Porphyromonas gingivalis é um dos principais organismos associados à periodontite crônica e apresenta intensa diversidade, que poderia refletir em sua virulência. A maioria dos estudos sobre a virulência de P. gingivalis foi realizada com cepas de referência, e pouco se conhece sobre este aspecto em isolados clínicos. A capacidade de indução de abscessos difusos em modelos animais experimentais parece estar associada a cepas capsuladas, enquanto a expressão de fímbrias e a capacidade de internalização em células epiteliais não fagocíticas, foram relacionadas à cepa não capsulada. Em P. gingivalis, o lócus de biossíntese do polissacarídeo capsular (BPC) apresenta características de ter sido adquirido por transferência horizontal de genes. O objetivo do presente estudo foi testar a hipótese de que a estrutura populacional de P. gingivalis relaciona-se com a variabilidade do lócus BPC e características fenotípicas como produção de cápsula e hidrofobicidade. Foram analisadas 28 cepas de P. gingivalis pertencentes aos 5 genótipos fimA quanto a, presença da cápsula por microscopia óptica, hidrofobicidade e detecção de genes do lócus BPC por PCR. A análise filogenética foi realizada por tipagem através de seqüenciamento de genes housekeeping (multilocus sequence typing, MLST). Dezesseis entre 28 amostras estudadas apresentaram cápsula, e não foram detectadas diferenças na hidrofobicidade dos isolados clínicos capsulados e não capsulados. O gene pg0106 foi detectado por PCR em 78% das amostras capsuladas e em 80% das amostras não capsuladas, enquanto pg0111 foi detectado em apenas 25% de amostras capsuladas. Apenas um isolado clínico e a amostra padrão W83 foram classificados como K1, por apresentarem o gene pg0118. Através do MLST foi possível identificar grande variabilidade entre as amostras de P.gingivalis. Foi observada relação entre STs e o tipo fimA ou hidrofobicidade, mas nenhum cluster foi associado à presença da cápsula ou aos genes do lócus de biossíntese do polissacarídeo capsular. Os dados indicam associação entre o lócus BPC e produção de cápsula, porém a diversidade deste lócus parece ser maior do que a relatada na literatura. O lócus BPC e a produção de cápsula não se relacionam com a origem filogenética da cepa, indicando a intensa recombinação que ocorre em P. gingivalis. / Porphyromonas gingivalis is one of main organisms associated with chronic periodontitis and is largely diverse, which could reflect in its virulence. Most studies on the virulence of P.gingivalis were performed with reference strains, and little is known about this aspect in clinical isolates. The ability to induce diffuse abscesses in experimental animal models seems to be associated with encapsulated strains, while expression of fimbriae and the ability to internalize into non phagocytic epithelial cells were related to noncapsulated strains. In P.gingivalis, the locus of biosynthesis of the capsular polysaccharide (GP) has characteristics of having been acquired by horizontal gene transfer. This study aimed to test the hypothesis that the structure population of P.gingivalis is related to the variability of the GPC locus and phenotypic characteristics such as capsule production and hydrophobicity. 28 P.gingivalis isolates representing fimA genotypes were screened for presence of capsule by light microscopy, hydrophobic properties and detection of genes in the GPC locus by PCR. The phylogenetic analysis was performed by sequencing of housekeeping genes typing (multilocus sequence typing, MLST). Sixteen of 28 studied samples had capsule, and there were no differences in the hydrophobic properties of capsulated and non capsulated clinical isolates. The pg0106 gene was detected by PCR in 78% of capsulated isolates and in 80% of not capsulated while pg011 was detected in only 25% of encapsulated isolates. One clinical isolate and reference strain W83 were classified as K1, due to the presence of pg0118 gene. MLST detected large variations within the P.gingivalis population. MLST clustering analysis revealed a relation between sequence type (STs) and fimA genotype or hydrophobic property, but there was no association of STs with the presence of capsule or the genes encoding for the biosynthesis of capsular polysaccharide. The data indicated an association between GPC locus and capsule production but the diversity of this locus appeared to be greater than that reported in the literature. The GPC locus and capsule production were not related to the phylogenetic origin of the strain, indicating intense recombination in P. gingivalis.

Made available in DSpace on 2017-07-21T19:59:53Z (GMT). No. of bitstreams: 1 Ricardo Oliveira Brito.pdf: 1923139 bytes, checksum: 08b643e5706fe65ed2608e5a4ed4e1e9 (MD5) Previous issue date: 2011-03-25 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The order Thysanoptera comprises small insects known as thrips, which present different life histories and habits; they reproduce by haplodiploidy, and reached the status of prague by their feeding damage to plants. Their systematics, taxonomy and phylogenetic relationships are complicated, due to the small morphological variation among the taxa. Thus, this study aimed to analyze three species of the family Phlaeothripidae: Gynaikothrips uzeli, Liothrips sp. and Pseudophilothrips gandolfoi through conventional and molecular cytogenetics giving emphasis to the evolutionary processes that have occurred during the differentiation karyotype in the group, reinforcing the importance of cytogenetic studies as a tool for taxonomy of order, in addition to estimate genetic population parameters of G. uzeli. To that end, we analyzed the diploid number, the chromosome morphology, the distribution pattern of the heterochromatic regions, the chromosomes bearing Ag-NORs and performed mapping physical for cístrons of rDNA 18S. In addition, it was estimated the genetic variability and population structure of G. uzeli through the RAPD marker. The results obtained with the probe of 18S rDNA revealed a pair chromosomal bearer of RON in the three species, however, in G. uzeli only one of the elements of chromosomal 13 pair was marked. In addition, the results of cytogenetic analysis indicate that the genera Pseudophilothrips and Liothrips display strong similarities in their chromosomal complements. The low genetic variability, observed with the RAPD marker for G. uzeli, and result of the system of sexual determination haplodiploidy and ecological features. The cytogenetic analysis revealed differences that are difficult to observe the morphological level, demonstrating the importance of this methodology for systematic and taxonomy and clarification of complex evolutionary patterns presented in the order. / A ordem Thysanoptera compreende pequenos insetos conhecidos como tripes, os quais apresentam diversas histórias de vida e hábitos, se reproduzem por haplodiploidia, e alcançaram o status de praga por sua alimentação danificar as plantas. Sua sistemática, taxonomia e relações filogenéticas são complicadas, devido à pequena variação morfológica entre os taxa. Assim, este trabalho teve como objetivo analisar três espécies da família Phlaeothripidae: Gynaikothrips uzeli, Liothrips sp. e Pseudophilothrips gandolfoi através de citogenética convencional e molecular dando ênfase aos processos evolutivos que ocorreram durante a diferenciação cariotípica do grupo, reforçando a importância da citogenética como ferramenta para taxonomia da ordem, além de estimar parâmetros genético populacionais de G. uzeli. Para tal, foi analisado o número diplóide, a morfologia cromossômica, o padrão de distribuição das regiões heterocromáticas, os cromossomos portadores de Ag-RONs e realizado o mapeamento físico dos cístrons de rDNA 18S. Além disso, foi estimada a variabilidade genética e a estrutura populacional de G. uzeli através do marcador RAPD. Os resultados obtidos com a sonda de rDNA 18S revelaram um par cromossômico portador da RON nas três espécies, no entanto, em G. uzeli somente um dos elementos cromossômicos do 13º par foi marcado. Em adição, os resultados da análise citogenética indicam que os gêneros Pseudophilothrips e Liothrips apresentam grandes semelhanças em seus complementos cromossômicos. A Baixa variabilidade genética, observada com o marcador RAPD, para G. uzeli, é resultado do sistema de determinação sexual haplodiplóide e de características ecológicas. As análises citogenéticas revelaram diferenças que são difíceis de observar no nível morfológico, demonstrando a importância desta metodologia para a sistemática e a taxonomia e no esclarecimento dos complexos padrões evolutivos apresentado pela ordem.

L'hyperchylomicronémie est une maladie rare et complexe impliquant plusieurs gènes qui sont eux-mêmes fortement régulés par plusieurs mécanismes et dont les voies métaboliques sont étroitement dépendantes de facteurs environnementaux. La survenue de la pathologie due à la présence de variants ou d'une association de variants sur ces gènes n'est pas toujours clairement définie. Ce qui suggère l'intervention d'autres mécanismes mal élucidés dans le développement des hyperchylomicronémies et la régulation du métabolisme des triglycérides. Nous avons essayé d'appréhender certains mécanismes causals dans la survenue de l hyperchylomicronémie en lien avec la présence de variants sur les gènes régulateurs APOC3 et LMF1 du métabolisme des triglycérides. Le gène APOC3 présente le variant SstI (rs5128) en région 3' non codante associée significativement à l'hypertriglycéridémie dans notre cohorte, nous avons cherché à caractériser sa régulation post-transcriptionnelle éventuelle par des microARN hépatiques ou intestinaux. Nos résultats ne confirment pas l'hypothèse d'une régulation du variant SstI du gène APOC3 par un microARN hépatique ou intestinal ciblant directement l'extrémité 3'UTR du gène APOC3. Le gène LMF1, nouveau gène candidat pour étudier les mécanismes des hyperchylomicronémies, est encore peu investigué. Nous avons mis en place son diagnostic génétique au sein du laboratoire ainsi qu'une technique in vitro permettant d'évaluer l'impact de la présence de certains variants codants de LMF1 sur l'activité post héparinique de la lipoprotéine lipase (LPL) par mesure de la lipolyse des triglycérides des VLDL. Nous avons mis en évidence des activités LPL significativement diminuées suggérant une dysfonction de LMF1 en présence des variants p.Gly172Arg (rs201406396), p.Arg354Trp (rs143076454), p.Arg364Gln (rs35168378), et des deux variants non-sens déjà décrits p.Tyr439Ter (rs121909397) et p.Trp464Ter (rs587777626). Ces travaux permettent de confirmer l'effet fonctionnel des variants LMF1 sur la régulation de la sécrétion de la LPL. Nous avons également retrouvé dans notre cohorte de 385 patients 18 variants sur la région 3' non codante du gène LMF1. Pour les trois variants : c*231C>A (rs75476513), c*512G>A (rs117039680), et c*530G>A (rs139657279), les résultats in vitro suggèrent une régulation post transcriptionnelle par les microARN. Ce qui pourrait ainsi expliquer le mécanisme de l'association de ces variants non traduits à l'hypertriglycéridémie. Ainsi, des interrelations des multiples gènes impliqués dans le métabolisme des triglycérides et leurs régulations à plusieurs niveaux simultanés modulent le phénotype d'hyperchylomicronémie. Il est nécessaire d'étudier tous les mécanismes complexes impliqués dans la régulation de la triglycéridémie afin de mieux appréhender la physiopathologie et de développer de nouvelles cibles thérapeutiques / Hyperchylomicronemia is a rare and complex disease involving several genes which are themselves highly regulated by several mechanisms and whose metabolic pathways are closely dependent on environmental factors. The occurrence of this disease due to the presence of variants or a combination of variants on these genes is not always clearly defined. This suggests the intervention of other ill-defined mechanisms in the development of hyperchylomicronemia and the regulation of triglyceride metabolism. We have tried to understand certain causal mechanisms in the occurrence of hyperchylomicronemia in relation to the presence of variants on the APOC3 and LMF1 known regulatory genes of triglyceride metabolism. APOC3 gene carries the SstI variant (rs5128) in the 3' untranslated region significantly associated with hypertriglyceridemia in our cohort. We sought to characterize its possible post-transcriptional regulation by hepatic or intestinal microRNA. Our results obtained in vitro do not support the hypothesis of a regulation of the SstI variant of the APOC3 gene by a hepatic or intestinal microRNA directly targeting the 3'UTR of APOC3 gene. LMF1 gene, a new candidate gene for studying the mechanisms of hyperchylomicronaemias, is still under investigation. We have established its genetic diagnosis in the laboratory and set up an in vitro method to evaluate the impact of LMF1 coding variants by measuring the release of post-heparin lipoprotein lipase (LPL) activity. We found decreased LPL activities suggesting a LMF1 dysfunction in the presence of variants p.Gly172Arg (rs201406396), p.Arg354Trp (rs143076454), p.Arg364Gln (rs35168378), and the two nonsense variants already described p.Tyr439Ter (rs121909397) and p.Trp464Ter (rs587777626). This study confirms the functional effect of LMF1 variants on the regulation of LPL secretion. In addition, we found 18 variants on the 3' untranslated region of LMF1 gene. For three variants : c*231C>A (rs75476513), c*512G>A (rs117039680), and c*530G>A (rs139657279), in vitro results suggest a post-transcriptional regulation by microRNA. These findings are an involvement of these untranslated variants in the occurrence of hypertriglyceridemia.Thus, complex interrelations of multiple genes involved in triglyceride metabolism and their simultaneous multi-level regulation modulate the phenotype of hyperchylomicronemic patients. It is necessary to study all the complex mechanisms involved in the regulation of triglyceridemia in order to better understand pathophysiology of hyperchylomicronemia and to develop new therapeutic targets

A cultura da batata (Solanum tuberosum L) está se tornando cada vez mais importante, tanto do ponto de vista dos produtores, dos pesquisadores e dos consumidores, por ser um dos alimentos protéicos mais consumidos em todo o mundo, entretanto, o Brasil depende de variedades importadas, originárias de clima temperado o que não condiz com as nossas condições, refletindo assim em valores inferiores de produtividade e de qualidade. Apesar da grande evolução que esta cultura apresentou em todos estes anos de cultivo se faz necessário a busca por materiais mais produtivos, adaptados e resistentes. Os programas de melhoramento convencionais são extremamente importantes para a seleção de novos progenitores, entretanto o tempo gasto para desenvolver e lançar uma variedade é bastante longo. Diante deste cenário, novas metodologias estão sendo cada vez mais utilizadas no processo de identificação de bancos de germoplasma e cultivares mais promissoras. O objetivo deste trabalho foi avaliar, por meio de marcadores microssatélites, 108 acessos de batata de cinco coleções contendo variedades comerciais, clones para programas de melhoramento e cultivo orgânico, visando a caracterização genética, a identificação de duplicatas e de possíveis parentais para uso nos programas de melhoramento. Para a caracterização molecular foram utilizados 10 iniciadores específicos (primers), gerando-se um total de 50 alelos (bandas) os quais foram analisados como dados binários, sendo que a partir destes dados foi obtida uma matriz de similaridade utilizando o coeficiente de similaridade de Jaccard. Com este coeficiente e com o método aglomerativo UPGMA, foram realizadas análises de agrupamento utilizando o software NTSYSpc e um método de reamostragens (bootstraps), gerando dendrogramas que permitiram a distinção genética entre os acessos. O conteúdo de informação de polimorfismo (PIC) e a heterogozidade esperada (He) foram significativos, sendo que os maiores valores foram 0,8594 e 0,8725, respectivamente, para o primer STM0019a. Em média, o número de alelos por loco foi cinco, variando de dois alelos para os primers STM 1053 e STM 1104 até 13 alelos por loco para o primer STM0019a. Para facilitar a visualização dos resultados, além de serem avaliados como um todo os 108 acessos foram divididos em grupos de acordo com as coleções (variedades comerciais, clones e cultivo orgânico), sendo que a maior variação pelo coeficiente de Jaccard foi de 0,39 a 0,93 para 57 acessos das coleções de cultivares orgânicos e comerciais. Ao se avaliar os 108 acessos juntos, o coeficiente de Jaccard variou de 0,42 a 0,93, mostrando a grande variabilidade genética entre os acessos das cinco coleções. Foram observadas seis possíveis duplicatas [ATLANTIC (Canadá) e ATLANTIC (Chile); 67-2 e 17-10 (clones CNPH1); COLORADO e ÁGATA (EPAMIG); 253 E 266 (clones CNPH2); MELODY e APTA 21-54 (cultivo orgânico); e 387-1 (E1) e VOYAGER], identificando-se também os acessos mais distantes geneticamente [clone 383-19 (EmbrapaCNPH1) e a cultivar comercial HPC-7B], permitindo desta forma a identificação de possíveis parentais para os programas de melhoramento. Os altos níveis de polimorfismo observados paraSolanum tuberosum sugerem que os marcadores microssatélites podem ser uma ferramenta útil para detectar as diferenças genéticas entre cultivares de batata. / The cultivation of potato (Solanum tuberosum L) is becoming increasingly important from the point of view of producers, researchers and consumers, for representing one of the food protein mostly consumed in the world. However, Brazil depends on imported varieties, originated from temperate climate which does not complies with our conditions, thus reflecting in lower productivity and quality. Despite the great progress that this crop presented in all these years of cultivation, it is necessary to search for more productive, adapted and resistant materials. The conventional breeding programs are important for the selection of new parents, but the time spent to develop and launch a new variety is quite long. In this scenario, new approaches are being increasingly used in the identification of germplasm banks and most promising cultivars. The objective of this study was to evaluate, using microsatellite markers, 108 accessions of five potato collections containing commercial varieties, clones for breeding programs and organic farming varieties, aiming at the genetic characterization, identification of duplicates and possible parents to be used in potato breeding programs. For the molecular characterization, 10 specific primers were used, generating a total of 50 alleles (bands) which were analyzed as binary data, and from this data a similarity matrix was obtained using the Jaccard coefficient of similarity. With this coefficient and the UPGMA method, cluster analysis were carried out using the NTSYSpc software and bootstraps analyses, generating dendrograms which allowed the genetic distinction between accessions. The polymorphism information content (PIC) and expected heterozygosity (He) were both significant, with the highest values (0.8594 and 0.8725, respectively) obtained for primer STM0019a. On average, the number of alleles per locus was five, ranging from two alleles for primers STM 1053 and STM 1104 to 13 alleles per locus for primer STM0019a. To facilitate the visualization of the results, in addition to being evaluated as a whole, the 108 accessions were divided into groups according to the collections (commercial varieties, clones and organic farming), where the highest variation for the Jaccard coefficient (0.39 - 0.93) was found for the 57 accessions of organic and commercial cultivars collections. When assessing the 108 accessions together, the Jaccard coefficient ranged from 0.42 to 0.93, showing a high genetic variability between accessions of the five collections. Six possible duplicates were found [\'ATLANTIC (Canada) and ATLANTIC (Chile); 67-2 and 17-10 (clones CNPH1); Color and AGATE (EPAMIG); 253 E 266 (clones CNPH2); MELODY and APTA 21-54 (organic farming); and 387-1 (E1) and VOYAGER], and also the more genetically distant accessions [clone 383-19 (EmbrapaCNPH1) and the commercial cultivar HPC- 7B] were identified, thereby enabling the identification of potential parents for breeding programs. High levels of polymorphism observed for Solanum tuberosum suggest that microsatellite markers can be a useful tool to detect the genetic differences between potato cultivars.

Heliothis virescens (Fabricius) é uma das pragas-alvo do algodão geneticamente modificado que expressa a proteína Cry1Ac de Bacillus thuringiensis Berliner (Bt). Estudos sobre a suscetibilidade de H. virescens à proteína Cry1Ac e sobre a estrutura genética e padrões de fluxo gênico nas escalas locais e regionais desse inseto são fundamentais para a implementação de programas de Manejo da Resistência de Insetos (MRI) no Brasil. Dessa forma, os principais objetivos do trabalho foram: (a) avaliar a suscetibilidade à proteína Cry1Ac em populações de H. virescens coletadas nas principais regiões produtoras de algodão no Brasil (Bahia, Goiás, Mato Grosso e Mato Grosso do Sul) durante as safras agrícolas 2007/08 e 2008/09; e (b) avaliar a variabilidade genética e fluxo gênico de populações de H. virescens provenientes das culturas de algodão (safras 2007/08, 2008/09 e 2009/10) e de soja (safra 2009/10) utilizando sequências de DNA mitocondrial (DNAmit). As linhas-básica de suscetibilidade à proteína Cry1Ac foram estabelecidas mediante o uso de lagartas neonatas, por meio de bioensaios de incorporação das diferentes concentrações da proteína em dieta artificial. Para avaliar a variabilidade genética e o fluxo gênico entre populações de H. virescens utilizou-se sequências de DNAmit das subunidades I e II da citocromo oxidase COI e COII e a subunidade 6 da desidrogenase dinucleotídica da adenina nicotinamida nad6. As CLs50 estimadas variaram de 0,18 a 0,66 µg de Cry1Ac/mL de dieta para as populações coletadas na safra 2007/08 (variação de 3,7 vezes). Da mesma forma, as concentrações efetivas médias para a inibição do desenvolvimento larval (CE50) variaram de 0,0053 a 0,0161 µg de Cry1Ac/mL dieta (variação de 3,0 vezes). A partir da análise conjunta dos dados de concentração-mortalidade de todas as populações avaliadas, foram definidas e validadas as concentrações diagnósticas de 3,1 e 5,6 µg de Cry1Ac/mL de dieta para programas de monitoramento da resistência de H. virescens à proteína Cry1Ac no Brasil. Baseadas em análises de agrupamento (Neighbor-Joining e Análise de Componentes Principais) e Bayesiana (Structure) foram verificadas uma baixa estruturação entre as populações de H. virescens de diferentes regiões, bem como para aquelas coletadas em plantas hospedeiras diferentes. As análises de AMOVA também indicaram baixa estruturação genética entre as populações de H. virescens estudadas independente da cultura (Fst= 0,019) ou escala geográfica (Fst= 0,012), sugerindo um nível significativo de fluxo gênico. Em média, a distância genética entre as amostras foi de 0,1%. Uma rede de haplótipos obtida com os dados combinados resultou em 35 haplótipos, com quatro haplótipos únicos presentes somente nas amostras coletadas em soja. A principal característica dessa rede é a forma de estrela na distribuição dos haplótipos, bem como a ocorrência de muitos alelos em baixa frequência. Esse tipo de rede é característico de populações que passaram por uma recente expansão populacional e, de fato, a história demográfica de H. virescens, baseada no teste de distribuição da diferença genética par-a-par entre haplótipos (distribuição de Mismatch) e nos resultados negativos nos testes de neutralidade seletiva indicam também um episódio de expansão populacional recente. As informações obtidas no presente trabalho serão fundamentais para o acompanhamento da efetividade das estratégias de manejo da resistência de H. virescens à proteína Cry1Ac no Brasil. / The tobacco budworm, Heliothis virescens (Fabricius), is one of target pests of genetically modified cotton expressing Cry1Ac insecticidal protein derived from Bacillus thuringiensis Berliner. Studies on susceptibility of H. virescens to Cry1Ac and the genetic structure and gene flow patterns at local and regional levels are crucial for establishing an Insect Resistance Management (IRM) program for Bt cotton in Brazil. Thus, the objectives of this study were (a) to evaluate the susceptibility of field-collected populations of H. virescens to Cry1Ac from major cotton-growing regions in Brazil (Bahia, Goiás, Mato Grosso and Mato Grosso do Sul) in the cropping seasons of 2007/08 and 2008/09; and (b) to evaluate the genetic variability and gene flow among H. virescens populations from cotton (2007/08, 2008/09 and 2009/10 cropping seasons) and soybean (2009/10 cropping season) with mitochondrial DNA markers. Baseline susceptibility data to Cry1Ac protein were estimated with neonate larvae thereby using diet incorporation bioassays. Genetic variation and gene flow among H. virescens populations were evaluated by using mitDNA sequences of cytochrome oxidase subunities I and II COI e COII and the subunity 6 of dinucleotide dehydrogenase of adenine nicotinamide nad6. The estimated LC50 values varied from 0.18 to 0.66 µg of Cry1Ac/mL of diet among the 2007/08 populations (3.7 fold variation). Similarly, the EC50 values based on growth inhibition ranged from 0.0053 to 0.0161 µg of Cry1Ac/mL of diet for the 2007/08 populations (3.0 fold variation). A joint analysis of the mortality data across all tested populations was used to develop candidate diagnostic concentrations for future monitoring programs. The proposed diagnostic concentrations of 3.1 and 5.6 µg of Cry1Ac/mL of diet were validated against field-collected populations from 2008/09 and will form the basis for future resistance monitoring programs with H. virescens. Based on cluster analysis (Neighbor-Joining and Principal Coordinate Analysis) and Bayesian analysis (Structure), a low structure was detected among H. virescens populations either by regions or host plants. AMOVA analysis also indicated low genetic structure among H. virescens populations across crops (Fst= 0.019) or geographic scale (Fst= 0.012), suggesting a significant gene flow. The mean genetic distance among samples was 0.1%. The haplotype network obtained with joint data resulted in 35 haplotypes, with four unique haplotypes present only in samples collected from soybean crop. The major characteristics of the haplotype network were the star-like pattern and the occurrence of many alleles at low frequencies. This type of network is typical for populations that passed through a recent population expansion and, in fact, the demographic history of H. virescens, based on distribution test of pair-wise genetic difference among haplotypes (Mismatch distribution) and negative results from tests of selective neutrality also indicate an episode of a recent population expansion.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-04-26T19:53:52Z No. of bitstreams: 1 2012 - Simone Quinelato Bezerra.pdf: 1703088 bytes, checksum: c39ad4f0bc5fb8b05085a7e4684060b9 (MD5) / Made available in DSpace on 2017-04-26T19:53:52Z (GMT). No. of bitstreams: 1 2012 - Simone Quinelato Bezerra.pdf: 1703088 bytes, checksum: c39ad4f0bc5fb8b05085a7e4684060b9 (MD5) Previous issue date: 2012-03-30 / FAPERJ / Aiming to decrease the chemicals acaricide use and their damages, new alternatives for ticks control has been studied. Metarhizium anisopliae s.l. is one of the most studied fungi in agricultural pest management programs, since it has great acaricide potential. Therefore, this study aimed to characterize molecular and morphologically, as well as evaluate the virulent potential of 30 M. anisopliae s.l. isolates from different geographical regions, hosts or substrates allowing the selection of virulent isolates in order to be further investigated for field programs of microbial control of pests. Initially, the analyses of morphological characterizations of the isolates were made to confirm their identification. Each isolate had its conidial potential production evaluated. The colonies studied showed morphological characteristics consistent with those described in the literature. The colonies diameter varied between 29.66 mm and 51.33 mm among isolates. There was both length and width variation in the conidia and phialides in the same isolate, as well as the presence of grouped and solitary phialides. The conidial production potential was variable among isolates, but both conidial size and colonies diameter did not influence the conidial production; isolates with low conidial production showed similar colony size in comparison to isolates with high potential. In a second stage of the study, the virulence of these isolates was evaluated to Rhipicephalus microplus larvae treated with one of the four different conidial concentrations (105, 106, 107 or 108 conidia.mL-1). The lethal action of Brazilian M. anisopliae s.l isolates to R. microplus larvae were confirmaded with high mortality among the isolates, which in general was proportional to the conidia concentration of the treatments. Most isolates killed larvae population with 107 conidia.mL-1 concentration, however the most virulent isolates presented lethal concentration of 106 conidia.mL-1 with main percentages of mortality nearly 100% at day 20 after treatment. In addition, the genetic variability of these isolates was performed to evaluate their relationship with other species of Metarhizium sp. through RFLP-PCR analysis and ITS1-5.8S-ITS2 rDNA sequencing. No specificity pattern was observed when isolates from the same region, host or substrate were grouped. Low genetic variability was observed among isolates, which were basically grouped into two groups. The CG 344 isolate was shown to be genetically distant from the remaining Brazilian isolates studied, but according to the ?GenBank? sequences comparison, it was related to the Metarhizium genus. It is suggested that this variation occured owing the lack of procedures that could generated morphological and molecular changes, which probably contribute to this low genetic variability. The present study allowed the detection of M. anisopliae s.l. isolates with highly virulence to R. microplus larvae, that may be considered potential biocontrol agents for this tick species, emphasizing the importance of molecular tools for identification and characterization of fungal isolates, ensuring the product quality, their success implement and the environmental track of the fungi at field biological control programs. / Na tentativa de diminuir a utiliza??o de produtos qu?micos e os danos por eles causados, novas alternativas para o controle de carrapatos vem sendo estudadas. O fungo Metarhizium anisopliae ? um dos mais estudados em programas agropecu?rios de manejo de pragas, pois apresenta grande potencial acaricida. Baseado nisso, o presente estudo objetivou a caracteriza??o morfol?gica, molecular e a avalia??o da virul?ncia de 30 isolados brasileiros de M. anisopliae s.l. provenientes de diferentes regi?es geogr?ficas, hospedeiros ou substratos, com a finalidade de selecionar isolados mais virulentos para utiliza??o em futuros programas de biocontrole de carrapatos. Inicialmente os isolados foram caracterizados morfologicamente para confirma??o de sua identifica??o, tamb?m sendo avaliado o potencial de produ??o de con?dios de cada isolado. As col?nias estudadas apresentaram caracter?sticas morfol?gicas compat?veis com as descritas na literatura. O tamanho das col?nias variou entre 29,66 mm e 51,33 mm de di?metro. Houve varia??o no comprimento e na largura de con?dios e fi?lides num mesmo isolado, assim como a presen?a de fi?lides agrupadas e solit?rias. O potencial de produ??o de con?dios foi vari?vel entre os isolados, por?m tanto o tamanho dos con?dios quanto o di?metro das col?nias n?o influenciaram a produ??o de con?dios. Numa segunda etapa do estudo, foi avaliada a virul?ncia destes isolados sobre larvas de Rhipicephalus microplus tratadas com uma das quatro diferentes concentra??es de con?dios (105, 106, 107 ou 108 con?dios/mL). Foi confirmada a a??o letal dos isolados brasileiros de M. anisopliae s.l. sobre larvas de R. microplus, geralmente ocorrendo de forma diretamente proporcional a concentra??o conidial dos tratamentos. A maioria dos isolados ocasionou a morte de metade da popula??o de larvas com a concentra??o de 107 con?dios/mL; os isolados mais virulentos apresentaram esta concentra??o letal com 106 con?dios/mL, com percentuais m?dios de mortalidade de larvas pr?ximos de 100% ao 20? dia ap?s tratamento. Al?m disso, buscou-se avaliar a variabilidade gen?tica destes isolados e sua rela??o com outras esp?cies do g?nero Metarhizium atrav?s da an?lise de RFLP-PCR e do sequenciamento da regi?o ITS1-5.8S-ITS2 do rDNA. N?o foi observado um padr?o de especificidade para o agrupamento entre isolados oriundos de mesma regi?o, hospedeiro ou substrato. Foi observada variabilidade gen?tica entre os isolados que basicamente se agruparam em dois grupos. O isolado CG 344 mostrou-se geneticamente distante de todos os outros, mas de acordo com a compara??o com sequ?ncias obtidas do ?GenBank? mostrou-se relacionado ao g?nero Metarhizium. Esta varia??o pode ser devido ao fato deste isolado ter sido poupado de processos que gerassem altera??es morfol?gicas e moleculares, o que possivelmente contribuiu para a pequena variabilidade gen?tica obsevada. O presente estudo possibilitou a detec??o de isolados brasileiros de M. anisopliae s.l. com elevada virul?ncia para larvas de R. microplus, podendo ser considerados potenciais agentes no biocontrole desta esp?cie de carrapato, ressaltando a import?ncia da utiliza??o de ferramentas moleculares para identifica??o e caracteriza??o destes isolados, contribuindo para a qualidade do produto, o sucesso de sua aplica??o e o monitoramento de um isolado introduzido no ambiente com finalidade de controle biol?gico.

Heliothis virescens (Fabricius) é uma das pragas-alvo do algodão geneticamente modificado que expressa a proteína Cry1Ac de Bacillus thuringiensis Berliner (Bt). Estudos sobre a suscetibilidade de H. virescens à proteína Cry1Ac e sobre a estrutura genética e padrões de fluxo gênico nas escalas locais e regionais desse inseto são fundamentais para a implementação de programas de Manejo da Resistência de Insetos (MRI) no Brasil. Dessa forma, os principais objetivos do trabalho foram: (a) avaliar a suscetibilidade à proteína Cry1Ac em populações de H. virescens coletadas nas principais regiões produtoras de algodão no Brasil (Bahia, Goiás, Mato Grosso e Mato Grosso do Sul) durante as safras agrícolas 2007/08 e 2008/09; e (b) avaliar a variabilidade genética e fluxo gênico de populações de H. virescens provenientes das culturas de algodão (safras 2007/08, 2008/09 e 2009/10) e de soja (safra 2009/10) utilizando sequências de DNA mitocondrial (DNAmit). As linhas-básica de suscetibilidade à proteína Cry1Ac foram estabelecidas mediante o uso de lagartas neonatas, por meio de bioensaios de incorporação das diferentes concentrações da proteína em dieta artificial. Para avaliar a variabilidade genética e o fluxo gênico entre populações de H. virescens utilizou-se sequências de DNAmit das subunidades I e II da citocromo oxidase COI e COII e a subunidade 6 da desidrogenase dinucleotídica da adenina nicotinamida nad6. As CLs50 estimadas variaram de 0,18 a 0,66 µg de Cry1Ac/mL de dieta para as populações coletadas na safra 2007/08 (variação de 3,7 vezes). Da mesma forma, as concentrações efetivas médias para a inibição do desenvolvimento larval (CE50) variaram de 0,0053 a 0,0161 µg de Cry1Ac/mL dieta (variação de 3,0 vezes). A partir da análise conjunta dos dados de concentração-mortalidade de todas as populações avaliadas, foram definidas e validadas as concentrações diagnósticas de 3,1 e 5,6 µg de Cry1Ac/mL de dieta para programas de monitoramento da resistência de H. virescens à proteína Cry1Ac no Brasil. Baseadas em análises de agrupamento (Neighbor-Joining e Análise de Componentes Principais) e Bayesiana (Structure) foram verificadas uma baixa estruturação entre as populações de H. virescens de diferentes regiões, bem como para aquelas coletadas em plantas hospedeiras diferentes. As análises de AMOVA também indicaram baixa estruturação genética entre as populações de H. virescens estudadas independente da cultura (Fst= 0,019) ou escala geográfica (Fst= 0,012), sugerindo um nível significativo de fluxo gênico. Em média, a distância genética entre as amostras foi de 0,1%. Uma rede de haplótipos obtida com os dados combinados resultou em 35 haplótipos, com quatro haplótipos únicos presentes somente nas amostras coletadas em soja. A principal característica dessa rede é a forma de estrela na distribuição dos haplótipos, bem como a ocorrência de muitos alelos em baixa frequência. Esse tipo de rede é característico de populações que passaram por uma recente expansão populacional e, de fato, a história demográfica de H. virescens, baseada no teste de distribuição da diferença genética par-a-par entre haplótipos (distribuição de Mismatch) e nos resultados negativos nos testes de neutralidade seletiva indicam também um episódio de expansão populacional recente. As informações obtidas no presente trabalho serão fundamentais para o acompanhamento da efetividade das estratégias de manejo da resistência de H. virescens à proteína Cry1Ac no Brasil. / The tobacco budworm, Heliothis virescens (Fabricius), is one of target pests of genetically modified cotton expressing Cry1Ac insecticidal protein derived from Bacillus thuringiensis Berliner. Studies on susceptibility of H. virescens to Cry1Ac and the genetic structure and gene flow patterns at local and regional levels are crucial for establishing an Insect Resistance Management (IRM) program for Bt cotton in Brazil. Thus, the objectives of this study were (a) to evaluate the susceptibility of field-collected populations of H. virescens to Cry1Ac from major cotton-growing regions in Brazil (Bahia, Goiás, Mato Grosso and Mato Grosso do Sul) in the cropping seasons of 2007/08 and 2008/09; and (b) to evaluate the genetic variability and gene flow among H. virescens populations from cotton (2007/08, 2008/09 and 2009/10 cropping seasons) and soybean (2009/10 cropping season) with mitochondrial DNA markers. Baseline susceptibility data to Cry1Ac protein were estimated with neonate larvae thereby using diet incorporation bioassays. Genetic variation and gene flow among H. virescens populations were evaluated by using mitDNA sequences of cytochrome oxidase subunities I and II COI e COII and the subunity 6 of dinucleotide dehydrogenase of adenine nicotinamide nad6. The estimated LC50 values varied from 0.18 to 0.66 µg of Cry1Ac/mL of diet among the 2007/08 populations (3.7 fold variation). Similarly, the EC50 values based on growth inhibition ranged from 0.0053 to 0.0161 µg of Cry1Ac/mL of diet for the 2007/08 populations (3.0 fold variation). A joint analysis of the mortality data across all tested populations was used to develop candidate diagnostic concentrations for future monitoring programs. The proposed diagnostic concentrations of 3.1 and 5.6 µg of Cry1Ac/mL of diet were validated against field-collected populations from 2008/09 and will form the basis for future resistance monitoring programs with H. virescens. Based on cluster analysis (Neighbor-Joining and Principal Coordinate Analysis) and Bayesian analysis (Structure), a low structure was detected among H. virescens populations either by regions or host plants. AMOVA analysis also indicated low genetic structure among H. virescens populations across crops (Fst= 0.019) or geographic scale (Fst= 0.012), suggesting a significant gene flow. The mean genetic distance among samples was 0.1%. The haplotype network obtained with joint data resulted in 35 haplotypes, with four unique haplotypes present only in samples collected from soybean crop. The major characteristics of the haplotype network were the star-like pattern and the occurrence of many alleles at low frequencies. This type of network is typical for populations that passed through a recent population expansion and, in fact, the demographic history of H. virescens, based on distribution test of pair-wise genetic difference among haplotypes (Mismatch distribution) and negative results from tests of selective neutrality also indicate an episode of a recent population expansion.

Species are one of the fundamental components of biology and the accurate delimitation of species is important in evolutionary, systematic and ecological studies, yet there is still confusion over how species can be recognised. Examining different characters allows multiple lines of evidence for successful and accurate species delimitation and identification. In this thesis, morphological, genetic and behavioural variation is investigated within an endemic species of ground weta, Hemiandrus pallitarsis, in the North Island, New Zealand. Twelve morphological characters were measured, and mitochondrial cytochrome oxidase I DNA sequences were analysed from populations across the distributional range of H. pallitarsis. Both methods provide no evidence of a species complex within H. pallitarsis. Instead, the morphometric results suggest females are significantly larger than males, and ground weta in Palmerston North are significantly smaller than weta further north. Additionally, genetic analyses found substantial population structuring, large genetic distances, and an historical south to north pattern of movement in the North Island. The pattern of vibratory drumming behaviour followed that predicted by morphology and geographic proximity – drumming signals were more similar between geographically close populations and did not match the patterns of genetic isolation. Overall, this thesis was able to show that H. pallitarsis is morphologically, genetically and behaviourally variable across the North Island.

The research field of genetics provides useful tools to investigate the biology of species that are difficult to observe and study and are especially valuable in guiding the conservation of endangered species. The Chatham Island Tāiko (Tchāik, Pterodroma magentae) is the world’s most endangered seabird with an estimated population size of just 120-150 birds, including only 8-15 breeding pairs. This thesis used genetic techniques to investigate aspects of Tāiko biology and relationships in order to aid Tāiko conservation. The mitochondrial cytochrome b gene and duplicated regions of domain I of the mitochondrial control region were DNA sequenced in almost the entire known Tāiko population. The level of genetic variation revealed in Tāiko was unexpectedly high considering endangered species typically exhibit low genetic diversity. Sequencing of ancient DNA from subfossil Tāiko bones allowed an investigation of the past level of genetic variation and the species’ previous geographic distribution. A large proportion of the genetic diversity of the extinct Tāiko populations was retained in the remnant population. However, genetic variation in Tāiko chicks was low, thus genetic diversity in the population could be lost in just a few generations. There are many nonbreeding Tāiko so DNA sexing was used to examine sex ratios in the population. Almost all unpaired birds were male, which signified a potential Allee effect (i.e. that a reduced density of potential mates is decreasing population productivity). Further understanding of the Tāiko mating system and behaviour was obtained by parentage, sibship and pairwise relatedness analyses of genotypes at eight microsatellite DNA loci. It is important that Tāiko are found so they can be protected from introduced predators. The results of mitochondrial DNA sequencing and microsatellite DNA genotyping indicated that there are likely to be more Tāiko breeding in undiscovered areas. Analysis of philopatry using both mitochondrial and nuclear markers can assist conservation by the identification of areas to search for these undiscovered individuals. Tāiko may have once and could still be found on islands near South America since DNA sequencing showed the Magenta Petrel type specimen (collected in 1867 in the South Pacific Ocean) is a Tāiko.

<p>In this thesis the genetic variation of two grouse species, the Chinese grouse (<i>Bonasa sewersowi</i>) and the Black grouse (<i>Tetrao tetrix</i>) was examined with neutral genetic markers: microsatellites. Habitat fragmentation and isolation leads to structuring among and loss of genetic variation within populations.</p><p>The Chinese grouse in a small population in Lianhuasan nature reserve was found to have undergone a population bottleneck and as a result of isolation and possible inbreeding showed genetic impoverishment hereof.</p><p>The Black grouse populations in Europe face various different conditions from widely distributed areas of suitable habitat in the northern and eastern parts of its range to highly naturally and anthropogenically fragmented habitat landscapes in the west.</p><p>Structure among populations was found in Great Britain where Wales, Scotland and England showed characteristics of three different genetic entities, indicating very little or no geneflow between these populations. </p><p>The Dutch population showed signs of loss of genetic variation as to be expected from a population that has historically decreased in population size from several thousands to tens of individuals in a matter of decades. However the possibility to spot signs of a bottleneck was impaired due to the short time-window in which this can be observed in a population with such a low effective population size (N_E).</p><p>The sampled populations in Europe clustered into five different groups of genetic identities. The different clusters were: Great Britain-, the Netherlands-, Fenno-Scandian-, Alpine- and lowland German-Austrian populations. The level of genetic variation when compared over all these different populations decreased as a sign of isolation and small N_E. However it was not feasible to separate the impact of these two factors.</p>