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Agradecemos a compreensão. on 2016-06-15T19:04:14Z (GMT) / Submitted by MELINA AULINO CAMPOS DE LIMA null (mel_linaa@hotmail.com) on 2016-07-28T09:31:04Z No. of bitstreams: 1 Campos, versão final.pdf: 6669108 bytes, checksum: bf58ca3048946ca88e3c4dd8fb99500a (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-07-28T14:10:56Z (GMT) No. of bitstreams: 1 lima_mac_dr_bot.pdf: 6669108 bytes, checksum: bf58ca3048946ca88e3c4dd8fb99500a (MD5) / Made available in DSpace on 2016-07-28T14:10:56Z (GMT). No. of bitstreams: 1 lima_mac_dr_bot.pdf: 6669108 bytes, checksum: bf58ca3048946ca88e3c4dd8fb99500a (MD5) Previous issue date: 2016-05-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Degradação florestal, alterações ambientais antropogênicas e mudanças climáticas são fatores que podem modificar a dinâmica populacional de anofelinos. Anopheles (Nyssorhynchus) dar- lingi é o principal vetor de malária no Brasil e em outros países na América do Sul. Estudos observaram o aumento da abundância do vetor An. darlingi e número de casos de malária após desflorestação e demais modificações ambientais antropogênicas. Além de ter ampla distribuição geográfica, essa espécie possui plasticidade comportamental e diversidade morfo- lógica, biológica e genética. Tendo em vista essa heterogeneidade, o presente estudo avaliou a diversidade genética populacional em An. darlingi ligada a distribuição geográfica, dinâmica sazonal e comportamento hematofágico, através de marcadores microssatélites e SNPs. Espé- cimes de An. darlingi foram coletados em dois assentamentos rurais próximos e, em uma área urbana à aproximadamente 600 km de distância. Além disso, as coletas foram realizadas no primeiro e segundo semestre e, dentro e fora das casas, durante o período de atividade hema- tofágica do vetor, ou seja, das 18-6 horas. Os resultados apresentaram subpopulações de An. darlingi em aspecto geográfico, em escalas macro e microgeográficas, acessadas com mais pro- fundidade com os dados dos SNPs. Além disso, o estudo corroborou o prévio achado de duas subpopulações de An. darlingi relacionadas ao regime de chuvas. Por fim, pela primeira vez, a heterogeneidade genética em populações simpátricas desse vetor foi encontrada e relacionada com o fenótipo de comportamento hematofágico, endo e exofagia. Esses achados demonstram a importância do entendimento e vigilância entomológica, pois possuem potencial impacto na transmissão de malária. Dado que cada localidade possui características ambientais peculia- res e portanto, diferentes composições populacionais dos vetores, intervenções específicas em menor escala passam a ser abordagens interessantes no controle da malária. / Forest degradation, human environmental alteration and climate changes are all influence anopheline populations. Anopheles darlingi is the main vector of malaria parasite in Brazil and other countries of South America. Deforestation and others anthropogenic activities have been accompanied by sharp increases in both abundance of the primary malaria vector Anopheles darlingi and numbers of malaria cases. Besides it is widely distributed, this species display great behavioral plasticity and morphological, biological and genetic diversity. The aim of this study was to analyze population genetic diversity of An. darlingi related to geographical distribution, seasonal dynamics and hematophagic behavior, using microsatellites and SNPs markers. An. darlingi specimens were collected in two close rural settlements and in an urban area about 600 km away. In addition, collections were performed in both semesters of the year and, indoor and outdoor during the biting activity period of the vector, i.e., 6pm-6am. The results showed subpopulations of An. darlingi related to geographical aspect, in a macro and micro geographic scales, better accessed with SNPs dataset. Moreover, this study corroborated with a previous finding showing two subpopulations of An. darlingi related to rainfall. Finally, for the first time, genetic heterogeneity was found in sympatric populations of this vector, and it was associated with a phenotype of hematophagic behavior, endo and exophagy. These outcomes demonstrate that genetic heterogeneity may represent an important vector phenotypic variation with potentially highly significant consequences for malaria transmission. Since each site has unique environmental characteristics and therefore, different population compositions of vectors, specific interventions on a smaller scale become interesting approaches for optimal targeted malaria transmission interventions. / CAPES: BEX 9230/12-2 / FAPESP: 2014/09461-3 / FAPESP: 2012/04881-9

Orientador: Paulo Eduardo Martins Ribolla / Resumo: Degradação florestal, alterações ambientais antropogênicas e mudanças climáticas são fatores que podem modificar a dinâmica populacional de anofelinos. Anopheles (Nyssorhynchus) dar- lingi é o principal vetor de malária no Brasil e em outros países na América do Sul. Estudos observaram o aumento da abundância do vetor An. darlingi e número de casos de malária após desflorestação e demais modificações ambientais antropogênicas. Além de ter ampla distribuição geográfica, essa espécie possui plasticidade comportamental e diversidade morfo- lógica, biológica e genética. Tendo em vista essa heterogeneidade, o presente estudo avaliou a diversidade genética populacional em An. darlingi ligada a distribuição geográfica, dinâmica sazonal e comportamento hematofágico, através de marcadores microssatélites e SNPs. Espé- cimes de An. darlingi foram coletados em dois assentamentos rurais próximos e, em uma área urbana à aproximadamente 600 km de distância. Além disso, as coletas foram realizadas no primeiro e segundo semestre e, dentro e fora das casas, durante o período de atividade hema- tofágica do vetor, ou seja, das 18-6 horas. Os resultados apresentaram subpopulações de An. darlingi em aspecto geográfico, em escalas macro e microgeográficas, acessadas com mais pro- fundidade com os dados dos SNPs. Além disso, o estudo corroborou o prévio achado de duas subpopulações de An. darlingi relacionadas ao regime de chuvas. Por fim, pela primeira vez, a heterogeneidade genética em populações... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Forest degradation, human environmental alteration and climate changes are all influence anopheline populations. Anopheles darlingi is the main vector of malaria parasite in Brazil and other countries of South America. Deforestation and others anthropogenic activities have been accompanied by sharp increases in both abundance of the primary malaria vector Anopheles darlingi and numbers of malaria cases. Besides it is widely distributed, this species display great behavioral plasticity and morphological, biological and genetic diversity. The aim of this study was to analyze population genetic diversity of An. darlingi related to geographical distribution, seasonal dynamics and hematophagic behavior, using microsatellites and SNPs markers. An. darlingi specimens were collected in two close rural settlements and in an urban area about 600 km away. In addition, collections were performed in both semesters of the year and, indoor and outdoor during the biting activity period of the vector, i.e., 6pm-6am. The results showed subpopulations of An. darlingi related to geographical aspect, in a macro and micro geographic scales, better accessed with SNPs dataset. Moreover, this study corroborated with a previous finding showing two subpopulations of An. darlingi related to rainfall. Finally, for the first time, genetic heterogeneity was found in sympatric populations of this vector, and it was associated with a phenotype of hematophagic behavior, endo and exophagy. These outcomes demon... (Complete abstract click electronic access below) / Doutor

Submitted by Milena Dias (milena.dias@ufpe.br) on 2015-03-11T17:49:05Z No. of bitstreams: 2 Tese Rafael Trindade Maia.pdf: 4595214 bytes, checksum: 9e51b91025a00458c05287160877d452 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-11T17:49:05Z (GMT). No. of bitstreams: 2 Tese Rafael Trindade Maia.pdf: 4595214 bytes, checksum: 9e51b91025a00458c05287160877d452 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2013 / O mosquito Anopheles gambiae (Diptera: Culicidae) é considerado o principal vetor do Plasmodium, o agente etiológico da malária, a doença parasitária que mais leva ao óbito em todo o mundo. O uso intensivo de alguns inseticidas químicos, entre os quais o DDT, direcionados para o controle desse vetor, levou à seleção de linhagens resistentes de An. gambiae. Desta forma, os mecanismos de resistência aos inseticidas vêm sendo amplamente estudados com o intuito de desenvolver novas estratégias de controle populacional do vetor. As glutationa s-transferases (GSTs) são enzimas de detoxificação celular que desempenham um importante papel biológico no metabolismo de xenobióticos através da conjugação da glutationa reduzida (GSH), tornandoos mais solúveis e facilmente excretados da célula. As GSTs da classe epsilon em An. gambiae (AgGSTE) apresentam atividade anti-DDT, especialmente a AgGSTE2, cuja estrutura encontra-se disponível no PDB. Também já foi demonstrado que a enzima AgGSTE5, cuja estrutura tridimensional ainda não foi elucidada, apresentou super expressão em presença do DDT. Assim, o objetivo do presente trabalho foi construir e validar um modelo tridimensional para elicidação da estrutura da AgGSTE5 através da modelagem comparativa e simular a dinâmica molecular da AgGSTE2 e AgGSTE5 e de uma isoforma mutante (AgGSTE2mut). Nas simulações de Dinâmica Molecular (DM) foram feitas por um período de 50 nanossegundos com e sem o ligante (GSH). Após a dinâmica, as três enzimas foram submetidas ao docking molecular contra os compostos DDT, CDNB, carbaril, malation e cipermetrina. Também foi analisado o polimorfismo genético e a taxa de mutação para os genes AgGSTE2 e AgGSTE5. A análise da seqüência dos genes apontou seleção purificadora para o AgGSTE2 e seleção positiva para o AgGSTE5 em populações de sete países da África. Os resultados demonstraram que as proteínas têm dinâmicas diferentes e interagem com os substratos de modo diferente. As mutações da AgGSTE2mut alteram sua dinâmica e modo de ação, sendo esta enzima particularmente capaz de se ligar ao DDT, com energia de ligação menor que as outras. Finalmente, os resultados do presente trabalho sugerem que estas enzimas desempenharam um papel crucial na adaptação de An. gambiae ao seu habitat e possivelmente a evolutibilidade destas GSTs teve participação neste processo evolutivo, sendo portanto alvos potenciais para o desenvolvimento de novas ferramentas de controle. Conclui-se que o papel da AgGSTE2 e AgGSTE5 na metabolização de inseticidas é importante para a adaptação do An. gambiae e o modo de ação destas enzimas deve ser entendido como uma importante via metabólica a ser interferida com o propósito de melhorar os inseticidas e métodos de controle. Os resultados permitem concluir que o modelo teórico é válido e que a AgGSTE2, AgGSTE2mut e AgGSTE5 apresentam diferenças na dinâmica e no modo de ligação aos compostos químicos estudados, o que provavelmente reflete em uma divergência funcional destas enzimas.

Made available in DSpace on 2018-08-01T21:35:45Z (GMT). No. of bitstreams: 1 tese_11995_Tese_28_mar_2018.pdf: 1434096 bytes, checksum: 78535cc2fc29ae622c421c0a525003aa (MD5) Previous issue date: 2018-03-22 / Nas regiões Sul e Sudeste do Brasil, casos de malária autóctone podem ser encontrados próximos a fragmentos de Mata Atlântica. No estado do Espírito Santo, tal doença é particularmente frequente, sendo Plasmodium vivax o parasito comumente reconhecido como o agente etiológico das infecções humanas. Porém, observadas as distâncias espaciais e temporais entre os casos relatados e o comportamento dos insetos vetores locais, especialmente Anopheles (Kerteszia) cruzii, o ciclo de transmissão parece não corresponder ao ciclo tradicional da malária. Sendo assim, a hipótese da existência de uma zoonose, com símios infectados mantendo a transmissão, é estabelecida. Considerando que há pontos obscuros em relação à malária-bromélia, como é chamada, a avaliação do comportamento dos componentes dessa cadeia de transmissão se faz necessária. No presente estudo, a transmissão da malária-bromélia na região rural endêmica do Espírito Santo é investigada com base em dois pilares principais: análise comportamental dos vetores do gênero Anopheles, principalmente os do subgênero Kerteszia, e a comparação do genoma mitocondrial completo do DNA extraído de diversos isolados de Plasmodium spp. que infectaram humanos, um símio do gênero Allouata e mosquitos Anopheles spp. da mesma região. Armadilhas luminosas com CO2 (CDC-CO2) foram instaladas nas áreas abertas, na margem e no interior da floresta, e armadilhas Shannon foram instaladas na margem da floresta para a captura dos mosquitos. O genoma mitocondrial dos Plasmodium spp. enontrados nos diferentes hospedeiros foi completamente sequenciado e comparado em uma rede de haplótipos que incluiu todas as sequências de genomas mitocondriais de P. vivax/simium de amostras de seres humanos e símios de outras regiões do Brasil. Foram capturados 1.414 anofelinos distribuídos em 13 espécies. Anopheles (Kerteszia) cruzii Dyar&Knab foi a espécie mais capturada na copa das árvores e foi também o vetor com maior prevalência de infecção por Plasmodium vivax, de acordo com técnicas moleculares de PCR. A rede de haplótipos nostrou que humanos e símios da Mata Atlântica estavam infectados pelo mesmo haplótipo, mas alguns isolados de seres humanos não eram idênticos ao isolado de símio. Além disso, o DNA de plasmódios extraído de mosquitos revelou sequências diferentes das obtidas de simios, mas semelhantes a de dois isolados de seres humanos. O estudo revelou, portanto, a maior prevalência de infecção em A. (K.) cruzii entre os anofelinos e o comportamento acrodendrofílico desses vetores, principalmente dos infectados, reforçando a hipótese de que a presença de P. vivax nesses espécimes surja pelos repastos sanguíneos em animais que vivem na copa das árvores, como os símios. A comparação dos genomas mitocondriais dos parasitos mostra que, na Mata Atlântica e especialmente no Espírito Santo, parasitos com estruturas moleculares semelhantes são compartilhados por seres humanos e símios. A reconhecida identidade entre P. vivax e P. simium ao nível de espécie, o compartilhamento de haplótipos, e a participação do mesmo vetor na transmissão da infecção para ambas as espécies hospedeiras indica transferência interespécies dos parasitos.

No description available.

Transposable elements (TEs) are mobile genetic elements. They are a significant component of many eukaryotic genomes. They are involved in chromosomal rearrangement by serving as substrates for homologous recombination, in creating new genes through a process of TE "domestication", and in modifying and shuffling existing genes by transducing neighboring sequences (Lander et al., 2001). Therefore, both active and inactive TEs are potentially potent agents for genomic change (Kidwell and Lisch, 2001, 2002; Rizzon et al., 2002; Petrov et al., 2003). In the meantime, active TEs are being explored as useful tools for genetic transformation and possible gene drive mechanisms to deliver genes in natural populations (Ashburner et al.,1998; Alphey et al.,2002; Handler and O'Brochta, 2004). My thesis project focuses on AGH1, a novel DNA-mediated TE in Anopheles gambiae and related mosquitoes. I have studied its genomic structure, insertion polymorphism, evolution, and transposition activity. As part of the sequence and structural characterization of AGH1 in the A. gambiae genome, the boundaries of AGH1were determined. The TA target site duplications flanking AGH1 were verified by comparing a genomic sequence that had an AGH1 insertion with the sequence of a corresponding empty site. AGH1 has relatively long, 350bp, TIRs (Terminal inverted repeats). In addition to the transposase ORF (ORF1) that contains a DD34E catalytic motif, it contains an unusual ORF2 with unknown function. Phylogenic analyses clearly suggest that unlike most DD34E transposons that are similar to the Tc1 family, AGH1 belongs to a different clade that is related to the previously characterized fungal TE Ant and protozoan TEC1 and TEC2. Truncated AGH1 and AGH1-related MITE (Miniature inverted-repeat TE) families were also identified. AGH1 insertion polymorphism was studied using 4 natural populations that belong to two molecular forms of A. gambiae, M and S. AGH1 insertions showed considerable differences between M and S forms and the insertions of AGH1 are highly variable in two populations of M. These results are potentially significant in light of the hypothesis that M forms are newly derived incipient species that are only found in West Africa. PCR and sequencing results showed more than 99% sequence identity between AGH1 sequences in A. gambiae, A. arabiensis, and A. melas, which may indicate either purifying selection or recent horizontal transfer. To assess whether AGH1 is currently active, inverse PCR was performed which provided evidence for extrachromosomal circular AGH1 that may be a product of imprecise excision. RT-PCR detected transcripts for both intact and truncated transposase. Preliminary TE display experiments using genomic DNA isolated from different passages of an A. gambiae Sua1B cell line showed possible new insertions and deletions of AGH1 related elements, which may have been mobilized by AGH1. In summary, the structural and genomic characteristics of AGH1 and the phylogenetic relationship between AGH1 and other known transposons in the IS630-Tc1-mariner superfamily have been determined. Significant divergence was shown between M and S forms of A. gambiae according to AGH1 insertion patterns. Observations of high level of insertion polymorphism and low insertion frequency per site in M populations are preliminary indications that AGH1 may be active in some populations. AGH1 has at least been recently transposing and there are also indications for its current activity in A. gambiae cell lines. If AGH1 is indeed active, it has the potential to be used as genetic tools to study mosquito biology and to spread refractory genes into the field populations to help control mosquito-borne diseases. Although a few active DNA transposons have been discovered in different insects and are being used as tools to transform mosquitoes, no DNA active transposons have been reported in mosquitoes. It is our hope that active endogenous DNA transposons may present new features that will help us overcome some of the deficiencies of current transformation tools developed based on exogenous transposons. In addition, the discovery of an active DNA transposon will help us understand how TEs spread in natural populations of mosquitoes, which is critical if we are to use TEs to drive refractory genes into mosquito populations to control vector-borne infectious diseases. The differential insertion patterns of AGH1 in M and S populations are consistent with the hypothesis that the M and S forms of A. gambiae are in the process of incipient speciation. AgH1 showed much higher levels of insertion polymorphisms in two west African populations of the M molecular form compared to two east African S populations. Similarly, the maximum level of chromosomal differentiation is observed in west African dry savannah areas, while a much lower degree of chromosomal polymorphism is observed in east Africa. Therefore our insertion data support the hypothesis that the speciation process is likely to be originated in west Africa, probably as the result of the need of ecological flexibility created by the greater ecological variability of this region. From a biomedical perspective, this type of analysis is critical because the genetic differences between M and S forms may directly impact the effectiveness of mosquito control measure and perhaps disease transmission. / Master of Science

Malaria parasite (Plasmodium spp.) infection in the mosquito Anopheles stephensi induces significant expression of A. stephensi nitric oxide synthase (AsNOS) in the midgut epithelium as early as 6 h post-infection and intermittently thereafter. This induction results in the synthesis of inflammatory levels of nitric oxide (NO) in the blood-filled midgut that limit parasite development. However, the Plasmodium-derived factors that can induce AsNOS expression and the signaling pathways responsible for transduction in A. stephensi have not been identified until completion of the work described herein. In my studies, I have determined that P. falciparum glycosylphosphatidylinositol (PfGPIs) can induce AsNOS expression in A. stephensi cells in vitro and in the midgut epithelium in vivo. Based on related work in mammals, I hypothesized that parasite-derived AsNOS-inducing factors signal through the insulin signaling pathway and the NF-kappaB-dependent Toll and Immune deficiency (Imd) signaling pathways. In support of this hypothesis, I have determined that signaling by P. falciparum merozoites and PfGPIs is mediated through A. stephensi protein kinase B (Akt/PKB) and DSOR1 (mitogen activated protein kinase kinase, MEK)/Extracellular signal-regulated protein kinase (ERK), kinases which are associated with the insulin signaling pathway. However, signaling by P. falciparum and PfGPIs is distinctively different from signaling by insulin and these parasite signals are not insulin-mimetic to A. stephensi cells. In other studies, treatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB, reduced AsNOS expression by P. falciparum merozoites in A. stephensi cells. This result suggested the involvement of Toll and Imd pathways in parasite signaling of mosquito cells. Knockout of Pelle, a proximal signaling protein in the Toll pathway, increased AsNOS expression following parasite stimulation, suggesting that the Toll pathway may negatively regulate signaling by Plasmodium-derived AsNOS-inducing factors. In contrast, knockout of TGF-beta-activated kinase 1 (Tak1), a proximal signaling protein in the Imd pathway, reduced AsNOS expression by 20% relative to the control, suggesting that the Imd pathway is required for signaling by Plasmodium-derived AsNOS-inducing factors. Despite the NO-rich environment of the midgut, Plasmodium development is not completely inhibited. This observation suggests that Plasmodium may have efficient detoxification systems during sexual development in A. stephensi. To identify Plasmodium defense genes that may defend parasites against nitrosative stress caused by AsNOS induction, expression of several antioxidant defense genes known to function in nitrosative stress defense in a variety of organisms were examined during sporogonic development. Notably, increased expression levels of P. falciparum peroxiredoxins containing 1 or 2 cysteines (1-cys or 2-cys PfPrx) were associated with periods of parasite development just prior to and during parasite penetration of midgut epithelium, an event associated with significant AsNOS induction in the midgut. The provision of N omega-L-arginine (L-NAME), a known inhibitor of NOS enzyme activity, to A. stephensi with Plasmodium culture by artificial bloodmeal significantly reduced expression of 1-cys and 2-cys PfPrx indicating that these gene products may function to protect parasites against nitrosative stress induced by AsNOS. / Ph. D.

Both vertebrates and anopheline mosquitoes inhibit <i>Plasmodium</i> spp. (malaria parasite) development via induction of nitric oxide (·NO) synthase. Expression of <i>Anopheles stephensi</i> ·NO synthase (<i>AsNOS</i>) is induced in the midgut epithelium beginning at 6 h following a <i>Plasmodium berghei</i>-infected blood meal. ·NO reacts readily with other biocompounds forming a variety of reactive nitrogen intermediates (RNIs) that may impose a nitrosative stress. These RNIs are proposed to be responsible for the AsNOS-dependent inhibition of <i>Plasmodium</i> development. In my studies, I identified several RNIs that are induced in the blood-filled midgut in response to <i>Plasmodium</i> infection. Stable end products of ·NO (NO₃⁻ and NO₂⁻), measured using a modified Griess assay, are elevated in infected midguts at 24 h post-blood meal (pBM). Further studies using chemical reduction-chemiluminescence with Hg displacement showed that infected midguts contained elevated levels of potentially toxic higher oxides of nitrogen (NO<SUB>x</SUB>), but <i>S</i>-nitrosothiol (SNO) and nitrite levels did not differ between infected and uninfected midguts at 12.5 and 24 h pBM. Thus, nitrates contributed to elevated NO<SUB>x</SUB> levels. SNO-biotin switch westerns indicated that <i>S</i>-nitrosated midgut proteins change over the course of blood meal digestion, but not in response to infection. Photolysis-chemiluminescence was used to release and detect bound ·NO from compounds in blood-filled midguts dissected from 0-33 h pBM. Results showed increased ·NO levels in <i>Plasmodium</i>-infected midgut lysates beginning at 8 h, with significant increases at 12.5-13.5 h and 24-25.5 h pBM and peak levels at 20-24 h. Photolyzed ·NO is derived from SNOs and metal nitrosyls. Since SNO concentrations did not change in response to infection, I proposed that metal nitrosyls, specifically Fe nitrosyl hemoglobin (nitrosylHb) based on the concentration of hemoglobin, were elevated in the infected midgut. At 12-24 h pBM, levels of midgut RNIs in infected mosquitoes were typical of levels measured during mammalian septic inflammation. The inverse relationship between AsNOS activity and parasite abundance indicates that nitrosative stress has a detrimental effect on parasite development. However, nitrosative stress may impact mosquito tissues as well in a manner analogous to mammalian tissue damage during inflammation. Elevated levels of nitrotyrosine (NTYR), a marker for nitrosative stress in many mammalian disease states, were detected in tissues of parasite-infected <i>A. stephensi</i> at 24 h pBM. Greater nitration of tyrosine residues was detected in the blood bolus, midgut epithelium, eggs and fat body. In the midgut, Hb remained in an oxygenated state for the duration of blood digestion. The reaction between ·NO and oxyhemoglobin (oxyHb) can result in the formation of nitrate and methemoglobin (metHb). Although nitrate levels were elevated in response to parasite infection, there was little to no metHb present in the mosquito midgut. The simultaneous presence of nitrates, nitrosylHb, oxyHb, and NTYR, together with a lack of elevated nitrites and metHb, suggested that alternative reaction mechanisms involving â ¢NO had occurred in the reducing environment of the midgut. In addition, I proposed that nitroxyl and peroxynitrite participated in reactions that yielded observed midgut RNIs. To cope with the parasite-induced nitrosative stress, cellular defenses in the mosquito may be induced to minimize self damage. I proposed that peroxiredoxins (Prx), enzymes that can detoxify peroxides and peroxynitrite, may protect <i>A. stephensi</i> from nitrosative stress. Six Prx genes were identified in the <i>A. gambiae</i> genome based on homology with known <i>D. melanogaster</i> Prxs. I identified one <i>A. stephensi</i> Prx, AsPrx, that shared 78% amino acid identity with a <i>D. melanogaster</i> 2-Cys Prx known to protect fly cells against various oxidative stresses. <i>AsPrx</i> was expressed in the midgut epithelium and is encoded by a single-copy, intronless gene. Quantitative RT-PCR analyses confirmed that induction of <i>AsPrx</i> expression in the midgut was correlated with malaria parasite infection and nitrosative stress. To determine whether AsPrx could protect against RNI- and ROS-mediated cell death, transient transfection protocols were established for AsPrx overexpression in <i>D. melanogaster</i> (S2) and <i>A. stephensi</i> (MSQ43) cells and for <i>AsPrx</i> gene silencing using RNA interference in MSQ43 cells. Viability assays in MSQ43 cells showed that AsPrx conferred protection against hydrogen peroxide, ·NO, nitroxyl and peroxynitrite. These data suggested that the ·NO-mediated defense response is toxic to both host and parasite. However, AsPrx may shift the balance in favor of the mosquito. / Ph. D.

The Anopheles gambiae complex is comprised of the most important vectors of malaria in Sub-Saharan Africa. Most current control methods involve the use of chemicals that help to limit potential contact with these mosquitoes. However, these control methods still have risks that include insect resistance, environmental toxicity, human health, as well as animal health. In order to develop new strategies that either produce novel targeted insecticides or transgenic mosquitoes that can replace current mosquito populations, it is important to acquire as much biological information about the vector as possible. The reduction in cost and speed of high-throughput sequencing has brought forth many new sequenced genomes that can provide a wealth of information about individual populations as well as their respective evolutionary histories. However, in order to fully understand a genome, these sequences must be assembled properly. One of the largest challenges toward fully assembling a genome is the abundance of repetitive sequences. These sequences, typically part of gene poor regions known as heterochromatin, are generally left as unassembled scaffolds that are neglected in many genomic studies. Heterochromatin is a biologically important chromatin state that has roles in gene regulation and genome stability. Exclusion of these chromatin domains from experimental assays can provide an incomplete picture in regards to organismal biology. A lack of information regarding heterochromatin, even in An. gambiae, necessitates further understanding and characterization of this chromatin type that can provide valuable information about the mosquito's biology. Heterochromatin is organized differently amongst different species. Some species with compact genomes, like Drosophila melanogaster, exhibit rigid organization of heterochromatin, with repetitive elements being confined to peri-centromeric and sub-telomeric regions of the chromosome. Larger genomes such as Aedes aegypti, have a much less structured heterochromatin pattern, with repetitive elements being dispersed across the genome. However, An. gambiae's genome is more intermediate in size as well as transposable element content. These factors may have an impact in controlling how heterochromatin is organized within the An. gambiae genome. Does An. gambiae compensate for the increased genome size by expanding past the peri-centromeric heterochromatin into new intercalary compartments? In An. gambiae, heterochromatin had yet to be identified separately from euchromatin. Morphologically, some regions of An. gambiae chromosomes exhibited characteristics similar to transcriptionally active puffs or peri-centromeric heterochromatin. We characterize these regions, as well as the rest of the genomic landscape, by using morphological and genetic features to identify various chromatin types. Peri-centromeric heterochromatin and new regions of intercalary heterochromatin were identified. Genomic coordinates representing the transition from euchromatin to heterochromatin were also identified. By finding these heterochromatin-euchromatin boundaries, various genetic features could be assigned to either heterochromatin or euchromatin. Critical genes associated with heterochromatin formation and basic genomic functions were identified. These data help to better understand features that are associated with the different environments created by chromatin compaction. This study also looks at the Piwi-interacting RNA (piRNA) pathway and its role in An. gambiae. The piRNA pathway is associated with transposable element (TE) suppression in many species, where clusters of vestigial TEs provide some of the RNA necessary for the pathway to function. These clusters are primarily associated with heterochromatin in Drosophila melanogaster. We identify piRNA clusters in An. gambiae and see a similar shift from primarily peri-centromeric compartmentalization toward the presence of intercalary regions located within the euchromatin. Transposable elements are maintained in secondary heterochromatin regions that exhibit similar morphology and features to peri-centromeric heterochromatin. The piRNA pathway also has implications in gene regulation, germline development, and anti-viral immunity. Three candidate genes associated with spermatogenesis and embryogenesis have been identified. These genes showed piRNA enrichment, and upon further analysis show up-regulation after a blood meal is taken. These genes could potentially prove useful in vector control as targets of transgenic experiments. Heterochromatin is an important, yet neglected aspect of the genome. These studies attempt to provide data to stimulate the study of heterochromatin through characterization of heterochromatin-related genomic features. / Ph. D.

Malaria is the main cause of approximately one million deaths every year that mostly affect children in south of Sub-Saharan Africa. The Anopheles gambiae complex consists of seven morphologically indistinguishable sibling species. However, their behavior, ecological adaptations, vectorial capacity, and geographical distribution differ. Studying the phylogenetic relationships among the members of the complex is crucial to understanding the genomic changes that underlie evolving traits. These evolutionary changes can be related to the gain or loss of human blood choice or to other epidemiologically important traits. In order to understand the phylogenetic relationships and evolutionary history of the members of the An. gambiae complex, breakpoints of the 2Ro and 2Rp inversions in An. merus and their homologous sequence in the outgroup species were analyzed using fluorescent in situ hybridization (FISH), library screening, whole-genome mate-paired sequencing and bioinformatics analysis. Molecular phylogenies of breakpoint genes were constructed afterwards. In addition, multigene phylogenetic analyses of African malaria vectors were performed. Our findings revised the chromosomal phylogeny, and demonstrated the ancestry of 2Ro, 2R+p and 2La arrangements.  Our new chromosomal phylogeny strongly suggests that vectorial capacity evolved repeatedly in members of the An. gambiae complex, and the most important vector of malaria in the world, An. gambiae, is more closely related to ancestral species than was previously thought. Our molecular phylogeny data were in agreement with chromosomal phylogeny, indicating that the position of the genetic markers with respect to chromosomal inversion is important for interpretation of the  phylogenetic trees. Multigene phylogenetic analysis revealed that a malaria mosquito from humid savannah and degraded rainforest areas, An. nili, belongs to the basal clade and is more distantly related to other major African malaria vectors than was assumed previously. Finally, for the first time a physical map of 12 microsatellite markers for the Asian malaria vector An. stephensi was developed. Knowledge about the chromosomal position of microsatellites was shown to be important for a proper estimation of population genetic parameters. In conclusion, our study improved understanding of genetics and evolution of some of the major malaria vectors in Africa and Asia. / Ph. D.