Análise proteômica e transcricional de Paracoccidioides em condições que mimetizam a infecção

Tese (doutorado)—Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Patologia Molecular, 2012.O fungo Paracoccidioides é um patógeno humano com ampla distribuição na América Latina. A infecção se inicia por inalação de conídios e ou propágulos da forma miceliana que ao atingirem o pulmão do hospedeiro se diferenciam na fase leveduriforme. Embora o perfil de expressão gênica em Paracoccidioides venha sendo estudado, pouco se conhece sobre o padrão de expressão de genes desta espécie durante o processo infeccioso. Durante a infecção, patógenos regulam o metabolismo em resposta a diferentes tipos de estresse e quantidades variadas de nutrientes disponíveis no hospedeiro, presumivelmente permitindo a sua adaptação e sobrevivência. Os íons metálicos são elementos essenciais para a manutenção de importantes vias metabólicas, e por esse motivo, a capacidade de adquiri-los pelos patógenos, a partir de fontes disponíveis no organismo hospedeiro, é considerada um fator de virulência. O zinco é considerado um íon metálico essencial para todos os organismos, por ser requerido para transcrição, tradução, replicação, resistência ao estresse oxidativo e virulência. Transportadores para este íons são altamente expressos em condições de infecção por Paracoccidioides. O presente trabalho descreve a análise de genes e proteínas diferencialmente expressos em células leveduriformes de Paracoccidioides em condições que mimetizam a infecção. Foi realizada a comparação do perfil proteômico de Paracoccidioides durante a privação de zinco e na disponibilidade desse metal com o objetivo de se descrever os mecanismos utilizados pelo fungo para sobreviver no ambiente com baixa disponibilidade de zinco, mimetizando a condição encontrada pelo patógeno durante a infecção. Adicionalmente, foi realizada a análise transcricional de Paracoccidioides recuperado de animais infectados. Os dados revelaram que proteínas relacionadas com resposta ao estresse, metabolismo de aminoácidos, gliconeogênese e ciclo do metilcitrato foram regulados durante a privação de zinco. Os transcritos induzidos durante a infecção no pulmão foram aqueles predominantemente relacionados com metabolismo de lipídios, ácidos graxos e isoprenóides, metabolismo de aminoácidos, resposta ao estresse e virulência e metabolismo de carboidratos. A expressão diferencial dos genes e proteínas identificados foi confirmada por ensaios de qRT-PCR. Os dados gerados podem facilitar estudos funcionais de novos genes e proteínas os quais podem ser importantes para estratégias de sobrevivência e adaptação do Paracoccidioides no hospedeiro. ______________________________________________________________________________ ABSTRACTParacoccidioides is a fungal human pathogen with a wide distribution in Latin America. The fungus causes infection through host inhalation of airborne propagules of the mycelial phase of the organism. These particles reach the lungs and differ in the yeast phase. Although gene expression in Paracoccidioides had been studied, little is known about the genome sequences expressed by this species during the infection process. During infection, pathogens regulate the metabolism in response to different types of stress and varying amounts of nutrients available in the host, presumably allowing its adaptation and survival. Metal ions are essential elements for the maintenance of important metabolic pathways, and therefore, the ability to acquire them by pathogens from sources available in the host organism, is considered a virulence factor. Zinc is considered a metal ion essential for all organisms, being required for transcription, translation, replication, resistance to oxidative stress and virulence. Here we describe the analysis of genes and proteins differentially expressed in yeast cells of Paracoccidioides under conditions mimicking of infection. It was performed a comparison of the proteomic profile of Paracoccidioides during zinc deprivation and in the availability of the metal in order to describe the mechanisms used by the fungus to survive in the environment with low vailability of zinc, mimicking the condition encountered by the pathogen during infection. Farther, was performed transcriptional analysis of Paracoccidioides recovered from lung of infected animals. The data revealed that proteins related to stress response, amino acid metabolism, gluconeogenesis and methylcitrate cycle were regulated during deprivation of zinc. The transcripts induced during infection in the lung were predominantly those related to lipid metabolism, fatty acid and isoprenoid, amino acid metabolism, carbohydrate metabolism and stress response and virulence. The differential expression of genes and proteins identified was confirmed by qRT-PCR assays. The data generated can facilitate functional studies of novel genes and proteins which may be important for survival strategies and adaptation of Paracoccidioides in the host

Hemoglobin Uptake by Paracoccidioides spp. Is Receptor-Mediated

Iron is essential for the proliferation of fungal pathogens during infection. The availability of iron is limited due to its association with host proteins. Fungal pathogens have evolved different mechanisms to acquire iron from host; however, little is known regarding how Paracoccidioides species incorporate and metabolize this ion. In this work, host iron sources that are used by Paracoccidioides spp. were investigated. Robust fungal growth in the presence of the iron-containing molecules hemin and hemoglobin was observed. Paracoccidioides spp. present hemolytic activity and have the ability to internalize a protoporphyrin ring. Using real-time PCR and nanoUPLC-MSE proteomic approaches, fungal growth in the presence of hemoglobin was shown to result in the positive regulation of transcripts that encode putative hemoglobin receptors, in addition to the induction of proteins that are required for amino acid metabolism and vacuolar protein degradation. In fact, one hemoglobin receptor ortholog, Rbt5, was identified as a surface GPI-anchored protein that recognized hemin, protoporphyrin and hemoglobin in vitro. Antisense RNA technology and Agrobacterium tumefaciensmediated transformation were used to generate mitotically stable Pbrbt5 mutants. The knockdown strain had a lower survival inside macrophages and in mouse spleen when compared with the parental strain, which suggested that Rbt5 could act as a virulence factor. In summary, our data indicate that Paracoccidioides spp. can use hemoglobin as an iron source most likely through receptor-mediated pathways that might be relevant for pathogenic mechanisms

Importância da escola no conhecimento empírico sobre infecções sexualmente transmissíveis e métodos contraceptivos: promoção da saúde na rede pública de ensino / Importance of the school in empirical knowledge about sexually transmitted infections and contraceptive methods: health promotion in the public school network

A sexualidade é um processo contínuo construído pelo indivíduo durante o desenvolvimento biológico, sendo influenciada por contextos sociais e culturais. A escola exerce papel fundamental na educação sexual dos discentes, sendo capaz de contribuir na construção do saber, refletindo na prevenção de Infecções Sexualmente Transmissíveis (IST) e métodos contraceptivos. Diante disso, o objetivo do presente estudo foi identificar as dificuldades dos estudantes quanto ao conhecimento das IST e métodos contraceptivos. Após a identificação do conhecimento empírico dos alunos foram realizadas atividades de intervenção como meio de promoção e prevenção de complicações à saúde. Um questionário foi aplicado para realizar levantamento das dúvidas dos participantes sobre a temática. Além disso, as intervenções embasadas nas dúvidas foram realizadas por meio de palestras e atividade dinâmica lúdica. Evidenciamos que os alunos de ambos os sexos apresentam pouco conhecimento em relação às IST, possuem mais conhecimento sobre HIV/AIDS e consideraram apenas os preservativos masculino e feminino como métodos contraceptivos. Em conjunto, esses dados revelam a importância da educação sexual na escola, principalmente, em relação às IST que causam diversas complicações como infertilidade, doença inflamatória pélvica (DIP), disfunção sexual, câncer de colo de útero e outros agravamentos à saúde

Influência familiar e de outras fontes de informações na construção dos conhecimentos dos adolescentes acerca da sexualidade / Influence of family and other sources of information in the construction of adolescents' knowledge about sexuality

O convívio familiar, relações interpessoais e as mídias digitais podem influenciar na construção do conhecimento dos adolescentes sobre a sexualidade. Apesar da importância das discussões no ambiente escolar e do impacto negativo na saúde pública, a educação sexual tem sido negligenciada devido a tabus e barreiras socioculturais. O objetivo do presente estudo foi identificar no ambiente escolar qual a fonte de informação que o adolescente está consumindo, bem como sua influência na educação e prática sexual. Foi aplicado um questionário aos alunos, levantando dados sociodemográficos, sexo e idade, bem como sobre fatores que influenciam a sexualidade. Nossos dados mostraram que o sexo feminino tem melhor contato familiar quando comparadas ao sexo masculino. Além disso, ambos os generos concordam sobre a importância de abordar a sexualidade na escola, caso contrário, as mídias digitais e os amigos passam a ser as principais fontes de informação sobre sexualidade. Por fim, os alunos de ambos os sexos se mostraram suscetíveis à intervenção de amigos na primeira relação sexual. Em conjunto, nossos resultados identificam que os adolescentes são influenciados por fontes inseguras, imprecisas e não científicas sobre sexualidade e práticas sexuais. Nesse cenário, as discussões sobre sexualidade com a família e na instituição escolar são essenciais, pois ajudará a promover a vivência consciente da prática sexual e da sexualidade

Measurement of the polarisation of W bosons produced with large transverse momentum in pp collisions at sqrt(s) = 7 TeV with the ATLAS experiment

This paper describes an analysis of the angular distribution of W->enu and W->munu decays, using data from pp collisions at sqrt(s) = 7 TeV recorded with the ATLAS detector at the LHC in 2010, corresponding to an integrated luminosity of about 35 pb^-1. Using the decay lepton transverse momentum and the missing transverse energy, the W decay angular distribution projected onto the transverse plane is obtained and analysed in terms of helicity fractions f0, fL and fR over two ranges of W transverse momentum (ptw): 35 < ptw < 50 GeV and ptw > 50 GeV. Good agreement is found with theoretical predictions. For ptw > 50 GeV, the values of f0 and fL-fR, averaged over charge and lepton flavour, are measured to be : f0 = 0.127 +/- 0.030 +/- 0.108 and fL-fR = 0.252 +/- 0.017 +/- 0.030, where the first uncertainties are statistical, and the second include all systematic effects.Comment: 19 pages plus author list (34 pages total), 9 figures, 11 tables, revised author list, matches European Journal of Physics C versio

Observation of a new chi_b state in radiative transitions to Upsilon(1S) and Upsilon(2S) at ATLAS

The chi_b(nP) quarkonium states are produced in proton-proton collisions at the Large Hadron Collider (LHC) at sqrt(s) = 7 TeV and recorded by the ATLAS detector. Using a data sample corresponding to an integrated luminosity of 4.4 fb^-1, these states are reconstructed through their radiative decays to Upsilon(1S,2S) with Upsilon->mu+mu-. In addition to the mass peaks corresponding to the decay modes chi_b(1P,2P)->Upsilon(1S)gamma, a new structure centered at a mass of 10.530+/-0.005 (stat.)+/-0.009 (syst.) GeV is also observed, in both the Upsilon(1S)gamma and Upsilon(2S)gamma decay modes. This is interpreted as the chi_b(3P) system.Comment: 5 pages plus author list (18 pages total), 2 figures, 1 table, corrected author list, matches final version in Physical Review Letter

Measurement of the inclusive and dijet cross-sections of b-jets in pp collisions at sqrt(s) = 7 TeV with the ATLAS detector

The inclusive and dijet production cross-sections have been measured for jets containing b-hadrons (b-jets) in proton-proton collisions at a centre-of-mass energy of sqrt(s) = 7 TeV, using the ATLAS detector at the LHC. The measurements use data corresponding to an integrated luminosity of 34 pb^-1. The b-jets are identified using either a lifetime-based method, where secondary decay vertices of b-hadrons in jets are reconstructed using information from the tracking detectors, or a muon-based method where the presence of a muon is used to identify semileptonic decays of b-hadrons inside jets. The inclusive b-jet cross-section is measured as a function of transverse momentum in the range 20 < pT < 400 GeV and rapidity in the range |y| < 2.1. The bbbar-dijet cross-section is measured as a function of the dijet invariant mass in the range 110 < m_jj < 760 GeV, the azimuthal angle difference between the two jets and the angular variable chi in two dijet mass regions. The results are compared with next-to-leading-order QCD predictions. Good agreement is observed between the measured cross-sections and the predictions obtained using POWHEG + Pythia. MC@NLO + Herwig shows good agreement with the measured bbbar-dijet cross-section. However, it does not reproduce the measured inclusive cross-section well, particularly for central b-jets with large transverse momenta.Comment: 10 pages plus author list (21 pages total), 8 figures, 1 table, final version published in European Physical Journal

Search for displaced vertices arising from decays of new heavy particles in 7 TeV pp collisions at ATLAS

We present the results of a search for new, heavy particles that decay at a significant distance from their production point into a final state containing charged hadrons in association with a high-momentum muon. The search is conducted in a pp-collision data sample with a center-of-mass energy of 7 TeV and an integrated luminosity of 33 pb^-1 collected in 2010 by the ATLAS detector operating at the Large Hadron Collider. Production of such particles is expected in various scenarios of physics beyond the standard model. We observe no signal and place limits on the production cross-section of supersymmetric particles in an R-parity-violating scenario as a function of the neutralino lifetime. Limits are presented for different squark and neutralino masses, enabling extension of the limits to a variety of other models.Comment: 8 pages plus author list (20 pages total), 8 figures, 1 table, final version to appear in Physics Letters

Measurement of the inclusive isolated prompt photon cross-section in pp collisions at sqrt(s)= 7 TeV using 35 pb-1 of ATLAS data

A measurement of the differential cross-section for the inclusive production of isolated prompt photons in pp collisions at a center-of-mass energy sqrt(s) = 7 TeV is presented. The measurement covers the pseudorapidity ranges |eta|<1.37 and 1.52<=|eta|<2.37 in the transverse energy range 45<=E_T<400GeV. The results are based on an integrated luminosity of 35 pb-1, collected with the ATLAS detector at the LHC. The yields of the signal photons are measured using a data-driven technique, based on the observed distribution of the hadronic energy in a narrow cone around the photon candidate and the photon selection criteria. The results are compared with next-to-leading order perturbative QCD calculations and found to be in good agreement over four orders of magnitude in cross-section.Comment: 7 pages plus author list (18 pages total), 2 figures, 4 tables, final version published in Physics Letters