No description available.

No description available.

Tigers (Panthera tigris tigris) can be viewed as a proxy for intact and healthy ecosystems. Their wild populations have plummeted to fewer than 3,200 individuals in the last four decades and threats to these apex predators are mounting rather than diminishing. Global conservation bodies (Global Tiger Initiative, World Wildlife Fund, Wildlife Conservation Society, Panthera etc.) have recently called for solidarity and scaling up of conservation efforts to save tigers from extinction. In South Asia, tiger habitat ranges from tropical evergreen forests, dry arid regions and sub-tropical alluvial floodplains, to temperate mixed deciduous forest. The churia habitat is relatively unstudied and is considered a young and geologically fragile mountain range in Nepal. The contribution of the churia habitat to tiger conservation has not been considered, since modern conservation started in 1970's. This study focuses on the ecology of the tiger with respect to population density, habitat use, and prey occupancy and density, in the churia habitat of Chitwan National Park. This study also includes the first assessment of genetic diversity, genetic structure, and gene flow of tigers across the Terai Arc Landscape- Nepal. The Terai Arc Landscape harbors the only remaining tiger population found across the foothills of the Himalayas in Nepal and northwest India. I used a combination of camera-trapping techniques, which have been a popular and robust method for monitoring tiger populations across the landscape, combined with a noninvasive genetic approach to gain information on tigers, thus adding new information relevant to global tiger conservation. I investigated tiger, leopard (Panthera pardus fusca), and prey densities, and predicted the tiger density across the Churia habitat in Chitwan National Park. I used a camera-trap grid with 161 locations accumulating 2,097 trap-nights in a 60 day survey period during the winter season of 2010-2011. Additionally, I used distance sampling techniques for estimating prey density in the churia habitat by walking 136 km over 81 different line transects. The team photographed 31 individual tigers and 28 individual leopards along with 25 mammalian species from a sampling area of 536 km² comprising Churia and surrounding areas. Density estimates of tigers and leopards were 2.2 (SE 0.42) tigers and 4.0 (SE 1.00) leopards per 100 km². Prey density was estimated at 62.7 prey animals per 100 km² with contributions from forest ungulates to be 47% (sambar Rusa unicolor, chital Axis axis, barking deer Muntiacus muntjak, and wild pigs Sus scrofa). Churia habitat within Chitwan National Park is capable of supporting 5.86 tigers per 100 km² based on applying models developed to predict tiger density from prey density. My density estimates from camera-traps are lower than that predicted based on prey availability, which indicates that the tiger population may be below the carrying capacity. Nonetheless, the churia habitat supports 9 to 36 tigers, increasing estimates of current population size in Chitwan National Park. Based on my finding, the Churia habitat should no longer remain ignored because it has great potential to harbor tigers. Conservation efforts should focus on reducing human disturbance to boost prey populations to potentially support higher predator numbers in Churia. I used sign surveys within a rigorous occupancy framework to estimate probability of occupancy for 5 focal prey species of the tiger (gaur Bos gaurus, sambar, chital, wild pig, and barking deer); as well as probability of tiger habitat use within 537 km² of churia habitat in Chitwan National Park. Multi-season, auto-correlation models allowed me to make seasonal (winter versus summer) inferences regarding changes in occupancy or habitat use based on covariates influencing occupancy and detection. Sambar had the greatest spatial distribution across both seasons, occupying 431-437 km² of the churia habitat, while chital had the lowest distribution, occupying only 100-158 km². The gaur population showed the most seasonal variation from 318- 413 km² of area occupied, with changes in occupancy suggesting their migration out of the lowland areas in the summer and into the churia in the winter. Wild pigs showed the opposite, moving into the churia in the summer (444 km² area occupied) and having lower occupancy in the winter (383 km²). Barking deer were widespread in both seasons (329 - 349 km²). Tiger probability of habitat use Ψ SE(Ψ) was only slightly higher in winter 0.63 (SE 0.11) than in summer 0.54 (SE 0.21), but confidence intervals overlapped and area used was very similar across seasons, from 337 - 291 km². Fine-scale variation in tiger habitat use showed that tigers intensively use certain areas more often than others across the seasons. The proportion of available habitat positively influenced occupancy for the majority of prey species and tigers. Human disturbance had a strong negative influence on the distribution of the majority of prey species but was positively related to tiger habitat use. Tigers appear to live in areas with high disturbance, thus increasing the risk of human-tiger conflict in the churia habitat. Thus, efforts to reduce human disturbance would be beneficial to reducing human wildlife conflict, enriching prey populations, and would potentially support more tigers in churia habitat of Nepal. Overall, I found high prey occupancy and tiger habitat use, suggesting that the churia is highly valuable habitat for tigers and should no longer be neglected or forgotten in tiger conservation planning. Thirdly, I assessed genetic variation, genetic structure, and gene flow of the tigers in the Terai Arc Landscape, Nepal. I opportunistically collected 770 scat samples from 4 protected areas and 5 hypothesized corridors across the Terai Arc Landscape. Historical landuse change in the Terai Arc was extracted from Anthrome data sets to relate landuse change to potential barriers and subsequent hypothesized bottleneck events in the landscape. I used standard genetic metrics (allelic diversity and heterozygosity) to estimate genetic variation in the tiger population. Using program Structure (non-spatial) and TESS (spatial), I defined the putative genetic clusters present in the landscape. Migrant analysis was carried out in Geneclass and Bayesass for estimating contemporary gene flow. I tested for a recent population bottleneck with the heterozygosity test using program Bottleneck. Of the 700 samples, 396 were positive for tiger (57% success). Using an 8 multilocus microsatellite assay, I identified 78 individual tigers. I found large scale landuse changes across the Terai Arc Landscape due to conversion of forest into agriculture in last two centuries and I identified areas of suspected barriers. I found low levels of genetic variation (expected heterozygosity = 0.61) and moderate genetic differentiation (F_ST = 0.14) across the landscape, indicative of sub-population structure and potential isolation of sub-populations. I detected three genetic clusters across the landscape consistent with three demographic tiger sub-populations occurring in Chitwan-Parsa, Bardia, and Suklaphanta protected areas. I detected 10 migrants across all study sites confirming there is still some dispersal mediated gene flow across the landscape. I found evidence of a bottleneck signature, especially around the lowland forests in the Terai, likely caused by large scale landuse change in last two centuries, which could explain the low levels of genetic variation detected at the sub-population level. These findings are highly relevant to tiger conservation indicating that efforts to protect source sites and to improve connectivity are needed to augment gene flow and genetic diversity across the landscape. Finally, I compared the abundance and density of tigers obtained using two non-invasive sampling techniques: camera-trapping and fecal DNA sampling. For cameras: I pooled the 2009 camera-trap data from the core tiger population across the lowland areas of Chitwan National Park. I sampled 359 km² of the core area with 187 camera-trap locations spending 2,821 trap-nights of effort. I obtained 264 identifiable photographs and identified a total of 41 individual tigers. For genetics, I sampled 325 km² of the core area along three spatial routes, walking a total of 1,173 km, collecting a total of 420 tiger fecal samples in 2011. I identified 36 tigers using the assay of 8 multilocus genotypes and captured them 42 times. I analyzed both data types separately for estimating density and jointly in an integrated model using both traditional, and spatial, capture-recapture frameworks. Using Program MARK and the model averaged results, my abundance estimates were 46 (SE 1.86) and 44 (SE 9.83) individuals from camera and genetic data, respectively. Density estimates (tigers per 100 km²) via traditional buffer strip methods using half of the Mean Maximum Distance Moved (½ MMDM) as the buffer surrounding survey grids, were 4.01 (SE 0.64) for camera data and 3.49 (SE 1.04) for genetic data. Spatially explicit capture recapture models resulted in lower density estimates both in the likelihood based program DENSITY at 2.55 (SE 0.59) for camera-trap data and 2.57 (SE 0.88) for genetic data, while the Bayesian based program SPACECAP estimates were 2.44 (SE 0.30) for camera-trap data and 2.23 (SE 0.46) for genetic data. Using a spatially explicit, integrated model that combines data from both cameras and genetics, density estimates were 1.47 (SD 0.20) tigers per 100 km² for camera-trap data and 1.89 (SD 0.36) tigers per 100 km² for genetic data. I found that the addition of camera-trap data improved precision in genetic capture-recapture estimates, but not visa-versa, likely due to low numbers of recaptures in the genetic data. While a non-invasive genetic approach can be used as a stand-alone capture-recapture method, it may be necessary to increase sample size to obtain more recaptures. Camera-trap data may provide a more precise estimates, but genetic data returns more information on other aspect of genetic health and connectivity. Combining data sets in an integrated modeling framework, aiding in pinpointing strengths and weaknesses in data sets, thus ultimately improving modeling inference. / Ph. D.

Over the last 100 years sexual selection has advanced into a vast field of theoretical and empirical research. While Darwin’s idea of female preference being an integral mechanism of sexual selection is no longer debated, our understanding of female preference is still very limited. For example, we know little about the genetic variation in female preference, and the costs of preference over and above the costs of mating with particular male phenotypes. Additionally, while costs of mate choice are well documented, the benefits of mate choice and their implications are still debated. For example, controversy exists over the inevitability of good gene benefits and their capability to promote adaptive sexual selection. Furthermore, the adaptiveness of sexual selection itself is debated. Our understanding of the traits involved in mate choice is also far from complete. Here I investigated aspects of sexual selection in Drosophila simulans, employing a range of behavioural approaches along with artificial selection and environmental manipulations. The findings presented here indicate that female preference can evolve when directly selected on, and that preference itself is not particularly costly. There was also no conclusive evidence for the good genes benefits of mate choice in D. simulans. These benefits are considered crucial in promoting the adaptiveness of sexual selection, and although we found sexual selection to be adaptive under some test conditions it was not adaptive in other conditions. Our investigations into traits involved in mate choice established sex-specific genetic variation in cuticular hydrocarbons and the genetic architecture of this trait was found to sex-specific evolution of cuticular hydrocarbons under natural and sexual selection. Additionally, we found that a secondary sexual character, the sex combs was positively allometric – just like most signalling and weapon traits, and there was no association between trait fluctuating asymmetry and trait size. These findings collectively indicate that sexual selection in D. simulans is consistent with classical models of this process.

O nematóide das lesões Pratylenchus coffeae é um dos principais parasitos do cafeeiro e de outras culturas e sua variabilidade biológica, que dificulta a adoção de métodos de controle, contribui para aumentar a sua importância no Brasil. Pela importância da cafeicultura e a falta de estudos com esse nematóide no Brasil, foram realizados experimentos com dois de seus isolados (K5 e M2), com os objetivos de correlacionar densidades populacionais do nematóide aos danos causados e estabelecer possíveis fontes de resistência de cafeeiros ao isolado K5. Foram testadas diferentes densidades populacionais iniciais do isolado M2 em plantas (seis pares de folhas) e plântulas (dois pares de folhas) do cafeeiro arábico ‘Catuaí Vermelho’. As densidades populacionais utilizadas foram de 0, 333, 1.000, 3.000 e 9.000 nematóides por plântula ou planta. A avaliação ocorreu aproximadamente cinco (plântulas) e sete (plantas) meses após a inoculação. Os resultados mostraram que houve uma acentuada redução do crescimento das plântulas, bem como massa fresca das raízes e massa seca da parte aérea, já a partir das densidades mais baixas. A variação populacional (Pf/Pi) foi menor que um (1,0) para todas as densidades de inóculo, indicando que esta cultivar, no estágio de plântulas com dois pares de folhas, mostrou-se intolerante ao parasitismo. Em relação à inoculação das plantas, já com seis pares de folhas, não houve diferenças significativas nas variáveis analisadas e ocorreram decréscimos populacionais do nematóide, indicando que, nessas condições, ‘Catuaí Vermelho’ mostrou-se resistente ao isolado M2. Em relação ao isolado K5, foram realizados cinco experimentos, visando caracterizar as reações de genótipos de Coffea canephora ('Robusta' e 'Conilon'), além de C. arabica ‘Mundo Novo’, comparado às reações frente ao nematóide de galhas Meloidogyne incognita raça 2. No Experimento 1, foram utilizadas plantas de C. arabica ‘Mundo Novo’, inoculadas com 1.480 nematóides por planta (isolado K5 e M. incognita). Após sete meses da inoculação foi feita a avaliação, mostrando que o crescimento populacional dos nematóides foi alto e a reação de suscetibilidade. Mesmo em mudas desenvolvidas de cafeeiro ‘Mundo Novo’, o isolado K5 destacou-se como tão agressivo quanto M. incognita. Os outros genótipos testados, de C. canephora, foram inoculados com 3.000 nematóides por planta. Nos Experimentos 2 e 3, as linhagens IAC 4804 e IAC 4810 de ‘Robusta’ foram suscetíveis ao isolado K5, mas em um deles (IAC 4804) ocorreu grande variação entre as repetições em relação à M. incognita. Apenas o isolado K5 promoveu redução do crescimento do cafeeiro, evidenciado na variável massa fresca das raízes, em ambas as linhagens, sendo que IAC 4810 comportou-se como resistente a M. incognita. No caso de C. canephora ‘Conilon’, ambas as linhagens testadas (IAC 4764 e IAC 4765) foram resistentes ao isolado K5 e suscetíveis a M. incognita. / The lesion-nematode Pratylenchus coffeae is a major pest of coffee and other economic crops and its biological variability, which often makes difficult the adoption of control methods, contributes to increase the importance of this parasite in Brazil. Due to the importance of coffee production and the lack of studies involving this nematode species in Brazil, experiments were set with two of its available isolates (K5 and M2) to correlate initial population densities with the damage caused on coffee plants and to establish possible resistance sources in relation to the isolate K5. Different population densities of isolate M2 were tested in plants (six pairs of leaves) and seedlings (two pairs of leaves) of Coffea arabica ‘Catuaí Vermelho’. The population densities (Pi) were: 0, 333, 1.000, 3.000 and 9.000 nematodes per seedling or plant. The evaluation was done at approximately five (seedlings) and seven (plants) months after inoculation. The results showed that there was a marked reduction of the height, as well as root fresh weight and shoot dry weight of the seedlings, starting from the lower Pi values. The nematode population decreased (Pf/Pi < 1), indicating that this cultivar, at the seedling stage, was intolerant to parasitism. In relation to the inoculation of older plants, there were no significant differences in the growth parameters and the nematode population also decreased allowing ‘Catuaí Vermelho’ to be rated as resistant to the isolate M2. In relation to isolate K5, five experiments (referred to as 1, 2, 3, 4, and 5) were set to characterize the reaction of different genotypes of Coffea canephora ('Robusta' and 'Conilon') and C. arabica ‘Mundo Novo', as compared with their reaction to the root-knot nematode Meloidogyne incognita race 2. In Experiment 1, plants of C. arabica ‘Mundo Novo’ were inoculated with 1,480 nematodes per plant (K5 and M. incognita). The final evaluation after seven months of the inoculation showed a high populational increase of the nematodes and that both were pathogenic at a same extent. The other genotypes tested, belonging to C. canephora, were inoculated with 3,000 nematodes per plant. The genotypes (IAC 4804 and IAC 4810) of ‘Robusta’ were susceptible to isolate K5, but in one of them (IAC 4804) there was great variation among the repetitions in relation to M. incognita. The isolate K5 caused marked reduction in the growth of coffee Robusta plants as evidenced particularly through the root fresh weight values in both tested genotypes; in addition, IAC 4810 was rated as resistant to M. incognita. With regard to C. canephora 'Conilon', both tested genotypes (IAC 4764 and IAC 4765) were resistant to isolate K5 and susceptible to M. incognita.

O Vírus Respiratório Sincicial Humano (HRSV) é considerado o principal causador de doenças agudas do trato respiratório inferior durante a infância, sendo o principal responsável por um elevado índice de hospitalização de crianças com até cinco anos de idade. Possui distribuição mundial, podendo acometer todas as faixas etárias, entretanto as crianças de 6 semanas a 9 meses são as que desenvolvem problemas mais sérios, como pneumonia e bronquiolite. A epidemia de HRSV apresenta uma sazonalidade bem clara, ocorrendo anualmente no período de outono tardio, inverno ou início da primavera, mas não durante o verão. No presente estudo foi realizada a análise da região G2 da glicoproteína G do HRSV. Um total de 44 amostras positivas para o HRSV do Hospital Universitário (HU) da Universidade de São Paulo (USP), nos anos de 2007-2008, foram seqüenciadas e posteriormente analisadas, sendo então comparadas com seqüências obtidas do NCBI/GeneBank. A análise filogenética mostrou que os genótipos GA2 e GA5, do grupo A, foram os predominantes nos anos de 2007 e 2008, alternando o padrão verificado nos anos anteriores, onde os genótipos do grupo B foram altamente predominantes. A comparação das mutações sinônimas e não sinônimas mostrou uma grande evidência de seleção positiva nos genótipos GA2 e GA5 do grupo A. / Human Respiratory Syncytial Virus (HRSV) is considered the most common cause of lower respiratory tract disease in infants and young children and are the main guilty for the elevated children hospitalizations rate under 5 years of age. The HRSV has a world-wide distribution, being able to attack all the ages however the 6 weeks to 9 months children of are the ones that develop more serious problems as pneumonia and bronquiolite. The HRSV outbreak presents a well defined season, occurring annually in the delayed falls period, winter or springs beginning, but not during the summer. In the present study, we performed a phylogenetic analysis from G2 region of HRSV G glycoprotein. Forty four samples positive for HRSV from University Hospital (UH) of University of Sao Paulo (USP) in 2007-2008, were submitted to sequencing by PCR and compared with GenBank sequences. Phylogenetic analysis revealed that HRSV group A genotypes GA2 and GA5 was the predominant in 2007-2008, alternating the standard verified in the previous years, where the group B genotypes had been highly predominant. Comparison of the synonymous/nonsynonymous mutation ratios showed greater evidence for positive selection pressure for group A genotypes GA2 and GA5.

A cana-de-açúcar é uma das principais culturas agroindustriais do Brasil, sendo amplamente cultivada para a produção de açúcar e etanol. Esta cultura se torna a cada dia mais importante no cenário mundial, devido à busca constante por fontes de energia alternativas e mais sustentáveis. Para atender a crescente demanda, é necessária a liberação frequente de novas variedades, mais adaptadas às regiões de cultivo e tolerantes às alterações ambientais. Assim, o estabelecimento da metodologia de transformação genética além de contribuir para o estudo funcional de genes de interesse é uma metodologia alternativa para obtenção de novas variedades. O processo de obtenção de transgênicos é dependente de um eficiente protocolo de regeneração de plantas in vitro, que geralmente envolve uma fase de formação de células indiferenciadas (calos). A indução e a manutenção dos calos são favoráveis ao aumento da atividade de elementos de transposição (ETs) os quais são muito freqüentes no genoma de cana e podem acarretar variabilidade no genoma vegetal pela alteração dos padrões e funções gênicas devido a essa mobilização, afrontando a fidelidade genética dos cultivares transgênicos obtidos. Baseando-se na importância de reduzir o período de cultura de tecidos e controlar a atividade dos ETs durante o desenvolvimento in vitro, o objetivo desse trabalho foi buscar alternativas no controle e na redução do tempo para regeneração de plantas, inclusive com a aplicação de peptídeos hormonais, assim como de transformar geneticamente as variedades RB835089 e RB835486 com o gene Ddm1 de Arabidopsis, visando o silenciamento dos elementos de transposição em cana-de-açúcar. Para isso, foram analisados os meios de cultura MS3c e ML1G1 e o efeito da água de coco na indução e formação de calos como também na regeneração de plantas. Foram testados os meios de regeneração de plantas MSAc, SRM, ML1R3 e ML1R4, obtendo-se em média 5,2 plantas por explante no meio MSAc, que foi superior aos demais meios. Este meio foi utilizado para testar o efeito individual dos peptídeos hormonais CLV3 e PSK- em calos embriogênicos, os quais apresentaram acréscimo na regeneração de plantas para 9,3 plantas por explantes com doses de 30 µM de PSK-a. A transformação genética por biolística através da cotransformação dos genes neo e AtDdm1 resultou em 34 plantas transgênicas. O estudo da mobilização dos ETs durante o desenvolvimento in vitro foi realizado para quatro retrotransposons. A expressão heteróloga do gene AtDdm1 em cana-de-açúcar mostrou atuar no controle da expressão do retroelemento TE010. O estudo da mobilização dos retrotransposons e do gene Ddm1 endógeno de cana (SsDdm1) durante o desenvolvimento in vitro confirmou que o gene SsDdm1 foi chave no controle da expressão dos retroelementos. A transformação genética com o gene AtDdm1 aliada a rápida regeneração de plantas a partir de discos foliares possibilitam condições que minimizam a expressão dos ETs em cana-de-açúcar. / Sugarcane is one of the major agro-industrial crops of Brazil being widely cultivated for the production of sugar and ethanol. This culture has become increasingly more important on the world stage each day due to the constant search for alternative and sustainable energy sources. In order to meet growing demand, it is necessary to often release new varieties, better adapted to cultivated expansion area and tolerant to environmental changes. Thus, the establishing of genetic transformation methodology beyond of contributing to the functional study of genes of interest and it is an alternative method for obtaining new varieties. The process of obtaining transgenic plants is dependent of an efficient protocol for in vitro plant regeneration, which generally involves a phase of undifferentiated cells (callus). The induction and maintenance of callus are favorable to increase the activity of transposable elements (TEs) which are very frequent in the genome of sugarcane and may cause variability in the plant genome by altering patterns and gene functions due to this mobilization, confronting the genetic fidelity of the transgenic cultivars obtained. Based on the importance of reducing the period of tissue culture and control the activity of TEs during in vitro development, the objective of this work was to seek alternatives to control and reduce the time for plant regeneration, including the use of peptides hormone, as well as to genetically transform sugarcane varieties RB835089 and RB835486 with the Ddm1 Arabidopsis gene to silence the transposable elements in cane sugar. For this, we tested the culture media MS3c and ML1G1and the effect of coconut water in callus induction and growth as well as on plant regeneration. We tested the plant regeneration media MSAc, SRM, ML1R3 and ML1R4, obtaining an average of 5.2 plants per explants using MSAC, superior to other medium tested. It was used to test the individual effect of peptides hormones such as CLV3 and PSK- in embryogenic callus, which showed an increase in plant regeneration to 9.3 plants per explant with doses of 30µM PSK-a. Genetic co-transformation with the neo and AtDdm1 genes by biolistic resulted in 34 transgenic plants. A study of TEs during in vitro development was performed for four retrotransposons. Heterologous expression of the AtDdm1 gene in sugarcane showed to control the expression of the retroelement TE010. The study of mobilization of retrotransposons and the endogenous Ddm1 gene (SsDdm1) during in vitro development confirmed that SsDdm1 was key gene in controlling the expression of retrotransposons. Genetic transformation with the AtDdm1 gene and the fast regeneration of plants provide positive conditions to minimize the expression of ETs in sugarcane.

O presente trabalho teve como objetivo o estudo da variabilidade genética e dos fatores de virulência de isolados de U. diversum. As cepas foram submetidas a sequenciamento dos genes da urease e 16S rRNA e a testes para verificar os fatores de virulência: cápsula, fosfolipase C, IgAse e adesão e invasão. A análise do sequênciamento parcial do gene 16S rRNA resultou na presença de polimorfismos em 44 posições da seqüência, que diferenciou as amostras em sete grupos. Em relação aos fatores de virulência, os dados mostraram que as cepas estudadas apresentaram uma camada densa ao redor da membrana celular dos microrganismos e atividade de fosfolipase C. No entanto, não foi observado a atividade de IgAse nas cepas. Em relação a atividade de invasão, observou-se que os ureaplasma estudados puderam ser visualizados no interior de células Hep-2 com apenas um minutos de infecção, sendo observados em uma região perinuclear, mas não no interior do núcleo. Além disto, pode verificar que entre 1% a 10% dos ureaplasmas estudos penetraram na célula pelo teste da gentamicina. / The aim of the present study was the study of genetic variability and virulence factors of U. diversum clinical isolates. The strains were submitted to sequencing for 16S rRNA and urease genes. Moreover, the strains were analyzed to the virulence factors: capsule, phospholipase C, IgA protease and adhesion and invasion into Hep-2 cells. The sequencing of parcial 16S rRNA gene showed polymorphic patterns into 44 positions. These polymorphisms clustered the strains in seven groups. For the virulence factors, ureaplasma cells showed a dense-stained external capsule-like structure surrounding the cell membrane. A high level of phospholipase C activity was also detected in 31 studied ureaplasma. However, no strains showed IgA protease activity. For the invasion assay, the isolates and strains used were detected inside the cells after infection of one minute. The invasions of the ureaplasmas surrounded the nuclear region but were not observed inside the nuclei. The gentamicin invasion assay detected that 1% to 10% of studied ureaplasmas were inside the infected cells.

Os genes de receptores olfatórios (OR) pertencem a uma família de proteínas de membrana formada por cerca de 1000 genes no genoma de camundongo. Os genes OR são expressos de forma monogênica e monoalélica nos neurônios olfatórios (OSNs). No entanto, ainda não está claro o mecanismo que permite essa forma de expressão peculiar, sobretudo, qual o papel da metilação de DNA nesse processo. Nosso estudo determinou o padrão de metilação de DNA da região promotora e codificadora do gene Olfr17. Em células de epitélio olfatório (MOE) de camundongos adultos, observamos na região codificadora (CDS) do gene uma frequência de metilação em dinucleotídeos CpG 58%, enquanto que na sua região promotora ela foi bem mais baixa. Os níveis de metilação do Olfr17 em MOE de embrião (E15.5) e fígado foram similares aos observados em MOE de animais adultos. Em seguida, analisamos se a metilação de DNA pode regular a expressão gênica do Olfr17. Utilizando animais transgênicos onde os neurônios olfatórios que expressam Olfr17 também expressam GFP, pudemos selecionar neurônios olfatórios GFP+ e analisar a metilação do gene Olfr17, que está ativo nestas células. Verificamos que o padrão geral de metilação do Olfr17, tanto na região CDS como na região promotora, não se altera quando este gene está ativo. Este resultado indica que alterações na metilação do gene Olfr17 não são necessárias para que este receptor seja expresso. Finalmente, verificamos que a região promotora do gene Olfr17, de duas linhagens de camundongos diferentes, a C57BL/6 e a 129, possuem dois polimorfismos de base única (SNPs) que alteram o conteúdo CpG. Devido a estes SNPs, a linhagem 129 apresenta dois sítios CpG adicionais, inexistentes na linhagem C57BL/6. Nossas análises mostraram que estes CpGs são frequentemente metilados, o que torna o promotor do Olfr17 de 129 significativamente mais metilado que o promotor de C57BL/6. Em seguida, nós analisamos o nível de expressão no MOE dos dois alelos de Olfr17, o 129 e o C57BL/6, utilizando ensaios de RT-qPCR. Estes experimentos demonstraram que o nível de expressão do alelo 129, que possui 3 CpGs metiladas em seu promotor, é menor que o do alelo C57BL/6, que apresenta apenas uma CpG que é pouco metilada em seu promotor. Nossos resultados sugerem que as alterações na região promotora influenciam a probabilidade com que o gene OR é escolhido para ser expresso no MOE. / Olfactory receptor (OR) genes belong to a large family of membrane proteins composed of 1000 genes in the mouse genome. The OR genes are expressed in the olfactory sensory neurons (OSNs) in a monogenic and monoallelic fashion. However, the mechanisms that govern OR gene expression are unclear. Here we asked whether DNA methylation plays a role in the regulation of OR gene expression. We first determined the DNA methylation pattern in the coding (CDS) and promoter regions of the odorant receptor gene Olfr17. In olfactory epithelium (MOE) cells, the CpG methylation level in the CDS is 58% but is much lower in the promoter region of the gene. In embryonic MOE (E15.5) and liver, the levels of Olfr17 DNA methylation are similar to the ones shown in adult MOE. We next analyzed whether DNA methylation is involved in Olfr17 regulation. We isolated GFP+ neurons from transgenic mice that coexpress GFP with Olfr17, and analyzed the DNA methylation pattern of the Olfr17, which is active in these cells. We found that the general methylation pattern, both, in the coding and promoter regions is not altered in the active gene. These results indicate that changes in DNA methylation are not required for the activation of Olfr17. Finally, we found that the Olfr17 promoter region from two different mouse strains, C57BL/6 and 129, has two single-nucleotide polymorphisms (SNPs) that alter the CpG content. The SNPs lead to the existence of two additional CpGs in the 129 allele, which are absent in the C57BL/6 allele. These CpGs are frequently methylated, making the 129 Olfr17 promoter significantly more methylated than the Olfr17 promoter from C57BL/6. We next performed RT-qPCR experiments to analyze the expression levels of the 129 and C57BL/6 Olfr17 alleles in the MOE. These experiments showed that the expression level of the 129 Olfr17 allele, which contains three methylated CpGs in its promoter region, is lower than the one from C57BL/6, which contains only one, undermethylated CpG, in its promoter. Our results suggest that these promoter modifications regulate the probability of the OR gene choice.

Os objetivos do presente trabalho compreenderam a obtenção de estimativas de parâmetros genéticos e fenotípicos, como variâncias, correlações, herdabilidades e respostas à seleção, em uma população derivada do cruzamento entre as linhagens 14 e 56 de soja, contrastantes para produção de grãos. Os tratamentos consistiram de uma amostra de 89 progênies F2:4 e 11 testemunhas comerciais, que foram avaliadas em experimentos em látice triplo 10 x 10 no ano agrícola 2007/08 na Estação Experimental Anhembi do Departamento de Genética da ESALQ/USP, localizada em Piracicaba, SP. A parcela experimental foi constituída de uma linha de 2 m de comprimento, espaçada de 0,5 m, contendo 35 plantas no estande ideal, após o desbaste. Foram avaliados os seguintes caracteres: dias até a maturação (DM), altura das plantas na maturação (AM), acamamento (AC) e produção de grãos (PG). As estimativas dos coeficientes de herdabilidade entre médias de progênies foram: elevada para AM (77,4%), mediana para DM (32,3%) e baixas para AC e PG (18,1% e 19,0%, respectivamente). Entretanto, os coeficientes de variação genética foram muito baixos para DM e AC (0,37% e 1,61%, respectivamente), e mais altos para AM e PG (6,80% e 6,83%, respectivamente), indicando a ocorrência de pouca variabilidade genética para DM e AC e, conseqüentemente, poucas perspectivas de resposta à seleção. O coeficiente de correlação genética entre AM e PG foi alto (rg = 0,67), indicando que a seleção para PG deve resultar em plantas mais altas. A estimativa da resposta com seleção das 20% progênies mais produtivas foi de 4,5%, e a resposta correlacionada em AM foi de 3,8%. Devido à baixa variabilidade genética, as respostas correlacionadas esperadas em DM e AC foram muito pequenas. Os resultados indicam que a população pode ser melhorada somente para os caracteres PG e AM. / This paper was carried out in order to estimate genetic and phenotypic parameters, such as variances, heritabilities, correlations and expected response to selection in a population derived from the cross between inbred lines 14 and 56 of soybeans, divergent for grain yield. Entries consisted of a sample of 89 F2:4 progenies and 11 commercial checks, evaluated in a triple 10 x 10 lattice design in the 2007/08 growing season at Anhembi Experimental Station of the Genetics Department (ESALQ/USP), near Piracicaba, SP. Plots were single 2-meter-long rows spaced 0.5 m apart, with 35 plants after thinning. The following traits were evaluated: days to maturity (DM), plant height at maturity (AM), lodging (AC) and grain yield (PG). Heritability estimates on a progeny mean basis were high for AM (77.4%), medium for DM (32.3%) and low for AC and PG (18.1% and 19.0%, respectively). However, genetic coefficients of variation were very low for DM and AC (0.37% and 1.61%, respectively) and higher for AM and PG (6.80% and 6.83%, respectively), which indicates low genetic variability for DM and AC and, consequently, small perspectives of response to selection. Genetic correlation coefficient between AM and PG was high (rg = 0.67), indicating that selection for higher PG will result in taller plants. Expected response based on selection of the 20% high yielding progenies was 4.5%, and the correlated response in AM was 3.8%. The correlated response in DM and AC were very low, due to the low genetic variability. General results have shown that the population can be improved only for the traits PG and AM.