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Aedes aegypti mosquitoes are the vectors of many significant arboviruses that cause tremendous social and economic impact. RNA interference (RNAi) plays a crucial role in the vector competence of mosquitoes and is often targeted in studies involving mosquito innate immunity, genetics-based vector control strategies, and the development of viral-resistant transgenic mosquitoes. In general, RNA interference is induced by double stranded RNA (dsRNA) and results in the inhibition of cognate gene expression. There are several different RNA interference pathways, with distinct functions and mechanisms. The micro RNA pathway is important for endogenous gene regulation and development. The endogenous small interfering RNA (endo-siRNA) pathway functions in gene regulation and protection of the genome from the deleterious effects of transposable elements. The exogenous siRNA (exosiRNA) pathway is a major contributor to mosquito innate immunity and vector competence by limiting viral replication during infection. Lastly, the piwi RNA (piRNA) pathway primarily functions in protecting the genome from the deleterious effects of transposable elements. While the structure and function of many genes involved in Drosophila RNAi have been characterized, the corresponding mosquito orthologs have only been peripherally described or remain unknown. Thus, the overall purpose of this study is to improve the understanding of mosquito RNAi mechanisms by identifying and analyzing genetic factors involved in the various pathways. This research especially focuses on characterizing and analyzing putative doubleiii stranded RNA binding proteins (dsRBPs) important to the function of the RNAi initiator and effector complexes. Two genes, r2d2 and r3d1 are orthologs of Drosophila genes known to have important roles in the RNAi initiator complex. A third member of the same family, which we refer to as extra loquacious (exloqs), appears to have no known orthologs outside of the Aedes genus. Structural characterization of these genes included identification of single nucleotide polymorphisms (SNPs), novel exons and alternative splice variants. RT-PCR assays were utilized to examine differential expression of all three genes in specific tissues and developmental stages. Sub-cellular fractionation assays enabled intracellular localization of the RNAi proteins within Ae. aegypti cells. Co-immunoprecipitation of tagged dsRBPs revealed protein-protein interactions between specific dsRBPs and known RNAi factors. In addition, an exo-siRNA sensor was designed and tested in-vivo and in-vitro with the purpose of facilitating the identification of novel genetic factors involved in this anti-viral pathway. Lastly, TALENbased gene disruption was successfully employed to knockout the exloqs gene in Ae. aegypti mosquitoes, enabling further analysis into the function of this gene. The research described in this document provides further insight into mosquito innate immunity and gene regulation, which is important to the advancement of genetics-based vector control strategies. / Ph. D.

Reduced access to atmospheric oxygen greatly increased early instar Aedes aegypti mortality. Data suggest that death was by drowning mainly because slow swimming speed and low visual acuity delayed larvae from finding access to air. Late instar larvae experienced developmental delays when food depletion in the immediate vicinity of the breathing site forced them to forage farther away. As the distance between feeding and breathing sites increased, feeding efficiency of fourth instars may have declined enough to reduce their pupation success and to cause increased pupal mortality, reduced adult emergence and sex ratio skewed in favour of males among emerging functional adults. Thus reduced access to atmospheric oxygen at the pre-imago level may reduce the vectorial capacity of emerging adult population.

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Submitted by Leon Diniz Alves (leonalves@fgvmail.br) on 2016-07-26T00:03:14Z No. of bitstreams: 1 Dissertação - Versão Final.pdf: 35080956 bytes, checksum: 344aceb862a811a2808e2fc8384f502a (MD5) / Approved for entry into archive by Maria Almeida (maria.socorro@fgv.br) on 2016-08-02T14:47:55Z (GMT) No. of bitstreams: 1 Dissertação - Versão Final.pdf: 35080956 bytes, checksum: 344aceb862a811a2808e2fc8384f502a (MD5) / Approved for entry into archive by Maria Almeida (maria.socorro@fgv.br) on 2016-08-02T14:48:11Z (GMT) No. of bitstreams: 1 Dissertação - Versão Final.pdf: 35080956 bytes, checksum: 344aceb862a811a2808e2fc8384f502a (MD5) / Made available in DSpace on 2016-08-02T14:48:54Z (GMT). No. of bitstreams: 1 Dissertação - Versão Final.pdf: 35080956 bytes, checksum: 344aceb862a811a2808e2fc8384f502a (MD5) Previous issue date: 2016-04-29 / O objetivo deste trabalho é prover um aplicativo de celular e um protocolo para aquisição de imagens para contagem dos ovos de Aedes aegypti com as seguintes características: facilidade de uso, alta acurácia e custo baixo. O mosquito Ae. aegypti, popularmente conhecido como mosquito da dengue, é um importante vetor de arboviroses como a própria dengue, a chikungunya, a zika e a febre amarela em seu ciclo urbano. O monitoramento entomológico é uma maneira de melhorar a capacidade de predição e na detecção precoce de epidemias das doenças mencionadas. Este monitoramento é majoritariamente baseado no índice larvário, o qual lista a quantidade de casas infectadas, ou a quantidade de ovos de Aedes coletados em palhetas em ovitrampas. Estas palhetas são normalmente de eucatex, mas existem pesquisas atuais testando o algodão.A contagem dos ovos coletados em ovitrampas é feita manualmente, a qual demanda tempo, profissionais qualificados para o manuseio de equipamento laboratorial (lupas e microscópios) e conhecimento entomológico. Buscou-se criar um método para acelerar o trabalho feito pelos profissionais em controle entomológico. A metodologia contou com a criação de um aplicativo e com um processo de contagem dos ovos, o qual consiste em quatro passos: a) Fotografar as palhetas em ovitrampas utilizando de uma câmera de celular; b) Transformar as fotos em uma imagem binarizada, removendo todos os elementos que não são ovos; c) Contar a área de cada elemento; d) A partir do uso de um classificador especialmente desenvolvido, estimar a quantidade de ovos baseado na área de cada elemento. Nos resultados, foi possível notar que houve uma disparidade na contagem de ovos em palhetas de algodão, a qual teve um erro médio próximo a zero, em relação às palhetas de eucatex, as quais tiveram erro médio acima de 5\%. Dos pontos mais importantes das conclusões, destacam-se a possibilidade de melhoria contínua do aplicativo por permanecer na nuvem, com possibilidade de avanços conforme novas descobertas, assim como o excelente custo-benefício obtido, por conseguir operar com baixo custo monetário.

Made available in DSpace on 2016-04-12T12:32:35Z (GMT). No. of bitstreams: 2 574.pdf: 4167257 bytes, checksum: 48c5b8ff9f1aae0fa414559c4af159c1 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, Brasil / Os altos índices de infestação pelo Aedes aegypti registrados em Santa Cruz do Capibaribe-PE (IIP = 5,8 a 13,2 por cento) durante os ciclos bimestrais do Programa de Controle da Dengue, desde 1997, mostraram a necessidade de se buscar novos métodos de abordagem objetivando resultados que apresentem maior impacto sobre este vetor. Neste sentido um Sistema de Monitoramento e Controle Populacional do Aedes aegypti (SMCP-Aedes), desenvolvido pela rede SAUDAVEL (Sistema de Apoio Unificado para Detecção e Acompanhamento em Vigilância Epidemiológica), vem sendo testado no município, desde 2008, em parceria com a Secretaria Estadual de Saúde e a Fiocruz/PE. O sistema inclui: a) coleta mensal de palhetas com ovos depositados em 262 ovitrampas-sentinela (OVT-S) georreferenciadas; b) contagem dos ovos, com repasse das informações para o banco de dados geográficos-SAUDAVEL que analisa e gera mapas de densidades; c) remoção mecânica massiva de ovos e de alados do ambiente, utilizando ovitrampas-controle (OVT-C) e aspiradores, em locais priorizados com base na vigilância entomológica; Os resultados da aplicação do SMCP-Aedes, como piloto, em Santa Cruz do Capibaribe (maio/2008 a abril/2010), permitiram uma compreensão mais clara da ocupação do território urbano pelo vetor, revelando altos níveis de infestação com áreas de maior concentração populacional do Aedes. O sistema, muito sensível para detectar e distinguir baixa e altíssimas densidades permitiu medir o impacto das ações integradas de controle mecânico, que resultaram em eliminação de 4,5 milhões de ovos de Aedes em seis meses e 10.819 culicídeos adultos do ambiente, causando redução importante da densidade do vetor. As ferramentas e estratégias empregadas neste sistema favorecem a compreensão da biologia do vetor pelos habitantes. A inserção dessas ferramentas permitiu melhor atuação daequipe responsável pelo controle da doença

BACKGROUND:The major Dengue virus vector Aedes aegypti requires nutrients obtained from blood meal proteins to complete the gonotrophic cycle. Although bioinformatic analyses of Ae. aegypti midgut serine proteases have provided evolutionary insights, very little is known about the biochemical activity of these digestive enzymes.RESULTS:We used peptide specific antibodies to show that midgut serine proteases are expressed as zymogen precursors, which are cleaved to the mature form after blood feeding. Since midgut protein levels are insufficient to purify active proteases directly from blood fed mosquitoes, we engineered recombinant proteins encoding a heterologous enterokinase cleavage site to permit generation of the bona fide mature form of four midgut serine proteases (AaET, AaLT, AaSPVI, AaSPVII) for enzyme kinetic analysis. Cleavage of the chromogenic trypsin substrate BApNA showed that AaET has a catalytic efficiency (kcat/KM) that is ~30 times higher than bovine trypsin, and ~2-3 times higher than AaSPVI and AaSPVII, however, AaLT does not cleave BApNA. To measure the enzyme activities of the mosquito midgut proteases using natural substrates, we developed a quantitative cleavage assay based on cleavage of albumin and hemoglobin proteins. These studies revealed that the recombinant AaLT enzyme was indeed catalytically active, and cleaved albumin and hemoglobin with equivalent efficiency to that of AaET, AaSPVI, and AaSPVII. Structural modeling of the AaLT and AaSPVI mature forms indicated that AaLT is most similar to serine collagenases, whereas AaSPVI appears to be a classic trypsin.CONCLUSIONS:These data show that in vitro activation of recombinant serine proteases containing a heterologous enterokinase cleavage site can be used to investigate enzyme kinetics and substrate cleavage properties of biologically important mosquito proteases.

Master of Science / Division of Biology / Rollie J. Clem / Caspases are a conserved family of cysteine proteases that play important roles in apoptosis and innate immunity as well as other cellular processes. Eleven caspase genes have been annotated in the mosquito Aedes aegypti. Amongst these, previous studies have demonstrated functional roles for AeDronc, CASPS7 and CASPS8 in the Ae. aegypti apoptosis pathway, while CASPS18 and CASPS19 have also been functionally characterized. A previous study from our research group showed that AeIAP1 has preferential binding for CASPS7 compared to CASPS8. In this study, it was confirmed that AeIAP1 has a higher capacity to inhibit CASPS7 than CASPS8. Furthermore, five of the remaining Ae. aegypti caspases, namely CASPS15, CASPS16, CASPS17, CASPS20 and CASPS21, were characterized. An attempt was made to classify these caspases as initiator or effector caspases, based on factors such as the length of their prodomain, sequence similarity to known Drosophila initiator and effector caspases, and their substrate specificity. The functions of these caspases in apoptosis was examined in the Ae. aegypti cell line Aag2, by using RNA interference to reduce their expression and test the effect on apoptosis. Recombinant CASPS16, 17, 20 and 21 were produced in bacteria and the abilities of these recombinant proteins to cleave different caspase substrates were examined. From the resulting data, it was concluded that CASPS17 and CASPS21 are likely to be effector caspases since they preferred a effector caspase substrate. When considering the prodomain length, CASPS17 has a short prodomain, but CASPS21 has a long prodomain, which is normally associated with initiator caspases. CASPS20 did not show preference for any specific substrate and has a short prodomain. Since it did not have a specific preference of substrate, it is likely to be an effector caspase based on prodomain length. CASPS16 showed a slightly higher preference for the initiator caspase substrate WEHD, and has a long prodomain. Based on these results, CASPS16 is likely an initiator caspase. To examine the potential roles of CASPS15, 16, 17, 20 and 21 in apoptosis, their expression in Aag2 cells was knocked down using RNA interference. Successful knockdown was verified by qRT-PCR. After silencing specific caspases, the cells were exposed to two different apoptotic stimuli, ultraviolet radiation (UV) or the RNA synthesis inhibitor actinomycin D (ActD). Following apoptotic treatment, apoptosis was measured by two methods; caspase activity was measured using an effector caspase substrate, and phosphatidyl serine exposure on the outer leaflet of the plasma membrane, which occurs in apoptotic cells, was measured by Annexin V staining and flow cytometry. In cells where CASPS15, 16, 17, 20 or 21 had been knocked down and the cells were then treated with UV or ActD, it was observed that effector caspase activity and Annexin V staining were both significantly lower than in UV- or ActD-treated cells that had received control double-stranded RNA. Together these results suggest that all of these caspases are involved in apoptosis in Aag2 cells. This study serves as a starting point for further research on Ae. aegypti caspases and their roles in specific cellular processes.