Transcriptômica de Trypanossoma cruzi em resposta a inibidores da síntese de esteróis

Orientadores : Dr. Marco Aurélio Krieger, Dr. Christian Macagnan ProbstDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 30/09/2010Inclui referênciasResumo: O Trypanosoma cruzi e o protozoario causador da doenca de Chagas, enfermidade que afeta cerca de 15 milhoes de pessoas no continente americano. Diferentemente de mamiferos que sintetizam colesterol, T. cruzi produz principalmente ergosterol, esterol caracteristico de fungos. Por este motivo, a via de sintese deste lipidio representa um potencial alvo quimioterapico da Doenca de Chagas. Dentro de uma etapa inicial do projeto Reguloma de T. cruzi, o presente trabalho teve como objetivo analisar o transcriptoma deste parasita em resposta a duas drogas inibitorias que agem em pontos distintos desta via: Cetoconazol e Lovastatina. Foram calculadas as doses das drogas capazes de inibir o crescimento das culturas celulares em 50% apos 3 dias de exposicao (IC50: 32ƒÊM Cetoconazol e 50ƒÊM Lovastatina); ou matar todas as celulas em 24 horas (DL100: 120ƒÊM Cetoconazol e 100ƒÊM Lovastatina). Foram extraidas amostras de RNA polissomal em tempos precoces de exposicao (30 e 60 minutos) a DL100 (triplicata) e em tempos tardios (1 a 5 dias) a IC50 (duplicata). Os cDNAs gerados a partir das fracoes poli-A+ foram analisados atraves de sequenciamento paralelo massivo (RNA-Seq) com a plataforma SOLiD 3. Foram gerados cerca de 320 milhoes de reads de 50 bases para os experimentos DL100, sendo que a analise dos dados evidenciou quase 3.000 genes diferencialmente expressos em resposta a Lovastatina e cerca de 1.500 frente a Cetoconazol, com FDR menor de 0,1% e foldchange (FC) de 1,5 vez. Analises de enriquecimento de ontologia genica (GO) mostraram que a resposta a DL100 envolve mecanismos estereotipados de expressao genica relacionados a ativacao de vias de catabolismo, principalmente de aminoacidos, e enriquecimento de genes envolvidos na sintese de ATP por fosforilacao oxidativa, alem de uma forte inibicao da sintese e processamento de RNA. Foi evidenciado tambem o aumento da expressao de cisteina-peptidases do tipo Calpaina e Catepsina na resposta a DL100, as quais podem estar envolvidas em morte celular necrotica. Esses resultados sao compativeis com experimentos de microscopia eletronica de transmissao e citometria de fluxo. Para os experimentos IC50, foram gerados cerca de 207 milhoes de reads, selecionando-se 863 genes diferencialmente expressos em resposta a Cetoconazol e 462 em resposta a Lovastatina, com FDR 5% e FC 1,5 vez. Experimentos de microscopia eletronica e de fluorescencia e citometria de fluxo evidenciaram um aumento do numero de reservossomos, enquanto os dados de RNA-Seq indicam o aumento de expressao de possiveis proteinas localizadas nessa organela, apontando para uma possivel funcao dessa estrutura celular na sintese de esterois. Analises de GO demonstraram um enriquecimento de vias envolvidas no transporte de aminoacidos e atividade peptidasica (para genes aumentados) e proteinas nucleossomais (genes diminuidos), alem de respostas droga-especificas. Os dados gerados constituem a primeira avaliacao global da resposta do parasitas a essas drogas, e sua analise em conjunto com os demais ensaios representa uma abordagem riquissima para o entendimento mais aprofundado das modificacoes moleculares que ocorrem com a inibicao da biossintese do ergosterol. Embora a quantidade de dados gerada no presente trabalho nao seja suficiente para analises robustas de reguloma, resultados preliminares demonstram que tal metodologia permite uma compreensao integrada e muito poderosa da biologia basica do Trypanosoma cruzi, principalmente com relacao a mecanismos pos-transcricionais da expressao genica. A inclusao dos dados gerados no presente trabalho na base maior do projeto reguloma representa um importante avanco na identificacao das redes de regulacao da expressao genica.Abstract: Trypanosoma cruzi is the protozoan that causes Chagas' disease, illness that affects about 15 million people in the Americas. Unlike mammals that synthesize cholesterol, T. cruzi produces mainly ergosterol, sterol characteristic of fungi. For this reason, the route of synthesis of this lipid represents a potential chemotherapic target for Chagas disease. Within an initial phase of T. cruzi Regulome project, the present study aim to analyze the transcriptome of this parasite in response to two inhibitory drugs that act at different points of the ergosterol biosynthesis pathway: ketoconazole and lovastatin. We calculated the drug doses capable of inhibiting the growth of cell cultures by 50% after 3 days of exposure (IC50: 32ìM ketoconazole and 50ìM lovastatin), or kill all cells at 24 hours (DL100: 120ìM ketoconazole and 100ìM lovastatin). Polysomal mRNA samples were extracted at very early exposure times (30 and 60 minutes) to the LD100 (triplicate) and at later times (1 to 5 days) to IC50 (duplicate). The cDNAs generated from poly A+ fractions were analyzed by massive parallel sequencing (RNA-Seq) with the SOLiD 3 platform. We generated about 320 million reads of 50 bases for the DL100 experiments, and the data analysis revealed nearly 3,000 genes differentially expressed in response to lovastatin and about 1,500 compared to ketoconazole, with FDR less than 0.1% and foldchange (FC) of 1.5. Analysis of gene ontology enrichment (GO) showed that the response to LD100 involves a stereotypical expression pattern that include mechanisms related to activation of catabolic pathways, especially amino acids, and enrichment of genes involved in ATP synthesis by oxidative phosphorylation, in addition to a strong inhibition of RNA synthesis and processing. It was also shown increased expression of cysteine peptidases of type calpain and cathepsin in response to LD100, which may be involved in necrotic cell death. These results are consistent with experiments of transmission electron microscopy and flow cytometry. For the IC50 experiments, we generated about 207 million reads, selecting 863 genes differentially expressed in response to ketoconazole and 462 in response to Lovastatin, with FDR 5% and FC 1.5. Experiments of electron and fluorescence microscopy and flow cytometry showed an increased number of reservosomes, while the RNA-Seq data indicate the increased expression of possible proteins located in this organelle, suggesting a possible role of these cell structure in the synthesis of sterols. GO analysis showed an enrichment of pathways involved in amino acid transport and peptidase activity (for increased genes) and proteins nucleosomes (genes decreased), as well as drug-specific responses. The data generated constitute the first comprehensive assessment of the response of parasites to these drugs, and their analysis in conjunction with other tests represents a rich approach to the deeper understanding of the molecular changes that occur through the inhibition of the ergosterol biosynthesis. Although the amount of data generated in this work is not sufficiently robust reguloma analysis, preliminary results show that this methodology allows a very powerful and integrated understanding of the basic biology of Trypanosoma cruzi, mainly in relation to posttranscriptional mechanisms of gene expression. The inclusion of data generated in this study on the larger reguloma base project represents an important advance in the identification of egulatory networks of gene expression

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

An Essential Nuclear Protein in Trypanosomes Is a Component of mRNA Transcription/Export Pathway

In eukaryotic cells, different RNA species are exported from the nucleus via specialized pathways. The mRNA export machinery is highly integrated with mRNA processing, and includes a different set of nuclear transport adaptors as well as other mRNA binding proteins, RNA helicases, and NPC-associated proteins. The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, a widespread and neglected human disease which is endemic to Latin America. Gene expression in Trypanosoma has unique characteristics, such as constitutive polycistronic transcription of protein-encoding genes and mRNA processing by trans-splicing. In general, post-transcriptional events are the major points for regulation of gene expression in these parasites. However, the export pathway of mRNA from the nucleus is poorly understood. The present study investigated the function of TcSub2, which is a highly conserved protein ortholog to Sub2/ UAP56, a component of the Transcription/Export (TREX) multiprotein complex connecting transcription with mRNA export in yeast/human. Similar to its orthologs, TcSub2 is a nuclear protein, localized in dispersed foci all over the nuclei —except the fibrillar center of nucleolus— and at the interface between dense and non-dense chromatin areas, proposing the association of TcSub2 with transcription/processing sites. These findings were analyzed further by BrUTP incorporation assays and confirmed that TcSub2 is physically associated with active RNA polymerase II (RNA pol II), but not RNA polymerase I (RNA pol I) or Spliced Leader (SL) transcription, demonstrating participation particularly in nuclear mRNA metabolism in T. cruzi. The double knockout of the TcSub2 gene is lethal in T. cruzi, suggesting it has an essential function. Alternatively, RNA interference assays were performed in Trypanosoma brucei. It allowed demonstrating that besides being an essential protein, its knockdown causes mRNA accumulation in the nucleus and decrease of translation levels, reinforcing that Trypanosoma-Sub2 (Tryp-Sub2) is a component of mRNA transcription/export pathway in trypanosomes

Differential Allocation of Constitutive and Induced Chemical Defenses in Pine Tree Juveniles: A Test of the Optimal Defense Theory

Optimal defense theory (ODT) predicts that the within-plant quantitative allocation of defenses is not random, but driven by the potential relative contribution of particular plant tissues to overall fitness. These predictions have been poorly tested on long-lived woody plants. We explored the allocation of constitutive and methyl-jasmonate (MJ) inducible chemical defenses in six half-sib families of Pinus radiata juveniles. Specifically, we studied the quantitative allocation of resin and polyphenolics (the two major secondary chemicals in pine trees) to tissues with contrasting fitness value (stem phloem, stem xylem and needles) across three parts of the plants (basal, middle and apical upper part), using nitrogen concentration as a proxy of tissue value. Concentration of nitrogen in the phloem, xylem and needles was found to be greater higher up the plant. As predicted by the ODT, the same pattern was found for the concentration of non-volatile resin in the stem. However, in leaf tissues the concentrations of both resin and total phenolics were greater towards the base of the plant. Two weeks after MJ application, the concentrations of nitrogen in the phloem, resin in the stem and total phenolics in the needles increased by roughly 25% compared with the control plants, inducibility was similar across all plant parts, and families differed in the inducibility of resin compounds in the stem. In contrast, no significant changes were observed either for phenolics in the stems, or for resin in the needles after MJ application. Concentration of resin in the phloem was double that in the xylem and MJ-inducible, with inducibility being greater towards the base of the stem. In contrast, resin in the xylem was not MJ-inducible and increased in concentration higher up the plant. The pattern of inducibility by MJ-signaling in juvenile P. radiata is tissue, chemical-defense and plant-part specific, and is genetically variable

Slaughter weight rather than sex affects carcass cuts and tissue composition of Bisaro pigs

Carcass cuts and tissue composition were assessed in Bisaro pigs (n=64) from two sexes (31 gilts and 33 entire males) reared until three target slaughter body-weights (BW) means: 17 kg, 32 kg, and 79 kg. Dressing percentage and backfat thickness increased whereas carcass shrinkage decreased with increasing BW. Slaughter weight affected most of the carcass cut proportions, except shoulder and thoracic regions. Bone proportion decreased linearly with increasing slaughter BW, while intermuscular and subcutaneous adipose tissue depots increased concomitantly. Slaughter weight increased the subcutaneous adipose tissue proportion but this impaired intramuscular and intermuscular adipose tissues in the loin primal. The sex of the pigs minimally affected the carcass composition, as only the belly weight and the subcutaneous adipose tissue proportions were greater in gilts than in entire males. Light pigs regardless of sex are recommended to balance the trade-offs between carcass cuts and their non-edible compositional outcomes.Work included in the Portuguese PRODER research Project BISOPORC – Pork extensive production of Bísara breed, in two alternative systems: fattening on concentrate vs chesnut, Project PRODER SI I&DT Medida 4.1 “Cooperação para a Inovação”. The authors are grateful to Laboratory of Carcass and Meat Quality of Agriculture School of Polytechnic Institute of Bragança ‘Cantinho do Alfredo’. The authors are members of the MARCARNE network, funded by CYTED (ref. 116RT0503).info:eu-repo/semantics/publishedVersio

An estimate of the number of tropical tree species

The high species richness of tropical forests has long been recognized, yet there remains substantial uncertainty regarding the actual number of tropical tree species. Using a pantropical tree inventory database from closed canopy forests, consisting of 657,630 trees belonging to 11,371 species, we use a fitted value of Fisher’s alpha and an approximate pantropical stem total to estimate the minimum number of tropical forest tree species to fall between ∼40,000 and ∼53,000, i.e. at the high end of previous estimates. Contrary to common assumption, the Indo-Pacific region was found to be as species-rich as the Neotropics, with both regions having a minimum of ∼19,000–25,000 tree species. Continental Africa is relatively depauperate with a minimum of ∼4,500–6,000 tree species. Very few species are shared among the African, American, and the Indo-Pacific regions. We provide a methodological framework for estimating species richness in trees that may help refine species richness estimates of tree-dependent taxa

The First Post-Kepler Brightness Dips of KIC 8462852

We present a photometric detection of the first brightness dips of the unique variable star KIC 8462852 since the end of the Kepler space mission in 2013 May. Our regular photometric surveillance started in October 2015, and a sequence of dipping began in 2017 May continuing on through the end of 2017, when the star was no longer visible from Earth. We distinguish four main 1-2.5% dips, named "Elsie," "Celeste," "Skara Brae," and "Angkor", which persist on timescales from several days to weeks. Our main results so far are: (i) there are no apparent changes of the stellar spectrum or polarization during the dips; (ii) the multiband photometry of the dips shows differential reddening favoring non-grey extinction. Therefore, our data are inconsistent with dip models that invoke optically thick material, but rather they are in-line with predictions for an occulter consisting primarily of ordinary dust, where much of the material must be optically thin with a size scale <<1um, and may also be consistent with models invoking variations intrinsic to the stellar photosphere. Notably, our data do not place constraints on the color of the longer-term "secular" dimming, which may be caused by independent processes, or probe different regimes of a single process

Consistent patterns of common species across tropical tree communities

Trees structure the Earth’s most biodiverse ecosystem, tropical forests. The vast number of tree species presents a formidable challenge to understanding these forests, including their response to environmental change, as very little is known about most tropical tree species. A focus on the common species may circumvent this challenge. Here we investigate abundance patterns of common tree species using inventory data on 1,003,805 trees with trunk diameters of at least 10 cm across 1,568 locations1,2,3,4,5,6 in closed-canopy, structurally intact old-growth tropical forests in Africa, Amazonia and Southeast Asia. We estimate that 2.2%, 2.2% and 2.3% of species comprise 50% of the tropical trees in these regions, respectively. Extrapolating across all closed-canopy tropical forests, we estimate that just 1,053 species comprise half of Earth’s 800 billion tropical trees with trunk diameters of at least 10 cm. Despite differing biogeographic, climatic and anthropogenic histories7, we find notably consistent patterns of common species and species abundance distributions across the continents. This suggests that fundamental mechanisms of tree community assembly may apply to all tropical forests. Resampling analyses show that the most common species are likely to belong to a manageable list of known species, enabling targeted efforts to understand their ecology. Although they do not detract from the importance of rare species, our results open new opportunities to understand the world’s most diverse forests, including modelling their response to environmental change, by focusing on the common species that constitute the majority of their trees.Publisher PDFPeer reviewe

Treatment of Trypanosoma cruzi with 2-bromopalmitate alters morphology, endocytosis, differentiation and infectivity

Abstract Background The palmitate analogue 2-bromopalmitate (2-BP) is a non-selective membrane tethered cysteine alkylator of many membrane-associated enzymes that in the last years emerged as a general inhibitor of protein S-palmitoylation. Palmitoylation is a post-translational protein modification that adds palmitic acid to a cysteine residue through a thioester linkage, promoting membrane localization, protein stability, regulation of enzymatic activity, and the epigenetic regulation of gene expression. Little is known on such important process in the pathogenic protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. Results The effect of 2-BP was analyzed on different developmental forms of Trypanosoma cruzi. The IC50/48 h value for culture epimastigotes was estimated as 130 μM. The IC50/24 h value for metacyclic trypomastigotes was 216 nM, while for intracellular amastigotes it was 242 μM and for cell derived trypomasigotes was 262 μM (IC50/24 h). Our data showed that 2-BP altered T. cruzi: 1) morphology, as assessed by bright field, scanning and transmission electron microscopy; 2) mitochondrial membrane potential, as shown by flow cytometry after incubation with rhodamine-123; 3) endocytosis, as seen after incubation with transferrin or albumin and analysis by flow cytometry/fluorescence microscopy; 4) in vitro metacyclogenesis; and 5) infectivity, as shown by host cell infection assays. On the other hand, lipid stress by incubation with palmitate did not alter epimastigote growth, metacyclic trypomastigotes viability or trypomastigote infectivity. Conclusion Our results indicate that 2-BP inhibits key cellular processes of T. cruzi that may be regulated by palmitoylation of vital proteins and suggest a metacyclic trypomastigote unique target dependency during the parasite development