This thesis describes the development and use of a new class of molecular tools called Selector probes, and its potential for investigations of genetic variation. The Selector technology provides multiplex amplification of targeted DNA sequences with a high specificity, and an enrichment factor in the same order of magnitude as PCR. A common feature in this thesis work is to focus the analysis on DNA regions of interest. For example, this technique can be implemented in analysing candidate regions found by whole genome studies that need validation (global to local analysis), and applications requiring detection of rare alleles (common to rare allele), important in for example cancer samples. An assay is presented that allows for fast and simple quantification of relative copy-number variations. The method was proven to be able to detect aneuploidy in chromosome 13, 18, 21 and X, with a resolution enough to distinguish between 4 and 5 copies. The method was successfully applied to solve a biological question regarding a copy-number variation, that explains the Ridge phenotype typical for the dog bread Rhodesian Ridgebacks. The Selector strategy was able to detect and map a tandem duplication with a size of 133 kb, which was characterized with base-pair resolution. A readout platform that facilitates simultaneous digital quantitative analysis of a large numbers of biomolecules is further introduced. The work involves arraying amplified product from successful selection and decoding each molecule by hybridization of fluorophore labeled oligonucleotides. Finally, a genome partitioning method which is applied upstream of next generation sequencing platforms is presented. It is shown that the method provides successful enrichment with 98 % coverage and 94 % specificity and high enrichment uniformity. The technique was applied for mutation analysis of 26 cancer-related genes in tumor cell-lines and tissue.

No description available.

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No description available.

Doctor of Philosophy (PhD) / The Australian cotton industry has developed high yielding and high quality fibre production systems and attributes a significant contribution of this achievement to highly innovative breeding programs, specifically focused on the production of premium quality lint for the export market. Breeding programs have recently shifted attention to the development of new germplasm with superior stress tolerance to minimise yield losses attributed to adverse environmental conditions and inputs such as irrigation, fertilisers and pesticides. Various contributors to yield, such as physiology, biochemistry and gene expression have been implemented as screening tools for tolerance to high temperatures under growth cabinet and laboratory conditions but there has been little extension of these mechanisms to field based systems. This study evaluates tools for the identification of specific genotypic thermotolerance under field conditions using a multi-level ‘top down’ approach from crop to gene level. Field experiments were conducted in seasons 1 (2006) and 3 (2007) at Narrabri (Australia) and season 2 (2006) in Texas (The United States of America) and were supplemented by growth cabinet experiments to quantify cultivar differences in yield, physiology, biochemical function and gene expression under high temperatures. Whole plants were subjected to high temperatures in the field through the construction of Solarweave® tents and in the growth cabinet at a temperature of 42 oC. The effectiveness of these methods was then evaluated to establish a rapid and reliable screening tool for genotype specific thermotolerance that could potentially improve the efficiency of breeding programs and aid the development to high yielding cultivars for hot growing regions. Cotton cultivars Sicot 53 and Sicala 45 were evaluated for thermotolerance using crop level measurements (yield and fibre quality) and whole plant measurements (fruit retention) to determine the efficacy of these measurements as screening tools for thermotolerance under field conditions. Sicot 53 was selected as a relatively thermotolerant cultivar whereas Sicala 45 was selected as a cultivar with a lower relative thermotolerance and this assumption was made on the basis of yield in hot and cool environments under the CSIRO Australian cotton breeding program. Yield and fruit retention were lower under tents compared with ambient conditions in all 3 seasons. Yield and fruit retention were highly correlated in season 1 and were higher for Sicot 53 compared to Sicala 45 suggesting that fruit retention is a primary limitation to yield in a hot season. Thus yield and fruit retention are good indicators of thermotolerance in a hot season. Temperature treatment and cultivar differences were determined for fibre quality in seasons 1 and 3; however, quality exceeded the industry minimum thereby indicating that fibre quality is not a good determinant of thermotolerance. Physiological determinants of plant functionality such as photosynthesis, electron transport rate, stomatal conductance and transpiration rate were determined for cultivars Sicot 53 and Sicala 45 under the tents and an index of these parameters was also analysed to determine overall plant physiological capacity in the field. Physiological capacity was also determined under high temperatures in the growth cabinet using a light response curve at various levels of photosynthetically active radiation (PAR). Photosynthesis and electron transport rate decreased, whilst stomatal conductance and transpiration rate increased under the tents as well as under high temperatures in the growth cabinet. Photosynthesis and electron transport rate were higher for Sicot 53 but stomatal conductance and transpiration rate were higher for Sicala 45 under the tents. No cultivar differentiation was evident for plants grown under high temperatures in the growth cabinet. Temperature treatment and cultivar differences in physiological function were greater in a hot year (season 1), thereby indicating the importance of cultivar selection for thermotolerance in the presence of stress. Electron transport rate was correlated with yield in season 1, thus suggesting the suitability of this method for broad genotypic screening for thermotolerance under field conditions. Biochemical processes such as membrane integrity and enzyme viability were used to determine cultivar specific thermotolerance under high temperature stress in the laboratory, field and growth cabinet. Electrolyte leakage is an indicator of decreased membrane integrity and may be estimated by the relative electrical conductivity or relative cellular injury assays. The heat sensitivity of dehydrogenase activity, a proxy for cytochrome functionality and capacity for mitochondrial electron transport, may be quantified spectrophotometrically. Cellular membrane integrity and enzyme viability decreased sigmoidally with exposure to increasing temperatures in a water bath. Membrane integrity was higher for Sicot 53 compared with Sicala 45 under the tents and under high temperatures in the growth cabinet. No temperature treatment or cultivar differences were found for enzyme viability under the tents; however, enzyme viability for Sicala 45 was higher in the growth cabinet compared with Sicot 53. Relative electrical conductivity was strongly correlated with yield under ambient field conditions and under the tents, suggesting impairment of electron flow through photosynthetic and/or respiratory pathways, thus contributing to lower potential for ATP production and energy generation for yield contribution. Thus, the membrane integrity assay was considered to be a rapid and reliable tool for thermotolerance screening in cotton cultivars. Gene expression was examined for cultivars Sicot 53 and Sicala 45 grown under high (42 oC) temperatures in the growth cabinet. Rubisco activase expression was quantified using quantitative real-time polymerase chain reaction analysis and was decreased under high temperatures and was lower for Sicala 45 than Sicot 53. Maximum cultivar differentiation was found after 1.0 h exposure to high temperatures and hence, leaf tissue sampled from this time point was further analysed for global gene profiling using cDNA microarrays. Genes involved in metabolism, heat shock protein generation, electron flow and ATP generation were down-regulated under high temperatures in the growth cabinet and a greater number of genes were differentially expressed for Sicala 45, thereby indicating a higher level of heat stress and a greater requirement for mobilisation of protective and compensatory mechanisms compared with Sicot 53. Cultivar specific thermotolerance determination using gene profiling may be a useful tool for understanding the underlying basis of physiological and biochemical responses to high temperature stress in the growth cabinet. There is future opportunity for profiling genes associated with heat stress and heat tolerance for identification of key genes associated with superior cultivar performance under high temperature stress and characterisation of these genes under field conditions. This research has identified cultivar differences in yield under field conditions and has identified multiple physiological and biochemical pathways that may contribute to these differences. Future characterisation of genes associated with heat stress and heat tolerance under growth cabinet conditions may be extended to field conditions, thus providing the underlying basis of the response of cotton to high temperature stress. Electron transport rate and relative electrical conductivity were found to be rapid and reliable determinants of cultivar specific thermotolerance and hence may be extended to broad-spectrum screening of a range of cotton cultivars and species and under a range of abiotic stress. This will enable the identification of superior cotton cultivars for incorporation into local breeding programs for Australian and American cotton production systems.

Doctor of Philosophy (PhD) / The Australian cotton industry has developed high yielding and high quality fibre production systems and attributes a significant contribution of this achievement to highly innovative breeding programs, specifically focused on the production of premium quality lint for the export market. Breeding programs have recently shifted attention to the development of new germplasm with superior stress tolerance to minimise yield losses attributed to adverse environmental conditions and inputs such as irrigation, fertilisers and pesticides. Various contributors to yield, such as physiology, biochemistry and gene expression have been implemented as screening tools for tolerance to high temperatures under growth cabinet and laboratory conditions but there has been little extension of these mechanisms to field based systems. This study evaluates tools for the identification of specific genotypic thermotolerance under field conditions using a multi-level ‘top down’ approach from crop to gene level. Field experiments were conducted in seasons 1 (2006) and 3 (2007) at Narrabri (Australia) and season 2 (2006) in Texas (The United States of America) and were supplemented by growth cabinet experiments to quantify cultivar differences in yield, physiology, biochemical function and gene expression under high temperatures. Whole plants were subjected to high temperatures in the field through the construction of Solarweave® tents and in the growth cabinet at a temperature of 42 oC. The effectiveness of these methods was then evaluated to establish a rapid and reliable screening tool for genotype specific thermotolerance that could potentially improve the efficiency of breeding programs and aid the development to high yielding cultivars for hot growing regions. Cotton cultivars Sicot 53 and Sicala 45 were evaluated for thermotolerance using crop level measurements (yield and fibre quality) and whole plant measurements (fruit retention) to determine the efficacy of these measurements as screening tools for thermotolerance under field conditions. Sicot 53 was selected as a relatively thermotolerant cultivar whereas Sicala 45 was selected as a cultivar with a lower relative thermotolerance and this assumption was made on the basis of yield in hot and cool environments under the CSIRO Australian cotton breeding program. Yield and fruit retention were lower under tents compared with ambient conditions in all 3 seasons. Yield and fruit retention were highly correlated in season 1 and were higher for Sicot 53 compared to Sicala 45 suggesting that fruit retention is a primary limitation to yield in a hot season. Thus yield and fruit retention are good indicators of thermotolerance in a hot season. Temperature treatment and cultivar differences were determined for fibre quality in seasons 1 and 3; however, quality exceeded the industry minimum thereby indicating that fibre quality is not a good determinant of thermotolerance. Physiological determinants of plant functionality such as photosynthesis, electron transport rate, stomatal conductance and transpiration rate were determined for cultivars Sicot 53 and Sicala 45 under the tents and an index of these parameters was also analysed to determine overall plant physiological capacity in the field. Physiological capacity was also determined under high temperatures in the growth cabinet using a light response curve at various levels of photosynthetically active radiation (PAR). Photosynthesis and electron transport rate decreased, whilst stomatal conductance and transpiration rate increased under the tents as well as under high temperatures in the growth cabinet. Photosynthesis and electron transport rate were higher for Sicot 53 but stomatal conductance and transpiration rate were higher for Sicala 45 under the tents. No cultivar differentiation was evident for plants grown under high temperatures in the growth cabinet. Temperature treatment and cultivar differences in physiological function were greater in a hot year (season 1), thereby indicating the importance of cultivar selection for thermotolerance in the presence of stress. Electron transport rate was correlated with yield in season 1, thus suggesting the suitability of this method for broad genotypic screening for thermotolerance under field conditions. Biochemical processes such as membrane integrity and enzyme viability were used to determine cultivar specific thermotolerance under high temperature stress in the laboratory, field and growth cabinet. Electrolyte leakage is an indicator of decreased membrane integrity and may be estimated by the relative electrical conductivity or relative cellular injury assays. The heat sensitivity of dehydrogenase activity, a proxy for cytochrome functionality and capacity for mitochondrial electron transport, may be quantified spectrophotometrically. Cellular membrane integrity and enzyme viability decreased sigmoidally with exposure to increasing temperatures in a water bath. Membrane integrity was higher for Sicot 53 compared with Sicala 45 under the tents and under high temperatures in the growth cabinet. No temperature treatment or cultivar differences were found for enzyme viability under the tents; however, enzyme viability for Sicala 45 was higher in the growth cabinet compared with Sicot 53. Relative electrical conductivity was strongly correlated with yield under ambient field conditions and under the tents, suggesting impairment of electron flow through photosynthetic and/or respiratory pathways, thus contributing to lower potential for ATP production and energy generation for yield contribution. Thus, the membrane integrity assay was considered to be a rapid and reliable tool for thermotolerance screening in cotton cultivars. Gene expression was examined for cultivars Sicot 53 and Sicala 45 grown under high (42 oC) temperatures in the growth cabinet. Rubisco activase expression was quantified using quantitative real-time polymerase chain reaction analysis and was decreased under high temperatures and was lower for Sicala 45 than Sicot 53. Maximum cultivar differentiation was found after 1.0 h exposure to high temperatures and hence, leaf tissue sampled from this time point was further analysed for global gene profiling using cDNA microarrays. Genes involved in metabolism, heat shock protein generation, electron flow and ATP generation were down-regulated under high temperatures in the growth cabinet and a greater number of genes were differentially expressed for Sicala 45, thereby indicating a higher level of heat stress and a greater requirement for mobilisation of protective and compensatory mechanisms compared with Sicot 53. Cultivar specific thermotolerance determination using gene profiling may be a useful tool for understanding the underlying basis of physiological and biochemical responses to high temperature stress in the growth cabinet. There is future opportunity for profiling genes associated with heat stress and heat tolerance for identification of key genes associated with superior cultivar performance under high temperature stress and characterisation of these genes under field conditions. This research has identified cultivar differences in yield under field conditions and has identified multiple physiological and biochemical pathways that may contribute to these differences. Future characterisation of genes associated with heat stress and heat tolerance under growth cabinet conditions may be extended to field conditions, thus providing the underlying basis of the response of cotton to high temperature stress. Electron transport rate and relative electrical conductivity were found to be rapid and reliable determinants of cultivar specific thermotolerance and hence may be extended to broad-spectrum screening of a range of cotton cultivars and species and under a range of abiotic stress. This will enable the identification of superior cotton cultivars for incorporation into local breeding programs for Australian and American cotton production systems.

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.

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 vírus sincicial respiratório humano (VSRH) é o agente viral mais freqüentemente relacionado a doenças do trato respiratório inferior em crianças abaixo de um ano de idade. Analíse da varibilidade antigênica e gênica mostraram que o VSRH pode ser divido em dois grupos: A e B. O vírus é um membro do gênero Pneumovirus pertencente a família Paramyxoviridea, e possui três principais proteínas que são: glicoproteina F (fusão), glicoproteina G (adesão), glicoproteina SH (pequena proteína hidrofóbica). A proteína F é responsável pela fusão da célula ao vírus, enquanto a proteína G tem papel fundamental na replicação do vírus, porém a função da proteína SH, ainda não está bem definida, estudos recentes mostram-na como responsável por inibir a sinalização do fator de necrose tumoral alfa (TNF-a). Neste estudo foram colhidas amostras de 965 crianças, entre os anos de 2004 e 2005, dentre as quais 424 foram positivas. 117 amostras foram seqüenciadas a proteína SH e G e comparadas com amostras que circularam mundialmente. A analíse filogenética mostrou uma baixa variabilidade entre os genótipos estudados tanto do grupo A quanto do B. / The human respiratory syncytial virus (HRSV) is the major cause of lawer respiratory tract infections in infantis, young children and elderly. Analysis of the antigenic and genetic variability has shown that there are two groups of the virus HRSV, A and B. The virus (HRSV) is a member of the genus pneumovirus in the paramyxoviridae family. The virus encodes three membrane-bound glicoproteins, namely the fusion (F) attachment (G) and small hydrophobic (SH) proteins. The F mediates fusion of the virus and cell membranes and the G proteins is involved in virus attachment. The biological properties of the F and G glicoproteins and role that they play during virus replication relatively well understood, however the functional significance of the SH protein during replication remains unclear, although recent study shown that it can inhibit TNF-alpha. In this study, HRSV strains were isolated from nasopharyngeal aspirates collected from 965 children between 2004 and 2005, yielding 424 positive samples. We sequenced the small hydrophobic protein (SH) gene and protein (G) of 117 samples and compared them with other viruses identified worldwide. The phylogenetic analysis showed a low genetic variably among the isolates but allowed us to classify the viruses into different genotypes for the A and B HRSV strains.

Este trabalho teve por objetivo caracterizar e comparar a estrutura genética de sete populações de cupuaçuzeiro, Theobroma grandiflorum (Willd. ex Spreng.) Schum., uma fruteira nativa da Amazônia brasileira, utilizando marcadores microssatélites e descritores botânico-agronômicos. Visou também conhecer, preliminarmente, o sistema reprodutivo do cupuaçuzeiro. A estrutura genética das sete populações, sendo três populações naturais, coletadas na suposta área de máxima diversidade da espécie, três populações estabelecidas em Banco Ativo de Germoplasma (BAG), e uma população coletada em plantios comerciais do município de Tomé açu - PA, foi analisada com auxílio de marcadores microssatélites. Foi observada alta variabilidade genética na espécie, ressaltado pelo elevado número de alelos por loco, alto nível de heterozigosidade e divergência entre as populações. A divergência foi mais acentuada entre as populações naturais, em comparação com as populações do Banco de Germoplasma. Essa divergência pode indicar um processo preliminar de diferenciação. Porém, foi mais acentuada entre as populações oriundas de Tucuruí e Nova Ipixuna, corroborando com as indicações que consideram essa região como o centro de máxima diversidade de T. grandiflorum. Estes resultados sugerem, como estratégia de conservação in situ, a necessidade de definição de mais de um local para reserva genética, bem como, em relação a conservação ex situ, as coletas devem ser realizadas em vários locais. A elevada diversidade genética observada nos plantios comerciais, permite recomendar essas plantações como uma fonte alternativa de genes e genótipos ao programa de melhoramento de T. grandiflorum. No BAG foi observada baixa divergência genética entre as populações, sendo que, a maior parte da variabilidade genética encontrava-se dentro das populações. Essa caracterização foi complementada com o emprego de descritores botânico-agronômicos, quando foi observada grande variabilidade para a maioria dos descritores empregados. Houve necessidade, inicialmente, de selecionar dentre as 53 variáveis, aquelas que melhor se prestavam para a caracterização dos acessos. Empregando análises univariada e multivariada por componentes principais, foi possível descartar 64% das variáveis iniciais, sendo sugerida uma lista mínima de 19 descritores para o cupuaçuzeiro. Com base nessa lista e o emprego da distância Euclideana média, foi obtida uma matriz de dissimilaridade entre os 31 acessos avaliados. Esses acessos foram agrupados pelo método de Tocher e UPGMA, tendo sido obtidos seis grupos de similaridade. A comparação entre as duas caracterizações realizadas no BAG, revelou uma correlação positiva e significativa entre distâncias genéticas e fenotípicas. Preliminarmente foi estudado o sistema de reprodução do cupuaçuzeiro, numa população natural de Nova Ipixuna – PA, sendo utilizadas oito progênies de polinização aberta, com dez indivíduos e oito locos microssatélites polimórficos. Baseado na estimativa da taxa de cruzamento multilocos ( $ t m =1,0) e individual por planta materna, o estudo nessa população sugere que o T. grandiflorum é uma espécie predominantemente alógama, com uma pequena percentagem (5,4%) de cruzamentos entre parentes. Esse fator tem implicações importantes nas estratégias de conservação in situ e na utilização de progênies oriundas de polinização aberta nos programas de melhoramento. / This work had the objectives to characterize and compare the genetic structure of seven populations of cupuassu, Theobroma grandiflorum (Willd. ex Spreng.) Schum., a fruit tree native to the Brazilian Amazon using microsatellite markers and botanic-agronomic descriptors; and to investigate the cupuassu mating system. The genetic structure of seven populations of cupuassu, with three originally collected at the putative center of maximum diversity of the species; three populations established at the active germplasm collection (BAG); and one population colected from commercial plantings were analyzed using microsatellite markers. High genetic variability was observed for the species, demonstrated by the elevated number of alleles per locus; high heterozigosity and divergence between populations. The divergence was more noticeable among natural populations than among populations from the germplasm collection. This divergence might indicate a preliminary process of diversification. However, it was more pronounced between the populations from Tucuruí and Nova Ipixuna, corroborating indications that this region is considered the center of maximum diversity of T. grandiflorum. These results suggested as strategy for in situ conservation, the requirement to define more than one site for genetic reserves, large enough to maintain rare alleles in the medium- to long term. For ex situ conservation, sample collection must be conducted in many sites, with low intensity in each site, due to the existing variability witihin population. The high genetic diversity observed in commercail plantings allow to recommend these areas as an alternative source for genes and genotypes for the breeding program of T. grandiflorum. The characterization of the germplasm populations was complemented using botanic-agronomic descriptors. The large observed variability for most of the evaluated descriptors indicated that the germplasm collection contained high phenotypic diversity. Initially, it was necessary to select from the 53 evaluated variables, those most suitable for characterization of the accessions. Using univariate and multivariate analyses of principal components, it was possible to discard 64% of the initial variables, and a minimum descriptor list with 19 traits was proposed for cupuassu. Based on the minimum descriptor list, a matrix of dissimilarity was constructed using Euclidean distances. The 31 evaluated cupuassu accessions were grouped using Tocher and UPGMA, into six groups. The comparison betwen the molecular and phenotypic characterization revealed a significant and positive correlation between the genetic and phenotypic distances. The mating system fo cupuassu was studied, based on a natural population from Nova Ipixuna – PA, using eight progenies derived from open-pollinated pods with ten individuals each and eight polymorphic microsatellite loci. Based on the estimation of the multilocus outcrossing rate ( $ t m =1,0) and individual outcrossing rate ( $ t =1.0), the results from this population suggested that T. grandiflorum is a predominatly outbreeding species, with a small percentage (5,4%) of biparental inbreeding. These results have important implications on the in situ conservation strategies and on the use of open-pollinated progenies in breeding programs.