O'nyong-nyong virus (ONNV) is an Alphavirus (family: Togaviridae) transmitted by Anopheles gambiae (An. gambiae) mosquitoes. Alphavirus transmission causes a number of severe diseases in vertebrates. However, in mosquito hosts, infection rarely causes pathology. To date, little is known about the cellular responses that privilege mosquitoes with the ability to cope with infection. This study evaluated the genetic responses of An. gambiae to ONNV infection. We adopted a proteomics approach to identify proteins and molecular pathways that respond to virus infection in the midgut, the primary physical and biological barrier to infection. The sensitive mass spec-based technique, Tandem Mass Tagging (TMT; Thermo Scientific), was used to quantify and compare protein expression changes induced by the virus. Several proteins were identified that mediated virus infection, including an FK506 binding protein (FKBP). Evidence suggests FKBPs are phylogenetically conserved virus resistance factors that may exert their function via the PI3K/Akt/TOR pathway. There are many powerful tools for investigating protein function, such as reverse genetics using RNA interference (RNAi). This technique, however, requires the ability to consistently and efficiently transfect cells, which has proven difficult with regard to mosquito studies; there is currently no acceptable in vitro model for studying gene function in An. gambiae. Therefore, having identified proteins of importance to virus infection, we next created an in vitro model system for future protein function studies. By evaluating different transfection reagents and cell culture methods, we developed a method for transfecting the immune-competent Sua5b (An. gambiae) cell line with very high efficiency. Finally, we evaluated the role of the PI3K/Akt/TOR pathway in o'nyong-nyong virus infection of both mammalian (Vero) and mosquito (Sua5b) cells. This pathway offered a window of understanding into how alphaviruses interact with their hosts, and perhaps more importantly, in what ways those interactions differ. We found that the PI3K/Akt/TOR pathway likely plays distinct, if not opposite roles in infection of the host cells studied. / acase@tulane.edu

Mosquito species of the Anopheles gambaie complex are the predominant vectors of malaria transmission throughout sub-Saharan Africa. These mosquitoes tend to be endophilic, as well as anthropophilic, making them prime candidates for disease transmission. Within the same region, related mosquito vectors play a significant role in the transmission of additional human and zoonotic diseases. Furthermore, mosquito nuisance biting is an immense issue that cannot be ignored in terms of its impact on African communities. Depending on the respective factors involved, mosquito control programs throughout the continent have attempted to tackle these issues in a multitude of ways. This research approached the issue by developing and integrating an American-style mosquito control district within the eastern Ugandan community of Papoli. The basic structure of such a district was blended with a community-based approach, employing local community members and leaders, thus ensuring an effective and sustainable program. A guide detailing all aspects and steps needed to properly develop and implement such a program is outlined.

Anopheles species composition and relative seasonal abundance were measured in an irrigated division (low historical malaria incidence) and in a rain-fed division (high historical malaria incidence) of southern Sri Lanka. Twelve species of anophelines were represented in adult and larval collections with Anopheles vagus Donitz being the most abundant. In cattle-baited net trap collections, Anopheles adults were significantly more abundant in the irrigated division than in the rain-fed division. In pyrethrum-spray sheet collections, cattle-baited but trap collections and larval collections, Anopheles abundance was significantly greater in the rain-fed division. Houses were of poorer construction in the rain-fed division, where pyrethrum-spray sheet collections consisted mainly of Anopheles subpictus Grassi (98%) and Anopheles culicifacies Giles (2%). Hut trap collections also consisted mainly of An. subpictus (88%) and An. culicifacies (7%). Net trap collections consisted mainly of An. vagus (43%) and Anopheles peditaeniatus Leicester (31%). Larval collections also consisted of An. peditaeniatus (24%) and An. vagus (21%). Weak associations were found between species abundance and environmental factors explored in this study (e.g., vegetation, water quality, sunlight exposure). The greater malaria risk in the rain-fed division is due in part to the occurrence of potential vectors in relatively higher numbers.

Malaria incidence in the province of KwaZulu-Natal is influenced by seasonal climatic variations, drug and insecticide resistance, and population mobility within the region. Current control methods are directed at the malaria vector, the Anopheles arabiensis mosquito, through indoor residual spraying. Control of the dominant malaria parasite, Plasmodium falciparum, is done by treatmenting the symptomatic and asymptomatic parasite carriers with prophylactic medications of. A ceiling of effectiveness with current control efforts have been reached, necessitating the search for supplementary methods. The study area is located in the Ingwavuma District of northern of KwaZulu-Natal which adjoins the Mozambique border to the north, and includes the malaria areas of Ndumu and Makanisdrift. Homestead location coordinates were obtained with Global Positioning System (GPS) receivers and linked to malaria case records through homestead numbers which have been allocated to all homes in the area. The study includes the cases reported during 1993 and 1994 as this was the only data available when the project commenced in 1995. A geographic information system was used to undertake the spatial analysis to test the hypothesis that the malaria vector, Anopheles arabiensis, is localised to certain breeding sites during the winter months in northern KwaZulu-Natal. Identification of these winter 'seed point' breeding sites from which the onset of transmission spreads during the following malaria season, will allow them to be targeted for winter larval control measures. This will contribute to limiting the distribution and lowering the levels of malaria intensity in the region as a whole. The analysis also provided evidence of the maximum likely flight distance of the female mosquito given an adequate host supply in close proximity, thereby identifying those areas requiring additional prevention and control activities. Understanding the local epidemiology of the disease was necessary to determine which monthly malaria cases to include in order to identify the winter breeding sites, due to seasonal variations in the length of the mosquitoes life cycle. Medical geography, as a sub-discipline of geography, combines investigating spatial patterns with the epidemiological principles of medicine and zoology through scientific methods. It is traditionally divided into two approaches, the first being the geography of disease, under which this research falls, and the second being the geography of health care. The integration of the two disciplines allowed the results of the analysis to be presented in maps, graphs and tables in order to describe, interpret, test and explain possible associations between the location of the potential breeding sites and the homesteads at which the malaria cases were reported. The potential breeding sites consisted of the perennial pans, non-perennial pans and dams. Zones were created in the GIS at one kilometre intervals from these sites up to a distance of four kilometres, and the number of cases within each zone determined and corrected for the population at risk per 1000 people for comparative purposes. This spatial analysis was followed by the statistical analysis of the results to verify the findings. The results of the spatial and statistical analysis indicated that the perennial pans were used as the winter 'seed point' breeding sites, and that the maximum likely flight distance of the female mosquito, given an adequate host supply in close proximity, is 4 kilometres. The results will be made available to the local malaria research and control community who will assess the feasibility of implementing supplementary control measures. / Thesis (M.Sc.)-University of Natal, Durban, 2001.

La lutte anti-vectorielle est une composante importante de la lutte contre les maladies à transmission vectorielle. La connaissance des populations vectorielles, tant dans leur composition spécifique que dans leur répartition spatio-temporelle, est fondamentale pour la conception des stratégies de lutte contre ce type de maladie.Cette thèse a modélisé la distribution spatiale des formes moléculaires M et S d'Anopheles gambiae s.s., des vecteurs majeurs du paludisme au Burkina Faso. La modélisation a été faite à partir de l'analyse d'une série d'observations portant à la fois sur les vecteurs et l'environnement. Elle procède d'une combinaison de concepts et méthodes de biogéographie avec des techniques et outils d'analyse spatiale, d'analyse des données et des systèmes d'informations géographiques.Nous retenons de ce travail de recherche que l'abondance de la forme moléculaire S d'Anopheles gambiae s.s., diminue au fur et à mesure que l'on évolue des régions humides du sud et du sud-ouest vers celles les plus arides du nord et du nord-est. Le NDVI, l'ETP, et l'insolation sont les facteurs les plus déterminants de sa distribution spatiale. Par contre, l'abondance de la forme moléculaire M d'Anopheles gambiae s.s. augmente des régions humides du sud et du sud-ouest vers celles plus arides du nord et du nord-est. L'altitude, la pression, l'insolation, la densité de végétation sont les facteurs les plus déterminants de cette répartition spatiale. De ces résultats, ont été dérivés deux modèles qui ont servi à l'élaboration des cartes de distribution des formes moléculaires M et S d'Anopheles gambiae s.s.Enfin, cette thèse révèle le rôle de l'approche géographique dans la réflexion sur les questions de santé et sa méthodologie pourrait être testée sur d'autres sites et pour d'autres vecteurs de maladies. Elle peut s'enrichir d'une analyse multi échelle et d'une modélisation de la variabilité temporelle.

Submitted by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2013-06-14T13:04:04Z No. of bitstreams: 1 dissertação_final_Fernanda Rezende1.pdf: 11790769 bytes, checksum: c513dea1d65a522e22458b1ce1b6e3e5 (MD5) / Made available in DSpace on 2013-06-14T13:04:04Z (GMT). No. of bitstreams: 1 dissertação_final_Fernanda Rezende1.pdf: 11790769 bytes, checksum: c513dea1d65a522e22458b1ce1b6e3e5 (MD5) Previous issue date: 2013 / Os mosquitos da espécie Anopheles (Nyssorhynchus) aquasalis, Curry 1932 são vetores da malária humana nas Américas. Desde o ano de 2004, esse vetor tem sido mantido em colônia no Laboratório de Malária do Centro de Pesquisas René Rachou (Lamal), Fiocruz-MG, onde tem sido utilizado para estudos de interação parasito-vetor. Na natureza, as larvas desses insetos desenvolvem-se em água salobra e, no insetário de criação do Lamal utiliza-se água do mar na proporção de 1:10 em água da torneira. No ano de 2008, observou-se uma queda significativa na produção de mosquitos da colônia, devido, principalmente, à mortalidade excessiva de larvas. As larvas, de 3º e 4º estádios, apresentavam características morfológicas semelhantes àquelas relacionadas com contaminação por bacilos entomopatogênicos. Como algumas bactérias do gênero Bacillussão patogênicas às larvas de mosquitos, o objetivo do presente trabalho foi identificar a (as) espécie (es) responsável (is) pela mortalidade das larvas de Anopheles aquasalis, bem como identificar as toxinas envolvidas nessa mortalidade. Através da caracterização morfológica e molecular foi possível identificar o Bacillus sphaericuscomo contaminante na colônia de criação de Anopheles aquasalis. Visando uma comparação inter-insetários, analisou-se ainda amostras de três insetários distintos, sendo a presença dos bacilos identificadas em dois desses. Pela técnica de PCR buscou-se amplificar as 5 toxinas mais frequentes das estirpes de B. sphaericus(BinA, BinB, Mtx1, Mtx2 e Mtx3 ), sendo que apenas 3 (família das MTxs) foram detectadas nas amostras estudadas. Os resultados dos ensaios biológicos confirmaram a toxidade destas toxinas para as larvas de 3º e 4º estádios. Os ensaios biológicos realizados identificaram as estirpes isoladas do Laboratório de Malária como de alta toxicidade para o Anopheles aquasalis. Em resumo, este trabalho foi o primeiro a demonstrar a ocorrência de contaminação natural por bacilos entomopatogênicos em colônias de mosquitos mantidas em insetário e a caracterizar as toxinas envolvidas nesta contaminação. Espera-se que estes resultados possam contribuir para traçar estratégias de monitoramento e controle de contaminação em insetários de experimentação. / Mosquitoes of the species Anopheles (Nyssorhynchus) aquasalis, Curry 1932 are vectors of human malaria in the American continent. Since 2004, this vector has been kept in colony at the Malaria Laboratory in René Rachou Research Center (Lamal), Fiocruz-Minas, where it has been used for studies on vector-parasite interaction. In nature, these insects' larvae grow in brackish water and, in the creation insectarium of Lamal sea water is used in the proportion 1:10 in tap water. In 2008, a significant decrease was observed in the production of mosquitoes of the colony, mostly due to the excessive mortality of larvae. These larvae, from 3rd and 4 th stage, showed morphologic characteristics similar to those related to contamination by entomopathogenic bacillus. As somebacteria from genus Bacillusare pathogenic to mosquitoes' larvae, the purpose of the present work was to identify the species responsible for the mortality of Anopheles aquasalislarvae, as well as identify the toxins included in this mortality. Through morphologic andmolecular characterization, it was possible to identify Bacillus sphaericusas contaminant in the creation colony of Anopheles aquasalis. Seeking an inter-insectariums comparison, samplesof three distinct insectariums were still analyzed, and the presence of Bacillus sphaericuswas identified in two of them. Using the technique of PCR, an amplification of the5 most frequent in the lineage of B. sphaericus(BinA, BinB, Mtx1, Mtx2 e Mtx3) was tried, with only 3 (MTxs families) being identified in the studied samples. The results of biologic trials confirmed the toxicity of these toxins for larvae in 3rd and 4th stage. In addition, the biologic trials identified the isolated lineages of the Malaria Laboratory as of high toxicity for Anopheles aquasalis. In short, this is a pioneer work, since it was the first to show the occurrence of natural contamination by entomopathogenic bacillus in colonies of mosquitoes maintained in insectariums and to characterize the toxins involved in this contamination. It is expected that these results might contribute to trace strategies in order to monitor and control contamination in experimentation insectariums.

Submitted by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2015-04-16T17:01:19Z No. of bitstreams: 1 Tese_ClaudiaMaríaRíosVelásquez.pdf: 10298436 bytes, checksum: a818292b9ed3715f2201a29b38afb273 (MD5) / Approved for entry into archive by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2015-04-16T17:01:33Z (GMT) No. of bitstreams: 1 Tese_ClaudiaMaríaRíosVelásquez.pdf: 10298436 bytes, checksum: a818292b9ed3715f2201a29b38afb273 (MD5) / Approved for entry into archive by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2015-04-16T17:01:41Z (GMT) No. of bitstreams: 1 Tese_ClaudiaMaríaRíosVelásquez.pdf: 10298436 bytes, checksum: a818292b9ed3715f2201a29b38afb273 (MD5) / Made available in DSpace on 2015-04-16T17:01:41Z (GMT). No. of bitstreams: 1 Tese_ClaudiaMaríaRíosVelásquez.pdf: 10298436 bytes, checksum: a818292b9ed3715f2201a29b38afb273 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Centro de Pesquisa René Rachou. Belo Horizonte, MG, Brasil / A malária é um problema de saúde pública. O Brasil é o país sul-americano que mais casos aporta todo ano, a maioria deles ocorridos na região Amazônica. Até o presente não há nenhuma vacina eficaz contra a malária. O controle dessa doença baseia-se principalmente no combate vetorial. Um dos desafios atuais é encontrar novas moléculas úteis para bloquear a transmissão da malária no vetor, sendo necessário para isso conhecer a biologia da interação entre os parasitos e seus hospedeiros. A maioria dos grupos de pesquisa utiliza como modelos de laboratórios combinações não naturais de Anopheles – Plasmodium. Neste trabalho foi avaliada a suscetibilidade ao P. vivax em espécies Amazônicas de Anopheles, fêmeas de Anopheles darlingi, An. albitarsis s.l., An. nuneztovari s.l. e An. triannulatus s.l. e An. aquasalis foram infectadas com P. vivax utilizando um sistema de infecção experimental por membrana artificial. Todas as espécies de Anopheles estudadas foram suscetíveis à infecção por P. vivax, porém a taxa de infecção e o numero de oocistos variaram significativamente entre elas. An. aquasalis (Spearman rho = 0.255, n = 386, p < 0.01) e An. darlingi (rho = 0.518; n = 54, p < 0.01) mostraram uma correlação positiva entre o número de gametócitos e o número de oocistos formados. Também foi avaliada a via JAK/STAT de resposta imune em A. aquasalis, durante a fase tardia da infecção, e em A. darlingi, no início da infecção. A expressão dos genes STAT, PIAS e NOS foi avaliada por q-PCR. Em An. aquasalis a expressão dos genes estudados foi induzida a partir de 8 dPI (PIAS e NOS) e 12 dPI (STAT), e começou a diminuir aos 14dPI, provavelmente indicando a indução transitória desses genes na fase tardia da infecção. Em An. darlingi não foi observada a ativação dessa via imune durante a fase inicial da infecção com P. vivax. Estudos futuros devem ser realizados para saber de que forma os genes regulados pela via JAK/STAT podem modular o desenvolvimento do P. vivax em An. aquasalis, e outras vias de sinalização devem ser estudadas na resposta de An. darlingi à infecção pelo Plasmodium. / Malaria is a public health concern. Brasil is the Latin American country with the higher number of cases registered, the most in the Amazon Region. Actually, there is not an effective vaccine against malaria and the disease control is based on vector control. New challenges include finding new molecules to block malaria transmission, making necessary to know the mechanisms involved in the Plasmodium – Anopheles interactions. The most studies have used laboratory parasite – vector pairs which does not resemble natural parasite – hst interactions. The goals of this work were 1) to compare the susceptibility of five Amazonian Anopheles mosquito species to Plasmodium vivax, and 2) to evaluate JAK/STAT immune pathway during the late-phase of A. aquasalis infection and the early phase of A. darlingi. To evaluate the P. vivax mosquito susceptibility we fed by membrane feeding assays field populations of Anopheles darlingi, An. albitarsis s.l., An. nuneztovari s.l. and An. triannulatus adult females and, An. aquasalis from colony. All the Anopheles species were susceptible to P. vivax infection, although the infection rate and oocyst numbers were significantly different among them. There was a positive correlation between the density of gametocytes and the infection rate in An. aquasalis (Spearman rho = 0.255, n = 386, p < 0.01) and An. darling (rho = 0.518; n = 54, p < 0.01). Anopheles aquasalis had high infection intensity, showing that the An. aquasalis - P. vivax pair is a feasible laboratory model. To evaluate the expression of STAT, PIAS and NOS in An. aquasalis and An. darlingi was used qPCR method. In An. aquasalis the studied genes were induzed since 8 dPI (STAT) and 12 dPI (both PIAS and NOS), and was diminishing at 14dPI, probably indicating that JAK/STAT activation in later phases of P. vivax infection is transient. In An. darlingi the genes STAT, PIAS and NOS were no expressed during the early phase of the infection with P. vivax. The role of JAK/STAT pathway, in later stages of P. vivax infection of An. darling, remains to be investigated.

Submitted by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2016-04-08T17:15:28Z No. of bitstreams: 1 Tese_BCM_LuisEduardoMartínezVillegas.pdf: 5786792 bytes, checksum: 41da46eaa9b4630d4ce0ce9181831739 (MD5) / Approved for entry into archive by Nuzia Santos (nuzia@cpqrr.fiocruz.br) on 2016-04-08T17:21:29Z (GMT) No. of bitstreams: 1 Tese_BCM_LuisEduardoMartínezVillegas.pdf: 5786792 bytes, checksum: 41da46eaa9b4630d4ce0ce9181831739 (MD5) / Made available in DSpace on 2016-04-08T17:21:29Z (GMT). No. of bitstreams: 1 Tese_BCM_LuisEduardoMartínezVillegas.pdf: 5786792 bytes, checksum: 41da46eaa9b4630d4ce0ce9181831739 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, Brasil / A malária humana é uma doença provocada por parasitas do gênero Plasmodium, os quais na natureza requerem de um mosquito anofelíno para completar o seu ciclo de vida e serem transmitidos a um hospedeiro humano. Nas Américas, o Brasil tem a maior incidência de malária, sendo responsável por 41% dos casos. Com o aparecimento do sequenciamento de nova geração e das ferramentas bioinformática relacionados, grandes avanços foram alcançados em relação à montagem de genomas e transcriptomas de anofelinos, assim como na exploração de estratégias de paratransgenesis para interromper a transmissão da malária. No entanto, os vetores neotropicais da malária encontram-se longe dos vetores da África e Ásia no que refere a estes conhecimentos. Este estudo é parte de um esforço contínuo para montar o genoma do Anopheles aquasalis, um vetor neotropical da malária humana, que atualmente posiciona-se como um excelente modelo de transmissão da malária no Brasil. Em paralelo ao sequenciamento do genoma, e para maximizar os dados gerados, optamos por focar em duas tarefas pontuais e viáveis: explorar a diversidade e composição do consórcio bacteriano associado ao anofelino; assim como montar e caracterizar o genoma mitocondrial desta espécie. O sequenciamento metagenômico shotgun ̈e o programa MG-RAST foram utilizados para fazer um ̈screening ̈ das bactérias associadas à pupas de A .aquasalis criadas em laboratório. O consórcio bacteriano predito é composto por 74 gêneros contendo bactérias marinhas e bioluminescentes. No nível taxonômico de família bacteriana, identificamos 14 OTUs compartilhadas entre anofelinos americanos e africanos. Além disso, foram comparadas cinco comunidades bacterianas associadas a duas espécies de anofelinos: A. aquasalis e Anopheles gambiae. Foi identificada uma associação significativa (NPMANOVA p <0,05) entre a composição da comunidade bacteriana e o ambiente aquático laboratório ou condições semi-naturais) nas quais cada hospedeiro anofelino foi criado. Atualmente, o entendimento da filogenia do gênero Anopheles é limitado e as informações sobre o tempo de divergência de dentro da linhagem de mosquitos é escassa. Apresentamos a sequencia de 15,393 pb correspondente ao genoma mitocondrial de A. aquasalis. Quando comparado com outros mitogenomas anofelinos relevantes, observou-se alta similaridade na composição dos genomas assim como características estruturais conservadas. Através de análises Bayesianas, reconstruímos as relações filogenéticas e estimamos a data de divergência entre 22 anofelinos e outras espécies de dípteros. Descobrimos que o mais recente ancestral entre as subfamílias Nyssorhynchus e Anopheles + Cellia existiu ~ 83 milhões anos atrás (MYA). Estimou-se que A. aquasalis divergiu do complexo do Anopheles albitarisis faz ~ 28 MYA, e faz ~ 38 MYA do Anopheles darlingi. A distribuição estreita e o peculiar nicho ecológico do A. aquasalis, além de considerar a sua adaptação a ambientes larvários com água salobra fizeram nos perguntar se a sua história evolutiva deixou uma marca na arquitetura do seu genoma, assim como sobre a estrutura da comunidade bacteria na associada a este anofelino. / Human malaria is a malady caused by Plasmodium parasites, which in nature, require an anopheline mosquito to complete their life cycle and be transmitted to a human host. In the Americas, Brazil has the largest incidence of malaria, accounting for 41% of the cases. With the advent of Next Generati on Sequencing and related bioinformatics’ tools, great leaps forward were attained regarding the assembly of anopheline genomes, transcriptomes; in addition to the exploration of paratransgenesis as means to interrupt malaria transmission. Nonetheless, Neotropical malaria vectors still lag behind those from Africa and Asia on such matters. This study is part of an ongoing effort to assemble the genome of Anopheles aquasalis , a Neotropical human malaria vector currently positioned as a key malaria transmission model in Brazil. In parallel to the genome sequencing study, and to maximize the NGS sequencing data generated, we opted to focus in two punctual a nd feasible tasks: exploring the diversity and composition of this anopheline’s associated b acterial consortium; plus, assemblying and characterizing, the mitochondrial genome of this species. Shotgun metagenomic sequencing and the MG-R AST suite were used to survey the bacteria associated to laboratory reared A. aquasalis pupae. The predicted bacterial consortium is composed of 74 genera and contai ns marine and biolumines cent bacteria. At the bacterial family rank, we identified 14 OTUs shared between African and American anophelines. In addition, we compared five Anopheles associated bacterial communities from two species: A. aquasalis and Anopheles gambiae . We found a significant association (NPMANOVA p < 0.05) between the bacteria l community composition and the aquatic environment (laboratory or semi-natural conditions) in which each Anopheles host was reared. The current understanding of the Anopheles phylogeny is limited and information regarding the time of deep lineage divergences w ithin mosquitoes is scarce. Here we also present the assembled 15,393 bp mitochondrial genome of A. aquasalis . When compared with other relevant anopheline mitogenomes, high com position similarity and conserved features were observed. Through Bayesian analyses, we reconstructed the phylogenetic relationships and estimated the date of divergence between 22 anopheline and other dipteran species. We found that the most r ecent ancestor between Nyssorhynchus and Anopheles + Cellia subfamilies was extant ~83 million y ears ago. It was estimated that A. aquasalis diverged from the Anopheles albitarisis complex ~28 MYA and ~38 MYA from Anopheles darlingi . The narrow distribution and peculiar niche of A. aquasalis , plus considering its adaptation to brackish-water larval environments makes us wonder if its evolutionary history left a mark upon its genome architecture, and also on the bacterial community structure associated to it.

Made available in DSpace on 2016-05-02T13:08:25Z (GMT). No. of bitstreams: 2 andre_veloso_ioc_dout_2015.pdf: 5077168 bytes, checksum: 9fce6d548bd0ac2fa5e83f9b028c4bf2 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Os dípteros Aedes aegypti, Anopheles albitarsis e Culex quinquefasciatus pertencem à família Culicidae e são considerados um problema de saúde pública quando atuam como vetores de parasitos e/ou arboviroses. O regime de alimentação de fêmeas dos mosquitos anautógenos influencia os processos que ocorrem no intestino e regula os genes envolvidos na reprodução. Além disso, apesar dos ciclos de vida dos parasitos veiculados por essas espécies de vetores serem distintos, todos eles são ingeridos durante a hematofagia e expostos ao ambiente do intestino médio (estômago) para em seguida atravessar o epitélio intestinal e chegarem ao tecido apropriado para seu desenvolvimento e/ou transmissão para um novo hospedeiro vertebrado. Neste trabalho, utilizamos uma abordagem proteômica para a identificação de proteínas totais, bem como ensaios em solução e enzimografia em gel copolimerizado com substrato para identificação de peptidases ativas presentes no intestino de fêmeas de Cx. quinquefasciatus e An. albitarsis s.s sob regime de alimentação com açúcar. As técnicas de enzimografia também foram utilizadas aqui para a caracterização de peptidases ativas de intestinos de fêmeas de Ae. aegypti alimentadas com açúcar ou sangue. Foi observado que o intestino de fêmeas das três espécies alimentadas com açúcar apresenta um complexo perfil de peptidases ativas, do tipo tripsina, composto por bandas migrando nas regiões entre ~ 24 a 40 kDa e em regiões de alto peso molecular Atividades proteolíticas foram detectadas entre pH 3,5 \2013 10, revelando diferenças quantitativas e qualitativas entre os perfis proteolíticos das três espécies. Em fêmeas de Ae. aegypti, o regime de alimentação com açúcar ou sangue alterou o perfil de SPtrip ativas do intestino de forma qualitativa e quantitativa. Um total de oito tripsinas em Cx. quinquefasciatus e dez tripsinas em An. albitarsis, foram identificadas por SDS-PAGE acoplado a LC-MS/MS. Apesar dessas SPtrip identificadas apresentarem características comuns às tripsinas digestivas de invertebrados, foram observadas diferenças nas sequências de aminoácidos, como por exemplo, em regiões de especificidade ao substrato e de ativação do zimogênio. Foi observado que os genes codificadores para SPtrip possuem diferentes tamanhos e organização, tais como, diferenças no número de éxons/introns. Com relação à composição de proteínas totais solúveis do intestino de fêmeas de Cx. quinquefasciatus e An. albitarsis, análise por LC-MS/MS e bioinformática revelou a presença de proteínas envolvidas em processos fisiológicos importantes, como exopeptidaes, glicosidades, enzimas detoxificantes e proteínas relacionadas com a interação parasito/vetor Em Cx. quinquefasciatus, foram identificadas 1397 proteínas, distribuídas em 1090 grupos, representando um total de ~ 7,5% das proteínas codificadas pelo genoma dessa espécie. Em An. albitarsis foram identificadas 916 proteínas distribuídas em 748 grupos. As centenas de proteínas presentes no intestino de fêmeas alimentadas com açúcar, de ambas as espécies, foram classificadas segundo sua ontologia gênica e o papel de algumas famílias de proteínas historicamente importante em mosquitos foram discutidas. Finalmente, foi demonstrado que abordagem proteômica combinada à enzimografia e bioinformática é uma ferramenta que pode auxiliar na anotação funcional dos genes de tripsinas expressos no intestino de fêmeas alimentadas com açúcar, bem como auxilia no mapeamento do repertório de proteínas totais presentes no intestino de Cx. quinquefasciatus e An. albitarsis / Culex quinquefasciatus, Anopheles albitarsis and Aedes aegypti are hematophagous insect from the Culicidae family that feeds on the blood of humans, dogs, birds and livestock. These species transmit a wide variety of pathogens between humans and animals. The midgut environment is the first location of pathogen-vector interaction for blood-feeding mosquitoes and the expression of specific peptidases in the early stages of feeding could influence the outcome of the infection. Trypsin-like serine peptidases belong to a multi-gene family that can be expressed in different isoforms under distinct physiological conditions. However, the confident assignment of the trypsin genes that are expressed under each condition is still a challenge due to the large number of trypsin-coding genes in the Culicidae family and most likely because they are low abundance proteins. We used zymography for the biochemical characterization of the peptidase profile of the midgut from Cx. quinquefasciatus, An. albitarsis, and Ae. aegypti females fed on sugar. We also caracterized the peptidses profile of the midgut from Ae. aegypti fed on blood, during different moments after fedding. Protein samples were also submitted to SDS-PAGE followed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis for peptidase identification. The peptidases sequences were analyzed by bioinformatics tools to assess their distinct features. Zymography revealed that trypsin-like serine peptidases were responsible for the proteolytic activity in the midgut of females fed on sugar or blood diet. In addition, we observed that the profile is influenced by the blood ingestion. After fractionation in SDS-PAGE, eight and ten trypsin-like serine peptidases were identified by LC-MS/MS in Cx. quinquefasciatus and An. albitarsis, respectively. Peptidases from Cx. quinquefasciatus were also analysed with bioinformatic tools revealing that they have structural features typical of invertebrate digestive trypsin peptidases but exhibited singularities at the protein sequence level such as: the presence of different amino acids at the autocatalytic motif and substrate binding regions as well as different number of disulfide bounds. Data mining revealed a group of trypsin-like serine peptidases that are specific to C. quinquefasciatus when compared to the culicids genomes sequenced so far. We demonstrated that proteomics approaches combined with bioinformatics tools and zymographic analysis can lead to the functional annotation of trypsin-like serine peptidases coding genes and aid in the understanding of the complexity of peptidase expression in mosquitoes. / 2016-10-11

Made available in DSpace on 2014-06-11T19:26:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-09-30Bitstream added on 2014-06-13T18:29:21Z : No. of bitstreams: 1 carlos_bc_me_botib.pdf: 1447163 bytes, checksum: bbee87d6f015501c72b499b26af6cd4a (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Estudos recentes vêm elucidando a importância de uma diversidade de proteínas do intestino médio de insetos vetores, tanto nos processos de digestão como em respostas imunológicas e interações parasita-hospedeiro. Este trabalho teve como objetivo analisar a expressão de genes do intestino médio de Anopheles aquasalis, um importante vetor de malária no Brasil, a partir de clones sequenciados de bibliotecas de cDNA de machos e fêmeas alimentados apenas com sacarose. Nas fêmeas, pôde-se notar a grande predominância de serino proteases, proteínas ligantes de quitina e fatores relacionados à imunidade. Os machos também apresentaram diversos peptídeos de defesa imune, porém apenas uma protease digestiva e uma glicosidase. Alguns genes foram selecionados das bibliotecas para estudo de suas expressões durante a vida de An. aquasalis. Tripsina 1, peritrofina 1 e quinurenina 3-monooxigense tiveram seus níveis de expressão aumentados 6h após a ingestão de sangue, analisados através de qRT-PCR. No entanto, o silenciamento desses genes não resultou em alterações na longevidade de fêmeas adulta. O gene da serpina foi expresso em todas as fases do desenvolvimento do mosquito, exceto em ovos; e o gene da cecropina foi expresso em trato digestivo e carcaça de machos e fêmeas, principalmente após alimentação de açúcar ou sangue. Considerando que a ingestão de alimentos é a principal porta de entrada a microorganismos durante a vida adulta destes mosquitos, a presença de diversos produtos antimicrobianos, bem como a precoce expressão de peritrofina, outra proteína relacionada com a proteção do trato digestivo, mostrou que An. aquasalis está bem preparado imunologicamente contra esses microorganismos. Esta proteção está envolvida com o hábito alimentar desta espécie e pode também estar associada à sua baixa capacidade vetorial com relação aos plasmódios. / The importance of midgut proteins of Anopheles aquasalis has been elucidated both in digestion process as in immune responses and parasite-host interactions. This project targeted to analyze the midgut genes expression from An. aquasalis, an important malaria vector in Brazil, selecting clones from midgut cDNA library of female and male mosquitoes fed only on sugar. Serine proteases were predominant in females besides chitin binding proteins and immunity factors. Male mosquitoes also showed immune defense peptides, however only one digestive protease and one glucosidase. Some genes were selected from these libraries to expression study during mosquito development stages. Trypsin 1, peritrophin 1 and kynurenine 3-monoxygenase expression were up regulated at the midgut 6h after blood feeding, analyzed by real time PCR. Nevertheless, the gene silencing did not change the survivorship of adult females. Serpin gene was expressed in all mosquito development stages but eggs; cecropin gene was expressed in midgut and carcass from male and female, mainly after sugar or blood feeding. Considering the alimentation is the main entrance way of pathogens, the presence of antimicrobial peptides as the early peritrophin expression showed that An. aquasalis is immunologically adapted against these microorganisms. This protection is involved in feeding behavior of this specie and can be also related to its low Plasmodium vector capacity.