Highly dynamic host actin reorganization around developing Plasmodium inside hepatocytes

Plasmodium sporozoites are transmitted by Anopheles mosquitoes and infect hepatocytes, where a single sporozoite replicates into thousands of merozoites inside a parasitophorous vacuole. The nature of the Plasmodium-host cell interface, as well as the interactions occurring between these two organisms, remains largely unknown. Here we show that highly dynamic hepatocyte actin reorganization events occur around developing Plasmodium berghei parasites inside human hepatoma cells. Actin reorganization is most prominent between 10 to 16 hours post infection and depends on the actin severing and capping protein, gelsolin. Live cell imaging studies also suggest that the hepatocyte cytoskeleton may contribute to parasite elimination during Plasmodium development in the liver.

Live and let die: manipulation of host hepatocytes by exoerythrocytic Plasmodium parasites

The generation of rodent Plasmodium strains expressing fluorescent proteins in all life cycle stages has had a big impact on malaria research. With this tool in hand, for the first time it was possible to follow in real time by in vivo microscopy the infection route of Plasmodium sporozoites transmitted to the mammalian host by Anopheles mosquitoes. Recently, this work has been extended to the analysis of both hepatocyte infection by Plasmodium sporozoites, as well as liver merozoite transport into blood vessels. The stunning results of these studies have considerably changed our understanding of hepatocyte invasion and parasite liberation. Here, we describe the most important findings of the last years and in addition, we elaborate on the molecular events during the intracellular development of Plasmodium exoerythrocytic forms that give rise to erythrocyte infecting merozoites.

Imaging liver-stage malaria parasites

Plasmodium parasites, the causative agents of malaria, first invade and develop within hepatocytes before infecting red blood cells and causing symptomatic disease. Because of the low infection rates in vitro and in vivo, the liver stage of Plasmodium infection is not very amenable to biochemical assays, but the large size of the parasite at this stage in comparison with Plasmodium blood stages makes it accessible to microscopic analysis. A variety of imaging techniques has been used to this aim, ranging from electron microscopy to widefield epifluorescence and laser scanning confocal microscopy. High-speed live video microscopy of fluorescent parasites in particular has radically changed our view on key events in Plasmodium liver-stage development. This includes the fate of motile sporozoites inoculated by Anopheles mosquitoes as well as the transport of merozoites within merosomes from the liver tissue into the blood vessel. It is safe to predict that in the near future the application of the latest microscopy techniques in Plasmodium research will bring important insights and allow us spectacular views of parasites during their development in the liver.

Clave ilustrada para los mosquitos anofelinos de Sudamérica oriental

Bibliography: p. 64.

CYP2D6: a global analysis of phenotypic and genotypic variation in search of radical cure of Plasmodium vivax malaria

Thesis (Master's)--University of Washington, 2016-06

Ultraconserved elements in insect genomes: A highly conserved intronic sequence implicated in the control of homothorax mRNA splicing

Recently, we identified a large number of ultraconserved (uc) sequences in noncoding regions of human, mouse, and rat genomes that appear to be essential for vertebrate and amniote ontogeny. Here, we used similar methods to identify ultraconserved genomic regions between the insect species Drosophila melanogaster and Drosophila pseudoobscura, as well as the more distantly related Anopheles gambiae. As with vertebrates, ultraconserved sequences in insects appear to Occur primarily in intergenic and intronic sequences, and at intron-exon junctions. The sequences are significantly associated with genes encoding developmental regulators and transcription factors, but are less frequent and are smaller in size than in vertebrates. The longest identical, nongapped orthologous match between the three genomes was found within the homothorax (hth) gene. This sequence spans an internal exon-intron junction, with the majority located within the intron, and is predicted to form a highly stable stem-loop RNA structure. Real-time quantitative PCR analysis of different hth splice isoforms and Northern blotting showed that the conserved element is associated with a high incidence of intron retention in hth pre-mRNA, suggesting that the conserved intronic element is critically important in the post-transcriptional regulation of hth expression in Diptera.

Molecular and biological characterisation of orthobunyaviruses

Orthobunyaviruses are the largest genus within the Bunyaviridae family, with over 170 named viruses classified into 18 serogroups (Elliott and Blakqori, 2001; Plyusnin et al., 2012). Orthobunyaviruses are transmitted by arthropods and have a tripartite negative sense RNA genome, which encodes 4 structural proteins and 2 non-structural proteins. The non-structural protein NSs is the primary virulence factor of orthobunyaviruses and potent antagonist of the type I interferon (IFN) response. However, sequencing studies have identified pathogenic viruses that lack the NSs protein (Mohamed et al., 2009; Gauci et al., 2010). The work presented in this thesis describes the molecular and biological characterisation of divergent orthobunyaviruses. Data on plaque morphology, growth kinetics, protein profiles, sensitivity to IFN and activation of the type I IFN system are presented for viruses in the Anopheles A, Anopheles B, Capim, Gamboa, Guama, Minatitlan, Nyando, Tete and Turlock serogroups. These are complemented with complete genome sequencing and phylogenetic analysis. Low activation of IFN by Tete serogroup viruses, which naturally lack an NSs protein, was also further investigated by the development of a reverse genetics system for Batama virus (BMAV). Recombinant viruses with mutations in the virus nucleocapsid protein amino terminus showed higher activation of type I IFN in vitro and data suggests that low levels of IFN are due to lower activation rather than active antagonism. The anti-orthobunyavirus activity of IFN-stimulated genes IFI44, IFITMs and human and ovine BST2 were also studied, revealing that activity varies not only within the orthobunyavirus genus and virus serogroups but also within virus species. Furthermore, there was evidence of active antagonism of the type I IFN response and ISGs by non-NSs viruses. In summary, the results show that pathogenicity in man and antagonism of the type I IFN response in vitro cannot be predicted by the presence, or absence, of an NSs ORF. They also highlight problems in orthobunyavirus classification with discordance between classical antigen based data and phylogenetic analysis.

Design and synthesis of novel quinolones directed to the Plasmodium falciparum bc1 protein complex

Tese de Doutoramento, Química, Especialização em Química Orgânica, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2016

Distribuição espaço - temporal da malária no Piauí de 2002 a 2013.

A malária é uma doença infecciosa, aguda, febril transmitida pela picada da fêmea de mosquito do gênero Anopheles. O estudo teve como objetivo realizar análise da distribuição espaço-temporal dos casos de malária no Piauí entre 2002 a 2013. Nesta pesquisa foi utilizada a taxa de incidência cumulativa para análise dos casos e o Sistema de Informação Geográfico (SIG) para mapeamento espacial. Os resultados obtidos por meio do cálculo da taxa de Incidência Cumulativa (IC) demonstraram que os municípios de Campo Largo do Piauí, Matias Olímpio, Luzilândia, Madeiro e Porto foram os que apresentaram maior IC de malária (>15,4/100.000 hab.). As pessoas mais acometidas foram as residentes na área rural, sendo pouquíssimos os casos ocorridos na zona urbana dos municípios. Através da utilização do SIG identificou-se que o padrão de distribuição espaçotemporal dos surtos de casos de malária está concentrado na porção noroeste do estado, na fronteira com o Maranhão. Após as análises, conclui-se que todos os 74 municípios que registraram malária no Piauí na série temporal do estudo, estão localizados e aglomerados em área vulnerável e receptiva para a transmissão da doença.

Opiliões (Arachnida: Opiliones) em remanescente florestal no município de Plácido de Castro, AC.

Os opiliões (Arachnida: Opiliones) são aracnídeos com ampla distribuição geográfica, sendo a maior parte das espécies registradas na região Neotropical. Esses organismos possuem baixa capacidade de dispersão, alto grau de endemismo e sensibilidade às mudanças ambientais, também participam da ciclagem da matéria orgânica do solo, visto que muitas espécies possuem o hábito alimentar detritívoro. Dada a importância desses organismos, objetivou-se com esse trabalho prospectar a fauna de opiliões em remanescente florestal localizado no município de Plácido de Castro, AC. Foram utilizados dois métodos de captura: armadilhas pitfall (captura passiva), dispostas em dois transectos, e procura livre noturna (coleta ativa). As armadilhas pitfall consistiram em copos plásticos de 500 mL, contendo uma solução de formaldeído a 1% e gotas de detergente neutro, sendo recolhidas semanalmente. As coletas ativas mensais foram realizadas por quatro pessoas, em um esforço de coleta de 1 h. As espécies de opiliões capturadas foram: Paecilaema marajoara, Paraprotus quadripunctatus, Taito kakera e Cynorta sp. (Cosmetidae); Geaya sp. (Sclerosomatidae) e quatro adultos e um imaturo, pertencentes à família Manaosbiidae. Todos os opiliões registrados nesse levantamento foram capturados pelo método de ?procura livre noturna?, sendo os gêneros Geaya e Cynorta, bem como a espécie P. quadripunctatus, registrados pela primeira vez no estado do Acre.