O objetivo do trabalho foi verificar se existe associação dos polimorfismos de lócus microssatélites com o desempenho e expressão gênica. Após genotipagem, os alelos 122 e 130 foram observados, com frequência de 0,94 e 0,06. A frequência gênica foi de 0,88 para animais 130/130 e de 0,12 para indivíduos 122/130. Não houve diferença para os dados fenotípicos entre animais de genótipos diferentes, embora o grupo de animais heterozigoto tenha apresentado uma leve superioridade. A taxa de expressão do Gh não é estatisticamente diferente entre os genótipos observados, porém o grupo homozigoto teve uma taxa de expressão de mRNA maior e mesmo assim menores valores de peso e comprimento, sugerindo uma correlação. O par de iniciadores desenhado amplificou e genotipou com eficiência o locus STR nos animais estudados. A falta de qualidade da água e o manejo inadequado dos animais podem ser os causadores das alterações histopatológicas encontradas no fígado dos animais estudados. Os peixes podem ser via de contaminação humana, devido ao seu consumo como alimento, portanto, medidas de controle devem ser iniciadas para minimizar esse processo. Estudos mais aprofundados com maior controle ambiental, maior variabilidade alélica, maior número amostral e análise de outros genes de interesse associados, seriam interessantes para complementar e enriquecer o presente trabalho. / The objective of this study was to verify if there is association of polymorphisms of microsatellite loci with the performance and gene expression. After genotyping, alleles 122 and 130 were observed, with a frequency of 0,94 and 0,06. The gene frequency was 0,88 for 130/130 animals and 0,12 for 122/130 individuals. There was no difference for phenotypic data among animals of different genotypes, although the group of heterozygous animals presented a slight superiority. The expression rate of GH is not statistically different among the observed genotypes, but the homozygote group had a higher mRNA expression rate and even lower values of weight and length, suggesting a correlation. The pair of primers designed amplified and efficiently genotyped the STR locus in the animals studied. The lack of water quality and inadequate management of the animals may be the cause of the histopathological changes found in the liver of the animals studied. Fish can be a way of human contamination, due to their consumption as food, therefore, control measures must be initiated to minimize this process. Further studies with greater environmental control, greater allelic variability, greater sample size and analysis of other associated genes of interest, would be interesting to complement and enrich the present work.

A embriogênese somática é uma técnica alternativa com potencial aplicação na propagação clonal de plantas, além de ser uma excelente ferramenta para estudos básicos e análise dos eventos moleculares e bioquímicos que ocorrem durante a embriogênese vegetal. Contudo, apesar de várias pesquisas relativas a embriogênese somática, a falta de conhecimento sobre os fatores que controlam o fenômeno, comprova que existem ainda muitos pontos a serem entendidos sobre o processo. Deste modo, o presente estudo foi concebido com o intuito de estudar a embriogênese somática de duas espécies de citros, as quais apresentam diferentes graus de eficiência na produção de embriões somáticos. Inicialmente, buscou-se avaliar comparativamente o processo de embriogênese somática de duas espécies de citros, observando-se as diferenças estruturais através de análises histológicas e histoquímicas, e as diferenças na expressão do gene AtSERK1 nos calos, através de hibridização in situ. Para instalação dos experimentos, foram utilizados calos provenientes de nucelos de duas espécies, Citrus sinensis L. Osbeck, variedade Valência, e Citrus limonia Osbeck, variedade limão ‘Cravo’. Amostras de calos em meio de multiplicação, em meio de obtenção de embriões, e embriões somáticos nas diferentes fases de desenvolvimento foram coletados para observações anatômicas e histoquímicas através de microscopia óptica e realização de estudo da expressão gênica através de hibridização in situ. Com relação à morfologia, os principais resultados obtidos demonstraram que existem diferenças anatômicas e histoquímicas entre calos de limão ‘Cravo’ e ‘Valência’. Em relação à expressão gênica, os resultados evidenciaram a expressão do gene AtSERK1 em calos das duas espécies de citros, tanto em meio de multiplicação, com sacarose, como em meio de indução, com maltose, indicando que o potencial embriogênico já está instalado no calo em meio de multiplicação. Pode-se concluir também que este gene é conservado entre Arabidopsis e Citrus. Desenvolveram-se, também, experimentos para estudar os efeitos de alguns tratamentos (frio, dessecação, agrupamentos celulares) na otimização da indução e sincronização da embriogênese somática de C. sinensis, var. Valência. Neste sentido, calos nucelares de laranja ‘Valência’, procedentes do meio de multiplicação foram mantidos no mesmo meio líquido em agitador orbital a 200 rpm durante 24h e posteriormente peneirados de acordo com os tratamentos. As peneiras utilizadas apresentaram malhas de 150 µm (P1), 300 µm (P2) e 600 µm (P3). Além do efeito das peneiras também foram observados os efeitos de exposição ao frio (4 o C por quatro semanas, ausência de luz) e dessecação (27 o C, em placas de Petri na ausência de meio de cultura, seis dias, ausência de luz), sendo posteriormente mantidos em B.O.D., a 26 ± 1 o C e intensidade luminosa de 300 lux. Os resultados obtidos permitiram concluir que em relação ao desenvolvimento e produção de embriões somáticos o fator peneira foi superior ao fator estresse na freqüência embriogênica. O desenvolvimento de embriões somáticos em Citrus sinensis ocorre mais eficientemente a partir de agrupamentos celulares de tamanhos específicos. / Somatic embryogenesis is an alternative technique with potential application for clonal propagation of plants, and an excellent tool for basic studies and analysis of the molecular and biochemical events that occur during embryogenesis. However, although many studies have been done, the factors that control somatic embryogenesis are not completely understood. Thus, the present study proposes to evaluate aspects of somatic embryogenesis of two citrus species, which differ in efficiency for the production of somatic embryos. Initially, a comparison of the embryogenic process was done in terms of structural and histochemical analysis and evaluation of the expression of the gene AtSERK1 in callus cultures through in situ hybridization. For the experiments, callus cultures obtained from nucelli of two species, Citrus sinensis L. Osbeck, cv. Valencia, and Citrus limonia Osbeck, cv. Rangpur lime were used for anatomical and histochemical observations, callus samples from cultures in multiplication medium, in embryo induction medium, and somatic embryos in different stages of development were collected. Callus and embryo samples were also collected for analysis of gene expression through in situ hybridization. The anatomical and histochemical analysis showed differences between Rangpur lime and Valencia callus cultures. The in situ hybridization results evidenced the expression of the gene AtSERK1 in cultures of both citrus species, and also in both culture conditions: multiplication medium, with sucrose, and embryo induction medium, with maltose, indicating that the embriogenic potential is already installed in the callus cultures in multiplication medium. The results also lead to the conclusion that the gene is conserved between Arabidopsis thaliana and Citrus. Experiments aiming to synchronize the somatic embryogenesis formation in C. sinensis, cv. Valencia, were installed testing the effect of cold, desiccation and cell cluster size on somatic embryo formation. For that, callus cultures of ‘Valência’ sweet orange were cultivated in liquid medium in an orbital shaker, at 200 rpm, for 24h, and sieved through the following screens: 150 µm (P1), 300 µm (P2) and 600 µm (P3). The cell aggregates were then exposed to cold (4 o C, for four weeks, in the dark) and desiccation (27 o C, in Petri plates without culture medium, six days, in the dark) treatments, and cultured in incubators at 26 ± 1 o C and 300 lux of light intensity. The results lead to the conclusion that the separation of cultures in clusters of different sizes and the stress treatments were effective for synchronization and embryogenesis frequency. The size of cell clusters was the most important factor.

Convergence of quantitative genetics and genomics is becoming the way that fundamental genetics and applied breeding will be carried out in the next decades. This study bridges the quantitative genetics of complex growth and wood properties traits with genomic technologies towards a more innovative approach to tree breeding. Planted forests play a major role to fulfill the growing world demand for wood products and energy. Eucalypts stand out for their high productivity and versatile wood resulting from the advanced breeding programs associated to clonal propagation and modern silviculture. Despite their fast growth, breeding cycles still take several years and wood properties assessment is limited to a sample of trees in the late stages of selection due to the costs involved in wood phenotyping, not exploitingthe range of genetic variation in wood properties. In this study, we examined fifteen traits including growth and wood chemical and physical properties in 1,000 individuals sampled from an elite Eucalyptus breeding population. Near-infrared spectroscopy (NIRS) models were developed and used for high-throughput phenotyping of wood traits.Highdensity data for 29,090 SNPs was used to obtain accurate pedigree-record-free estimates of trait variance components, heritabilities, genetic and phenotypic correlations, based on a realized relationship matrix, comparing them to pedigree-based estimates. To the best of our knowledge, this is the first study to do this in plants. NIRS predictions were accurate for wood chemical traits and wood density, and variably successful for physical traits. Heritabilities were medium for growth (0.34 to 0.44), high for wood chemical traits (0.56 to 0.85) and variable for wood physical traits (0.11 to 0.63). High positive correlations among growth traits and negative between cellulose and lignin content were observed, while correlations between wood chemical and physical traits and between growth and wood quality traits were low although significant. Phenotypes and SNP markers were then used to build genomic predictive models using a marker density higher than any previous genomic selection study in trees (1 SNP/21 kbp). Two models (RR-BLUP and Bayesian LASSO) that differ regarding the assumed distribution of marker effects were used for genomic predictions. Predictions were compared to those obtained by phenotypic BLUP. Predictive abilities very similar by the two models and strongly correlated to the heritabilities. Accurate genomic-enabled predictions were obtained for wood chemical traits related to lignin, wood density and growth, although generally 15 to 25% lower than those achieved by phenotypic BLUP prediction. Nevertheless, genomic predictions yielded a coincidence above 70% in selecting the top 30 trees ranked by phenotypic selection for growth, wood density and S:G ratio, and 60% when tandem selection was applied. The results of this study open opportunities for an increased use of highthroughput NIRS phenotyping and genome-wide SNP genotyping in Eucalyptus breeding, allowing accurate pedigree-record-free estimation of genetic parameters and prediction of genomic breeding values for yet to be phenotyped trees. These applications should become routine in tree breeding programs for the years to come, significantly reducing the length of breeding cycles while optimizing resource allocation and sustainability of the breeding endeavor. / A convergência da genética quantitativa com a genômica está se tornando a maneira pela qual a genética fundamental e aplicada serão conduzidas nas próximas décadas. Este estudo buscou conectar a genética de fenótipos complexos de crescimento e propriedades de madeira às tecnologias genômicas, em uma abordagem inovadora para o melhoramento florestal. Florestas plantadas têm papel fundamental para satisfazer a crescente demanda mundial por produtos madeireiros e energia. O eucalipto,com sua alta produtividade e madeira versátil, é resultado de programas avançados de melhoramento associados à propagação clonal e silvicultura moderna. Apesar de seu rápido crescimento, ciclos de melhoramento ainda levam muitos anos e a avaliação detalhada de propriedades da madeira é limitada a apenas uma amostra das árvores em estágios avançados de seleção, devido aos altos custos de fenotipagem, não explorando assim toda a variação genética disponível. Neste estudo, examinamos quinze caracteres, incluindo crescimento e propriedades químicas e físicas da madeira, em 1000 indivíduos amostrados de uma população elite de melhoramento. Modelos de espectroscopia de infravermelho próximo (NIRS) foram desenvolvidos e utilizados para fenotipagem de alto desempenho de propriedades de madeira. Genotipagem de alta densidade com 29.090 SNPs foi utilizada para obter estimativas acuradas de componentes de variância, herdabilidades e correlações genéticas baseadas em uma matriz de parentesco realizado, ou seja,sem o uso de pedigree. Este é o primeiro estudo de que temos conhecimento a fazer isso em plantas. Predições NIRS foram precisas para caracteres químicos da madeira e densidade, e apresentaram sucesso variável para caracteres físicos. As herdabilidades foram médias para crescimento (0,34 a 0,44), altas para caracteres químicos de madeira (0,56 a 0,85) e variáveis para caracteres físicos da madeira (0,11 a 0,63). Altas correlações positivas entre caracteres de crescimento e negativas entre celulose e lignina foram observadas, enquanto correlações entre caracteres químicos e físicos da madeira foram baixas, porém significativas. Fenótipos e marcadores SNP foram em seguida utilizados na construção de modelos preditivos com a maior densidade de marcadores já utilizada em estudos de seleção genômica em espécies florestais (1 SNP/21 kpb). Dois modelos de predição (RR-BLUP e LASSO Bayesiano)foram usados nas predições genômicas e comparados ao BLUP fenotípico. Os modelos apresentaram capacidades preditivas similares, fortemente correlacionadas às herdabilidades. Predições genômicas precisas foram obtidas para caracteres relacionados à lignina, densidade e crescimento, embora geralmente 15 a 25% menores do que as predições obtidas por BLUP fenotípico. Contudo, predições genômicas alcançaram coincidências acima de 70% na seleção das melhores 30 árvores ranqueadas pela seleção fenotípica para crescimento, densidade e relação S:G, e de 60% quando seleção em tandem foi aplicada. Os resultados deste estudo abrem enormes oportunidades para o uso combinado de fenotipagem NIRS e genotipagem com SNPs no melhoramento do eucalipto, permitindo estimativas acuradas de parâmetros genéticos e a predição de valores genéticos genômicos para plantas jovens ainda não fenotipadas. Estas aplicações deverão se tornar rotineiras nos programas de melhoramento florestal nos próximos anos, reduzindo significativamente a duração dos ciclos de seleção e, consequentemente, otimizando a alocação de recursos e a sustentabilidade do melhoramento.

Trypanosamas de diversas espécies podem infectar mamíferos de interesse econômico em todo o mundo. Trypanosoma vivax, T. evansi, T. equiperdum, T. congolense, T. b. brucei e T. simiae geram importantes doenças em ungulados na África, Ásia e Américas Central e do Sul; enquanto T. theileri e espécies relacionadas são pouco patogênicas. Compreender a epidemiologia e as interações parasita-vector-hospedeiro requer estudos de estrutura populacional, filogeográficos e de diversidade e relações filogenéticas entre isolados. Sequências de microssatelites e dos genes SSUrRNA, gGAPDH, CatL, ITS, SL, 5S, Cytb e ESAG6 mostraram ampla diversidade biológica e diferenciaram genótipos com associação geográfica e restrição de hospedeiros em T. theileri e espécies relacionadas. T. evansi mostrou importante heterogeneidade de sequências no gene ESAG6, enquanto populações de T. vivax muito divergentes foram observadas em regiões preservadas e com transmissão cíclica quando comparadas com a microheterogeneidade biológica de áreas de transmissão mecânica. / Trypanosames can infect diverse species of mammals of economic interest worldwide. Trypanosoma vivax, T. evansi, T. equiperdum, T. congolense, T. brucei brucei and T. simiae cause important diseases in ungulates in Africa, Asia and Central and South America, while T. theileri and related species are of low pathogenicity. Understanding the epidemiology and host-parasite-vector interactions requires studies of population structure, phylogeography and diversity and phylogenetic relationships among isolates. Microsatellite loci and sequences from genes SSUrRNA, gGAPDH, CatL, ITS, SL, 5S, Cytb and ESAG6 revealed high biological diversity and allowed differentiation of genotypes with geographic structure and host-restriction event in T. theileri and related species. T. evansi showed significant heterogeneity in ESAG6 sequences, while populations of T. vivax widely divergent were observed in pristine regions and cyclical transmission compared to the microheterogeneity showed by isolates from mechanical transmission areas.

Molecular markers for seed quality traits including erucic acid content genes, seed coat color genes in Brassica napus and seed coat color genes in B. rapa were developed. A single base change in the Bn-FAE1.1 gene in the A genome and a two-base deletion in the Bn-FAE1.2 gene in the C genome produce the nearly zero content of erucic acid observed in canola. The single base change was detected as single nucleotide polymorphic (SNP) marker with an ABI SNaPshot kit. A multiplexing primer set was designed by adding a polyT to the 5´ primer end to increase SNP detection throughput through sample pooling. The two-base deletion in the C genome gene was detected as a sequence characterized amplified region (SCAR) marker in an ABI 3100 Genetic analyzer. To increase the throughput, one genome specific primer was labeled with four fluorescence dyes and combined with 20 different primers to produce PCR products with different fragment sizes. These multiplexed high throughput molecular markers have been successfully implemented in our canola/rapeseed breeding programs. Trigenic inheritance was observed for seed coat color in B. napus. Three Sequenced Related Amplified Polymorphism (SRAP) markers very closely linked to the three different seed coat color genes were developed. Chromosome-walking technology was used to convert the SRAP marker into a SCAR marker and a SNP marker. Subsequently, the first seed coat color gene (Bn1) marker was converted into a SCAR marker, and the second seed coat color gene (Bn2) marker was converted into a SNP marker. Digenic inheritance was observed for seed coat color genes in B. rapa. A SRAP marker was identified as being tightly linked to the major seed coat color gene (Br1). The SRAP marker was sequenced and extended sequences were obtained using chromosome-walking technology. The flanking sequences of the SRAP marker contained 24 SNPs and a 12-bp deletion position that allowed the marker to be converted into a co-dominant SNP marker and a co-dominant SCAR marker, respectively. The SCAR marker was detected in the ABI 3100 genetic analyzer with four fluorescently labeled M13 primers integrated with different SCAR primers, which permitted pooling of PCR samples for high throughput detection. / October 2007

Cucurbit yellow stunting disorder virus (CYSDV) is a relatively new virus affecting cantaloupe production in South Texas and worldwide. No resistant commercial cultivars are available. A cross of ‘Dulce’ (susceptible) x ‘TGR1551’ (resistant) was made and populations were developed for screening. Although no complete resistance was recovered, ‘TGR1551’ showed some tolerance and may be useful in breeding efforts. Sugar components such as sucrose, fructose, glucose, and total soluble solids are major factors in determining mature melon fruit sweetness, and Vitamin C is important for human health. A F2 population was developed from the melon cross ‘Dulce’ (high values) x ‘TGR1551’ (low values) and bulked segregant analysis was used to detect random amplified polymorphic DNA (RAPD) markers associated with quantitative trait loci (QTL) for each trait. Out of 500 primers, fifteen RAPD markers were found to be significantly associated with fruit quality QTL. These markers could be useful in a marker assisted selection program to transfer these genes into a low quality cultivar or breeding line to enhance fruit quality. Gummy stem blight (Didymella brioniae) affects melon production in South Texas as well as other melon production areas in the U.S. A cross between ‘TMS’ (susceptible) and PI 140471 (resistant) was made and a F2 population was screened with a strain of the disease from South Texas. F2 plants exhibited symptoms ranging from resistant to susceptible. PI 140471 may be useful in developing commercial varieties of melon resistant to the disease in Texas. Monosporascus root rot and vine decline (Monosporascus cannonballus) affects melon production in South Texas as well as other melon production areas in the US. A cross was made between ‘TGR1551’ (moderately resistant) and ‘Deltex’ (resistant) to develop a F2 population. Both parents and the F2 were planted in infested soil. Once symptoms appeared, plant roots were removed from the soil and rated. ‘TGR1551’ showed greater resistance than ‘Deltex’ and should be utilized in breeding to develop improved resistant cultivars.

The fungus Fusarium graminearum (Schwabe) causes Gibberella ear rot in maize, resulting in accumulation of harmful mycotoxins in the grain. Disease severity and pericarp/aleurone dehydrodiferulic acid content are negatively correlated. Furthermore, quantitative trait locus mapping (QTL) identified colocalization between QTL for both traits. A candidate gene approach was employed to identify the genes responsible for the observed colocalization. Candidate genes selected on the basis of their putative involvement in various aspects of cell wall DFA accumulation were mapped in silico using the maize genome sequence. Polymorphisms were discovered in putative genes and converted to molecular markers. The in silico mapping effort was successful in predicting map locations of the analyzed sequences, and the segregation of certain marker alleles could explain variation for Gibberella ear rot severity and pericarp-aleurone DFA content.

Molecular markers for seed quality traits including erucic acid content genes, seed coat color genes in Brassica napus and seed coat color genes in B. rapa were developed. A single base change in the Bn-FAE1.1 gene in the A genome and a two-base deletion in the Bn-FAE1.2 gene in the C genome produce the nearly zero content of erucic acid observed in canola. The single base change was detected as single nucleotide polymorphic (SNP) marker with an ABI SNaPshot kit. A multiplexing primer set was designed by adding a polyT to the 5´ primer end to increase SNP detection throughput through sample pooling. The two-base deletion in the C genome gene was detected as a sequence characterized amplified region (SCAR) marker in an ABI 3100 Genetic analyzer. To increase the throughput, one genome specific primer was labeled with four fluorescence dyes and combined with 20 different primers to produce PCR products with different fragment sizes. These multiplexed high throughput molecular markers have been successfully implemented in our canola/rapeseed breeding programs. Trigenic inheritance was observed for seed coat color in B. napus. Three Sequenced Related Amplified Polymorphism (SRAP) markers very closely linked to the three different seed coat color genes were developed. Chromosome-walking technology was used to convert the SRAP marker into a SCAR marker and a SNP marker. Subsequently, the first seed coat color gene (Bn1) marker was converted into a SCAR marker, and the second seed coat color gene (Bn2) marker was converted into a SNP marker. Digenic inheritance was observed for seed coat color genes in B. rapa. A SRAP marker was identified as being tightly linked to the major seed coat color gene (Br1). The SRAP marker was sequenced and extended sequences were obtained using chromosome-walking technology. The flanking sequences of the SRAP marker contained 24 SNPs and a 12-bp deletion position that allowed the marker to be converted into a co-dominant SNP marker and a co-dominant SCAR marker, respectively. The SCAR marker was detected in the ABI 3100 genetic analyzer with four fluorescently labeled M13 primers integrated with different SCAR primers, which permitted pooling of PCR samples for high throughput detection.

Molecular markers for seed quality traits including erucic acid content genes, seed coat color genes in Brassica napus and seed coat color genes in B. rapa were developed. A single base change in the Bn-FAE1.1 gene in the A genome and a two-base deletion in the Bn-FAE1.2 gene in the C genome produce the nearly zero content of erucic acid observed in canola. The single base change was detected as single nucleotide polymorphic (SNP) marker with an ABI SNaPshot kit. A multiplexing primer set was designed by adding a polyT to the 5´ primer end to increase SNP detection throughput through sample pooling. The two-base deletion in the C genome gene was detected as a sequence characterized amplified region (SCAR) marker in an ABI 3100 Genetic analyzer. To increase the throughput, one genome specific primer was labeled with four fluorescence dyes and combined with 20 different primers to produce PCR products with different fragment sizes. These multiplexed high throughput molecular markers have been successfully implemented in our canola/rapeseed breeding programs. Trigenic inheritance was observed for seed coat color in B. napus. Three Sequenced Related Amplified Polymorphism (SRAP) markers very closely linked to the three different seed coat color genes were developed. Chromosome-walking technology was used to convert the SRAP marker into a SCAR marker and a SNP marker. Subsequently, the first seed coat color gene (Bn1) marker was converted into a SCAR marker, and the second seed coat color gene (Bn2) marker was converted into a SNP marker. Digenic inheritance was observed for seed coat color genes in B. rapa. A SRAP marker was identified as being tightly linked to the major seed coat color gene (Br1). The SRAP marker was sequenced and extended sequences were obtained using chromosome-walking technology. The flanking sequences of the SRAP marker contained 24 SNPs and a 12-bp deletion position that allowed the marker to be converted into a co-dominant SNP marker and a co-dominant SCAR marker, respectively. The SCAR marker was detected in the ABI 3100 genetic analyzer with four fluorescently labeled M13 primers integrated with different SCAR primers, which permitted pooling of PCR samples for high throughput detection.

This research carries out a survival analysis for patients with breast cancer. The influence of clinical and pathologic features, as well as molecular markers on survival time are investigated. Special attention focuses on whether the molecular markers can provide additional information in helping predict clinical outcome and guide therapies for breast cancer patients. Three outcomes, breast cancer specific survival (BCSS), local relapse survival (LRS) and distant relapse survival (DRS), are examined using two datasets, the large dataset with missing values in markers (n=1575) and the small (complete) dataset consisting of patient records without any missing values (n=910). Results show that some molecular markers, such as YB1, could join ER, PR and HER2 to be integrated into cancer clinical practices. Further clinical research work is needed to identify the importance of CK56. The 10 year survival probability at the mean of all the covariates (clinical variables and markers) for BCSS, LRS, and DRS is 77%, 91%, and 72% respectively. Due to the presence of a large portion of missing values in the dataset, a sophisticated multiple imputation method is needed to estimate the missing values so that an unbiased and more reliable analysis can be achieved. In this study, three multiple imputation (MI) methods, data augmentation (DA), multivariate imputations by chained equations (MICE) and AREG, are employed and compared. Results shows that AREG is the preferred MI approach. The reliability of MI results are demonstrated using various techniques. This work will hopefully shed light on the determination of appropriate MI methods for other similar research situations.