Localization of MRNA storage complexes in Plasmodium berghei throughout the life cycle

Tese de mestrado. Biologia (Microbiologia Aplicada). Universidade de Lisboa, Faculdade de Ciências, 2011Plasmodium is the causative agent of malaria, a disease that caused 781 thousands deaths during the year of 2009. The apicomplexan responsible for this disease have shown to be very well adapted to both mosquito and Vertebrate hosts, regulating its gene expression often in a posttranscriptional manner. Recently the protein HoMu and the translation initiation factor eIF4E (4E) were found forming complexes with translationally arrested mRNAs (e.g. transcripts encoding the surface proteins p25 and p28). Storage of these mRNAs in the female gametocyte will permit the parasite to have them readily available to be translated once it enters into the mosquito. Using bioinformatics tools we showed that HoMu and 4E are conserved proteins. Their homologs are distributed in a wide range of species and participate in both translation initiation and arrest of transcripts containing specific sequences in their 3’ untranslated regions (UTRs). The main purpose of this study was to understand when those proteins are expressed during the Plasmodium berghei life cycle and where they are localized. We also show that parasite lines expressing GFP fused to HoMu and 4E are able to establish infection with normal parasitological patterns compared to the wild type. By life cell imaging of these mutant lines we find that HoMu and 4E are strongly expressed in gametocytes, oocysts, and sporozoites suggesting that they are important during the transmission phases of the P.berghei life cycle.A malária é uma doença causada pelo parasita unicelular Plasmodium e transmitida pela picada do mosquito Anopheles. Segundo os dados da organização mundial de saúde (OMS) esta doença vitimou em 2009 cerca de 780 mil pessoas, sendo a maioria crianças que habitavam na África subsariana. São quatro as espécies deste parasita que infectam o Homem: Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, e Plasmodium falciparum, sendo a última a causadora da forma mais severa da doença conhecida como malária cerebral. Actualmente a investigação em parasitologia molecular procura desvendar quais os processos celulares que estão por de trás da interacção deste parasita com o hospedeiro. Como tal e visto ser ainda difícil reproduzir todo o ciclo de vida de Plasmodium em laboratório é necessário utilizar um hospedeiro passível de ser mantido em cativeiro e que sirva de modelo para o estudo da malária. A malária infecta também roedores e é sabido que as espécies do parasita que os infectam partilham elevado número de genes com as espécies da malária humana. Como tal é muitas vezes utilizado em investigação o parasita de roedores Plasmodium berghei. O parasita da malária caracteriza-se pela sua rápida adaptação ao hospedeiro. Durante a fase sanguínea o parasita invade os eritrócitos e diferencia-se em células precursoras dos gâmetas, os gametócitos. Estas células caracterizam-se por possuir complexos contendo mRNA e proteínas específicos no seu citoplasma onde a tradução é inibida. Pensa-se que estes complexos estão relacionados com o controlo da expressão génica no parasita da malária. De facto poucos factores de transcrição são ainda conhecidos em Plasmodium, por outro lado, as proteínas de ligação ao RNA são particularmente abundantes neste parasita. Estas observações reforçam a hipótese de que, em Plasmodium a regulação génica ser feita preferencialmente ao nível pós-transcricional. Em gametócitos femininos de Plasmodium foram identificadas ribonucleoproteínas (mRNP) como DOZI e CITH formando complexos onde o mRNA é armazenado. Ao gerar-se linhas celulares mutantes do parasita sem estas proteínas codificadas no seu genoma verificou-se que este não se diferenciava em oocinetos após a formação do zigoto. Fazendo parte dos mesmos complexos foram encontradas outras proteínas, entre elas uma proteína homóloga de Musashi (HoMu) e um factor de iniciação da tradução eIF4E (4E). Estas proteínas são o alvo deste estudo e possuem homologia com proteínas de diferentes espécies de seres vivos. HoMu por exemplo possui homologia com a proteína Musashi do sapo Xenopus. Neste animal a proteína Musashi liga-se ao mRNA mos de oócitos activando a sua tradução. Noutras espécies, no entanto, Musashi caracteriza-se por inibir a tradução de certos mRNAs, armazenando-os no citoplasma em complexos de mRNP. Já 4E por outro lado é um factor de iniciação da tradução em células eucariotas que juntamente com outros factores permite activar o mRNA para ser traduzido. No entanto esta proteína participa também em processos de inibição da tradução, integrando complexos de mRNP onde o mRNA é armazenado. Em Drosophila por exemplo a proteína Cup liga-se a 4E impedindo a formação do complexo de iniciação da tradução. Este projecto teve como objectivo principal identificar o padrão de expressão das proteínas HoMu e 4E durante o ciclo de vida do parasita P. berghei. Para isso foram utilizadas duas linhas celulares contendo os genes homu e eif4e de fusão com o gene gfp (green fluorescent protein) de forma a possibilitar a identificação da expressão de HoMu e 4E por análise da fluorescência. As duas proteínas revelaram ser expressas na fase sanguínea bem como no mosquito, sugerindo a importância de ambas no ciclo de vida de Plasmodium. Foi demonstrado neste projecto que as linhas celulares GFP possuem um desenvolvimento normal quando comparado com a linha selvagem. A cinética de crescimento do parasita mutante durante a fase sanguínea foi idêntica à do selvagem e os números de esporozoítos nas glândulas salivares do mosquito foram normais. Isto comprova que as linhas celulares GFP utilizadas neste projecto são viáveis para o estudo das proteínas HoMu e 4E. Finalmente, a análise bioinformática das regiões 3’ UTR (untranslated regions) dos quase 5000 genes codificantes de proteínas identificados até então em P. berghei revelou que muitos deles possuem sequências consensus nas quais as proteínas Musashi de outros seres vivos se ligam. Isto leva-nos a sugerir que o homologo de Musashi em Plasmodium possa também ligar-se às regiões 3’ UTR de diversos mRNAs inibindo ou não a tradução dos mesmos

Biodiversidade de fungos filamentosos em grãos de café cultivados em sistema orgânico convencional

The fruits of organically and conventionally produced coffee are subject to contamination from several species of fungi, and that may be related to poor beverage quality and mycotoxin production. The aim of this study was to identify the biodiversity of isolated filamentous fungi in the coffee beans produced on organic and conventional farms within the same area. Two hundred and twelve isolates belonging to eleven different genders were identified from the fifteen samples analyzed. The main gender found was Aspergillus, with isolation of fungi from the Sections Circumdati, Nigri, Flavi and Versicolores. The samples that obtained the greatest index of contamination were those that didn’t pass through the disinfection process with 1% sodium hypochlorite. The samples of coffee beans from organic cultivation exhibited the greatest degree of richness and diversity within a single location with very similar climatic conditions. Thus, organic coffee production requires greater care due to the increased incidence of filamentous fungi.Os frutos de café produzidos de forma orgânica ou convencional estão sujeitos à contaminação de diversas espécies de fungos que podem estar relacionados à má qualidade da bebida e à produção de micotoxinas. Realizou-se este estudo para identificar a biodiversidade de fungos filamentosos isolados nos grãos de café produzidos em fazendas orgânicase convencionais de uma mesma localidade. Das 15 amostras analisadas, foram identificados 212 isolados, pertencentes a 11 diferentes gêneros. O principal gênero encontrado foi o Aspergillus, sendo isolados fungos das Seções Circumdati, Nigri, Flavi e Versicolores. As amostras que obtiveram o maior índice de contaminação foram as que não passaram pelo processo de desinfecção com hipoclorito de sódio a 1%. As amostras de grãos de café de cultivo orgânico apresentaram o maior índice de riqueza e diversidade dentro de uma mesma localidade, com condições climáticas muito próximas. Sendo assim, a produção de café orgânico necessita de maiores cuidados devido ao aumento na incidência de fungos filamentosos

A list of land plants of Parque Nacional do Caparaó, Brazil, highlights the presence of sampling gaps within this protected area

Brazilian protected areas are essential for plant conservation in the Atlantic Forest domain, one of the 36 global biodiversity hotspots. A major challenge for improving conservation actions is to know the plant richness, protected by these areas. Online databases offer an accessible way to build plant species lists and to provide relevant information about biodiversity. A list of land plants of “Parque Nacional do Caparaó” (PNC) was previously built using online databases and published on the website "Catálogo de Plantas das Unidades de Conservação do Brasil." Here, we provide and discuss additional information about plant species richness, endemism and conservation in the PNC that could not be included in the List. We documented 1,791 species of land plants as occurring in PNC, of which 63 are cited as threatened (CR, EN or VU) by the Brazilian National Red List, seven as data deficient (DD) and five as priorities for conservation. Fifity-one species were possible new ocurrences for ES and MG states

COVID-19 outcomes in people living with HIV: Peering through the waves

Objective: To evaluate clinical characteristics and outcomes of COVID-19 patients infected with HIV, and to compare with a paired sample without HIV infection. Methods: This is a substudy of a Brazilian multicentric cohort that comprised two periods (2020 and 2021). Data was obtained through the retrospective review of medical records. Primary outcomes were admission to the intensive care unit, invasive mechanical ventilation, and death. Patients with HIV and controls were matched for age, sex, number of comorbidities, and hospital of origin using the technique of propensity score matching (up to 4:1). They were compared using the Chi-Square or Fisher's Exact tests for categorical variables and the Wilcoxon for numerical variables. Results: Throughout the study, 17,101 COVID-19 patients were hospitalized, and 130 (0.76%) of those were infected with HIV. The median age was 54 (IQR: 43.0;64.0) years in 2020 and 53 (IQR: 46.0;63.5) years in 2021, with a predominance of females in both periods. People Living with HIV (PLHIV) and their controls showed similar prevalence for admission to the ICU and invasive mechanical ventilation requirement in the two periods, with no significant differences. In 2020, in-hospital mortality was higher in the PLHIV compared to the controls (27.9% vs. 17.7%; p = 0.049), but there was no difference in mortality between groups in 2021 (25.0% vs. 25.1%; p > 0.999). Conclusions: Our results reiterate that PLHIV were at higher risk of COVID-19 mortality in the early stages of the pandemic, however, this finding did not sustain in 2021, when the mortality rate is similar to the control group

Geographic patterns of tree dispersal modes in Amazonia and their ecological correlates

Aim: To investigate the geographic patterns and ecological correlates in the geographic distribution of the most common tree dispersal modes in Amazonia (endozoochory, synzoochory, anemochory and hydrochory). We examined if the proportional abundance of these dispersal modes could be explained by the availability of dispersal agents (disperser-availability hypothesis) and/or the availability of resources for constructing zoochorous fruits (resource-availability hypothesis). Time period: Tree-inventory plots established between 1934 and 2019. Major taxa studied: Trees with a diameter at breast height (DBH) ≥ 9.55 cm. Location: Amazonia, here defined as the lowland rain forests of the Amazon River basin and the Guiana Shield. Methods: We assigned dispersal modes to a total of 5433 species and morphospecies within 1877 tree-inventory plots across terra-firme, seasonally flooded, and permanently flooded forests. We investigated geographic patterns in the proportional abundance of dispersal modes. We performed an abundance-weighted mean pairwise distance (MPD) test and fit generalized linear models (GLMs) to explain the geographic distribution of dispersal modes. Results: Anemochory was significantly, positively associated with mean annual wind speed, and hydrochory was significantly higher in flooded forests. Dispersal modes did not consistently show significant associations with the availability of resources for constructing zoochorous fruits. A lower dissimilarity in dispersal modes, resulting from a higher dominance of endozoochory, occurred in terra-firme forests (excluding podzols) compared to flooded forests. Main conclusions: The disperser-availability hypothesis was well supported for abiotic dispersal modes (anemochory and hydrochory). The availability of resources for constructing zoochorous fruits seems an unlikely explanation for the distribution of dispersal modes in Amazonia. The association between frugivores and the proportional abundance of zoochory requires further research, as tree recruitment not only depends on dispersal vectors but also on conditions that favour or limit seedling recruitment across forest types

Geography and ecology shape the phylogenetic composition of Amazonian tree communities

AimAmazonia hosts more tree species from numerous evolutionary lineages, both young and ancient, than any other biogeographic region. Previous studies have shown that tree lineages colonized multiple edaphic environments and dispersed widely across Amazonia, leading to a hypothesis, which we test, that lineages should not be strongly associated with either geographic regions or edaphic forest types.LocationAmazonia.TaxonAngiosperms (Magnoliids; Monocots; Eudicots).MethodsData for the abundance of 5082 tree species in 1989 plots were combined with a mega-phylogeny. We applied evolutionary ordination to assess how phylogenetic composition varies across Amazonia. We used variation partitioning and Moran's eigenvector maps (MEM) to test and quantify the separate and joint contributions of spatial and environmental variables to explain the phylogenetic composition of plots. We tested the indicator value of lineages for geographic regions and edaphic forest types and mapped associations onto the phylogeny.ResultsIn the terra firme and várzea forest types, the phylogenetic composition varies by geographic region, but the igapó and white-sand forest types retain a unique evolutionary signature regardless of region. Overall, we find that soil chemistry, climate and topography explain 24% of the variation in phylogenetic composition, with 79% of that variation being spatially structured (R2 = 19% overall for combined spatial/environmental effects). The phylogenetic composition also shows substantial spatial patterns not related to the environmental variables we quantified (R2 = 28%). A greater number of lineages were significant indicators of geographic regions than forest types.Main ConclusionNumerous tree lineages, including some ancient ones (>66 Ma), show strong associations with geographic regions and edaphic forest types of Amazonia. This shows that specialization in specific edaphic environments has played a long-standing role in the evolutionary assembly of Amazonian forests. Furthermore, many lineages, even those that have dispersed across Amazonia, dominate within a specific region, likely because of phylogenetically conserved niches for environmental conditions that are prevalent within regions

Geography and ecology shape the phylogenetic composition of Amazonian tree communities

Aim: Amazonia hosts more tree species from numerous evolutionary lineages, both young and ancient, than any other biogeographic region. Previous studies have shown that tree lineages colonized multiple edaphic environments and dispersed widely across Amazonia, leading to a hypothesis, which we test, that lineages should not be strongly associated with either geographic regions or edaphic forest types. Location: Amazonia. Taxon: Angiosperms (Magnoliids; Monocots; Eudicots). Methods: Data for the abundance of 5082 tree species in 1989 plots were combined with a mega-phylogeny. We applied evolutionary ordination to assess how phylogenetic composition varies across Amazonia. We used variation partitioning and Moran\u27s eigenvector maps (MEM) to test and quantify the separate and joint contributions of spatial and environmental variables to explain the phylogenetic composition of plots. We tested the indicator value of lineages for geographic regions and edaphic forest types and mapped associations onto the phylogeny. Results: In the terra firme and várzea forest types, the phylogenetic composition varies by geographic region, but the igapó and white-sand forest types retain a unique evolutionary signature regardless of region. Overall, we find that soil chemistry, climate and topography explain 24% of the variation in phylogenetic composition, with 79% of that variation being spatially structured (R$^{2}$ = 19% overall for combined spatial/environmental effects). The phylogenetic composition also shows substantial spatial patterns not related to the environmental variables we quantified (R$^{2}$ = 28%). A greater number of lineages were significant indicators of geographic regions than forest types. Main Conclusion: Numerous tree lineages, including some ancient ones (>66 Ma), show strong associations with geographic regions and edaphic forest types of Amazonia. This shows that specialization in specific edaphic environments has played a long-standing role in the evolutionary assembly of Amazonian forests. Furthermore, many lineages, even those that have dispersed across Amazonia, dominate within a specific region, likely because of phylogenetically conserved niches for environmental conditions that are prevalent within regions

Mapping density, diversity and species-richness of the Amazon tree flora

Using 2.046 botanically-inventoried tree plots across the largest tropical forest on Earth, we mapped tree species-diversity and tree species-richness at 0.1-degree resolution, and investigated drivers for diversity and richness. Using only location, stratified by forest type, as predictor, our spatial model, to the best of our knowledge, provides the most accurate map of tree diversity in Amazonia to date, explaining approximately 70% of the tree diversity and species-richness. Large soil-forest combinations determine a significant percentage of the variation in tree species-richness and tree alpha-diversity in Amazonian forest-plots. We suggest that the size and fragmentation of these systems drive their large-scale diversity patterns and hence local diversity. A model not using location but cumulative water deficit, tree density, and temperature seasonality explains 47% of the tree species-richness in the terra-firme forest in Amazonia. Over large areas across Amazonia, residuals of this relationship are small and poorly spatially structured, suggesting that much of the residual variation may be local. The Guyana Shield area has consistently negative residuals, showing that this area has lower tree species-richness than expected by our models. We provide extensive plot meta-data, including tree density, tree alpha-diversity and tree species-richness results and gridded maps at 0.1-degree resolution