biol & Saude, Vicosa, MG, BrazilInst Butantan, Lab Especial Ciclo Celular, São Paulo, BrazilUniv São Paulo, Dept Biol, Fac Filosofia Ciencias & Letras Ribeirao Preto, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilWeb of Scienc

Evolutionary analyses of myosin genes in trypanosomatids show a history of expansion, secondary losses and neofunctionalization

Submitted by Manoel Barata ([email protected]) on 2019-02-14T13:49:21Z No. of bitstreams: 1 s41598-017-188.pdf: 5208060 bytes, checksum: 284ab473d765391a581c2ad147ddbd1a (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2019-02-15T18:18:59Z (GMT) No. of bitstreams: 1 s41598-017-188.pdf: 5208060 bytes, checksum: 284ab473d765391a581c2ad147ddbd1a (MD5)Made available in DSpace on 2019-02-15T18:18:59Z (GMT). No. of bitstreams: 1 s41598-017-188.pdf: 5208060 bytes, checksum: 284ab473d765391a581c2ad147ddbd1a (MD5) Previous issue date: 2018Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Programa de Pós-Graduação em Biociências e Biotecnologia. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Programa de Pós-Graduação em Biociências e Biotecnologia. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Programa de Pós-Graduação em Biociências e Biotecnologia. Curitiba, PR, Brasil. / Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Laboratório de Genômica Funcional. Curitiba, PR, Brasil. / Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Myosins are motor proteins that comprise a large and diversified family important for a broad range of functions. Two myosin classes, I and XIII, were previously assigned in Trypanosomatids, based mainly on the studies of Trypanosoma cruzi, T. brucei and Leishmania major, and important human pathogenic species; seven orphan myosins were identified in T. cruzi. Our results show that the great variety of T. cruzi myosins is also present in some closely related species and in Bodo saltans, a member of an early divergent branch of Kinetoplastida. Therefore, these myosins should no longer be considered "orphans". We proposed the classification of a kinetoplastid-specific myosin group into a new class, XXXVI. Moreover, our phylogenetic data suggest that a great repertoire of myosin genes was present in the last common ancestor of trypanosomatids and B. saltans, mainly resulting from several gene duplications. These genes have since been predominantly maintained in synteny in some species, and secondary losses explain the current distribution. We also found two interesting genes that were clearly derived from myosin genes, demonstrating that possible redundant or useless genes, instead of simply being lost, can serve as raw material for the evolution of new genes and functions

Characterization of TcSTI-1, a homologue of stress-induced protein-1, in Trypanosoma cruzi

The life cycle of the protozoan Trypanosoma cruzi exposes it to several environmental stresses in its invertebrate and vertebrate hosts. Stress conditions are involved in parasite differentiation, but little is known about the stress response proteins involved. We report here the first characterization of stress-induced protein-1 (STI-1) in T. cruzi (TcSTI-1). This co-chaperone is produced in response to stress and mediates the formation of a complex between the stress proteins HSP70 and HSP90 in other organisms. Despite the similarity of TcSTI-1 to STI-1 proteins in other organisms, its expression profile in response to various stress conditions, such as heat shock, acidic pH or nutrient starvation, is quite different. Neither polysomal mRNA nor protein levels changed in exponentially growing epimastigotes cultured under any of the stress conditions studied. Increased levels of TcSTI-1 were observed in epimastigotes subjected to nutritional stress in the late growth phase. Co-immunoprecipitation assays revealed an association between TcSTI-1 and TcHSP70 in T. cruzi epimastigotes. Immunolocalization demonstrated that TcSTI-1 was distributed throughout the cytoplasm and there was some colocalization of TcSTI-1 and TcHSP70 around the nucleus. Thus, TcSTI-1 associates with TcHSP70 and TcSTI-1 expression is induced when the parasites are subjected to stress conditions during specific growth phase

Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics

BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. METHODS The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses

Profiling the Trypanosoma cruzi phosphoproteome

Submitted by Manoel Barata ([email protected]) on 2019-12-03T17:47:47Z No. of bitstreams: 1 journal.pone.002538ok.PDF: 411332 bytes, checksum: 1c64dda8f58ad34d9bd06e475d53ef4a (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2020-02-19T14:39:34Z (GMT) No. of bitstreams: 1 journal.pone.002538ok.PDF: 411332 bytes, checksum: 1c64dda8f58ad34d9bd06e475d53ef4a (MD5)Made available in DSpace on 2020-02-19T14:39:34Z (GMT). No. of bitstreams: 1 journal.pone.002538ok.PDF: 411332 bytes, checksum: 1c64dda8f58ad34d9bd06e475d53ef4a (MD5) Previous issue date: 2011Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil / Department of Proteomics and Signal Transduction. Max Planck Institute of Biochemistry. Martinsried, Germany.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Department of Proteomics and Signal Transduction. Max Planck Institute of Biochemistry. Martinsried, Germany.Department of Proteomics and Signal Transduction. Max Planck Institute of Biochemistry. Martinsried, Germany.Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Protein phosphorylation is a reversible post-translational modification essential for the regulation of several signal transduction pathways and biological processes in the living cell. Therefore, the identification of protein phosphorylation sites is crucial to understand cell signaling control at the molecular level. Based on mass spectrometry, recent studies have reported the large-scale mapping of phosphorylation sites in various eukaryotes and prokaryotes. However, little is known about the impact of phosphorylation in protozoan parasites. To in depth characterize the phosphoproteome of Trypanosoma cruzi, a parasite of the Kinetoplastida class, protein samples from cells at different phases of the metacyclogenesis--differentiation process of the parasites from non-infective epimastigotes to infective metacyclic trypomastigotes--were enriched for phosphopeptides using TiO(2) chromatography and analyzed on an LTQ-Orbitrap mass spectrometer. In total, 1,671 proteins were identified, including 753 phosphoproteins, containing a total of 2,572 phosphorylation sites. The distribution of phosphorylated residues was 2,162 (84.1%) on serine, 384 (14.9%) on threonine and 26 (1.0%) on tyrosine. Here, we also report several consensus phosphorylation sequence motifs and as some of these conserved groups have enriched biological functions, we can infer the regulation by protein kinases of this functions. To our knowledge, our phosphoproteome is the most comprehensive dataset identified until now for Kinetoplastida species. Here we also were able to extract biological information and infer groups of sites phosphorylated by the same protein kinase. To make our data accessible to the scientific community, we uploaded our study to the data repositories PHOSIDA, Proteome Commons and TriTrypDB enabling researchers to access information about the phosphorylation sites identified here

Trypanosoma cruzi transcriptome during axenic epimastigote growth curve

Submitted by Manoel Barata ([email protected]) on 2019-02-14T12:34:54Z No. of bitstreams: 1 0074-0276-mioc-113-5.pdf: 3046522 bytes, checksum: ca70ac4a197b74c287651a9f0359a489 (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2019-02-14T17:36:01Z (GMT) No. of bitstreams: 1 0074-0276-mioc-113-5.pdf: 3046522 bytes, checksum: ca70ac4a197b74c287651a9f0359a489 (MD5)Made available in DSpace on 2019-02-14T17:36:01Z (GMT). No. of bitstreams: 1 0074-0276-mioc-113-5.pdf: 3046522 bytes, checksum: ca70ac4a197b74c287651a9f0359a489 (MD5) Previous issue date: 2018Universidade Federal do Paraná. Centro Politécnico. Programa de Pós-Graduação em Biologia Celular e Molecular. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas, Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas, Curitiba, PR, Brasil. / Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas, Curitiba, PR, Brasil. / Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Instituto Carlos Chagas, Curitiba, PR, Brasil. / Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Universidade Federal do Paraná. Centro Politécnico. Programa de Pós-Graduação em Biologia Celular e Molecular. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas, Curitiba, PR, Brasil. / Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Universidade Federal do Paraná. Centro Politécnico. Programa de Pós-Graduação em Biologia Celular e Molecular. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas, Curitiba, PR, Brasil.Universidade Federal do Paraná. Centro Politécnico. Programa de Pós-Graduação em Biologia Celular e Molecular. Curitiba, PR, Brasil. / Fundação Oswaldo Cruz. Instituto Carlos Chagas, Curitiba, PR, Brasil.Trypanosoma cruzi is an important protozoan parasite and the causative agent of Chagas disease. A critical step in understanding T. cruzi biology is the study of cellular and molecular features exhibited during its growth curve. We aimed to acquire a global view of the gene expression profile of T. cruzi during epimastigote growth. RNA-Seq analysis of total and polysomal/granular RNA fractions was performed along the 10 days T. cruzi epimastigote growth curve in vitro, in addition to cell viability and cell cycle analyses. We also analysed the polysome profile and investigated the presence of granular RNA by FISH and western blotting. We identified 1082 differentially expressed genes (DEGs), of which 220 were modulated in both fractions. According to the modulation pattern, DEGs were grouped into 12 clusters and showed enrichment of important gene ontology (GO) terms. Moreover, we showed that by the sixth day of the growth curve, polysomal content declined greatly and the RNA granules content appeared to increase, suggesting that a portion of mRNAs isolated from the sucrose gradient during late growth stages was associated with RNA granules and not only polyribosomes. Furthermore, we discuss several modulated genes possibly involved in T. cruzi growth, mainly during the stationary phase, such as genes related to cell cycle, pathogenesis, metabolic processes and RNA-binding proteins

The zinc finger protein TcZFP2 binds target mRNAs enriched during Trypanosoma cruzi metacyclogenesis

Trypanosomes are parasitic protozoa in which gene expression is primarily controlled through the regulation of mRNA stability and translation. This post-transcriptional control is mediated by various families of RNA-binding proteins, including those with zinc finger CCCH motifs. CCCH zinc finger proteins have been shown to be essential to differentiation events in trypanosomatid parasites. Here, we functionally characterise TcZFP2 as a predicted post-transcriptional regulator of differentiation in Trypanosoma cruzi. This protein was detected in cell culture-derived amastigotes and trypomastigotes, but it was present in smaller amounts in metacyclic trypomastigote forms of T. cruzi. We use an optimised recombinant RNA immunopreciptation followed by microarray analysis assay to identify TcZFP2 target mRNAs. We further demonstrate that TcZFP2 binds an A-rich sequence in which the adenosine residue repeats are essential for high-affinity recognition. An analysis of the expression profiles of the genes encoding the TcZFP2-associated mRNAs throughout the parasite life cycle by microarray hybridisation showed that most of the associated mRNAs were upregulated in the metacyclic trypomastigote forms, also suggesting a role for TcZFP2 in metacyclic trypomastigote differentiation. Knockdown of the orthologous Trypanosoma brucei protein levels showed ZFP2 to be a positive regulator of specific target mRNA abundance