Prospecção de marcadores moleculares sexo-específicos e análise de estrutura populacional de pirarucu (Arapaima gigas) na região de Santarém, Pará

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, 2012.O pirarucu (Arapaima gigas) é considerado um dos maiores peixes de água doce, podendo atingir três metros e peso aproximado de 200 quilos. Este peixe possui grande importância econômica para populações ribeirinhas da Bacia Amazônica, por esse motivo vem sofrendo uma grande exploração comercial resultando na drástica redução de espécimes e consequentemente na variabilidade genética desta espécie. O presente trabalho visa associar duas metodologias, com o objetivo de gerar mais conhecimentos sobre a biologia da espécie que sejam úteis para o desenvolvimento de tecnologias para a criação do pirarucu em cativeiro e sua conservação nas bacias fluviais onde pode ser encontrado. O pirarucu possui o comportamento de formar casais para acasalamento, o que proporciona uma barreira para sua criação em cativeiro, pois a identificação do sexo só é possível no período de maturidade sexual e por meio de métodos inviáveis economicamente e ao bem estar do peixe. Na tentativa de solucionar esse problema foi realizada uma varredura do genoma desta espécie utilizando a metodologia BSA (“Bulk Segregant Analysis”) associada a marcadores RAPD (“Random Amplified Polymorphic DNA”) a fim de prospectar sequências sexo-específicas para o desenvolvimento de um marcador molecular para sexagem de peixes pré-púberes. Foram combinadas amostras de machos e fêmeas para formar bulks sexo-específicos e estes foram avaliados com 566 primers RAPD (Operon®). Foram amplificados 2.609 fragmentos com uma cobertura estimada de um marcador a cada 714Kb. Os resultados sugerem que o pirarucu possui um sistema de determinação sexual não cromossômico ou, alternativamente, que a espécie sofreu perda recente do cromossomo sexual. O pirarucu tem passado por uma drástica redução populacional e conseqüente diminuição da variabilidade genética em algumas regiões da Bacia Amazônica. A fim de determinar o estado dessas populações e sua variabilidade, estudos de dinâmica populacional e estrutura genética têm sido realizados. Nesses estudos, normalmente se trabalha com um N amostral reduzido e uma maior abrangência geográfica. No entanto, com base nos dados obtidos neste trabalho, foi possível observar que uma amostragem maior de apenas uma região pode fornecer resultados semelhantes. A metodologia utilizada fundamentou-se no sequenciamento e análise de duas regiões do mtDNA, ATPase (n=95) e citocromo oxidase (n=97) em indivíduos de quatro populações, geograficamente próximas, na região Santarém – PA. Também foi possível concluir que em se tratando dessa espécie a variabilidade genética encontrada é baixa, no entanto, entre as populações estudadas é possível encontrar certa diferenciação. Além disso, de acordo com a distribuição das redes de haplótipos observada, é possível sugerir uma recente expansão populacional de peixes oriundos da região Santa Maria. Esses resultados são compatíveis com os poucos trabalhos reportados na literatura sobre essa espécie, comprovando que é possível aumentar o N amostral e reduzir a representatividade geográfica, e assim melhorar a realização de estratégias de manejo locais a partir dos resultados de estudos filogenéticos para análise da estrutura populacional da espécie. ______________________________________________________________________________ ABSTRACTThe arapaima (Arapaima gigas) is considered one of the largest freshwater fish and can reach three meters and weighing approximately 200 pounds. This fish has great economic importance to coastal communities in the Amazon Basin, for this reason has been undergoing a major commercial operation resulting in drastic reduction of specimens and thus genetic variability in this species. The present work aims to link two methodologies, in order to generate more knowledge about the biology of the species that are useful for the development of technologies for the creation of arapaima in captivity and conservation in river basins where it can be found. The arapaima has behavior to form mating pairs, which provides a barrier to their breeding in captivity, because the sex identification is possible only in the period of sexual maturity and by methods uneconomical and welfare of the fish. In an attempt to solve this problem, we scanned the genome of this species using the methodology BSA ("Bulk Segregant Analysis") associated with RAPD ("Random Amplified Polymorphic DNA") to sex-specific sequences prospect for the development of a molecular marker for sexing fish prepubescent. Samples were combined to form male and female sex-specific bulks and these were evaluated with 566 RAPD primers (Operon ®). 2609 fragments were amplified with an estimated coverage of one marker every 714Kb. The results suggest that pirarucu has a system for determining sex chromosome not or, alternatively, that it has suffered sudden loss of the sex chromosome. The arapaima has undergone a drastic population reduction and consequent reduction of genetic variability in some regions of the Amazon Basin. In order to determine the status of these populations and their variability, studies of population dynamics and genetic structure have been performed. In these studies, usually working with a small sample N and greater geographic coverage. However, based on data obtained in this study we observed that a larger sample of one region can provide similar results. The methodology is based on the sequencing and analysis of two regions of mtDNA, ATPase (n = 95) and cytochrome oxidase (n = 97) in individuals from four populations, geographically close, in the Santarém - PA. It was also concluded that when it comes to this kind of genetic variability found is low, however, among the studied populations is possible to find some differentiation. Moreover, according to the distribution networks of haplotypes observed, it is possible to suggest a recent population expansion of fish from the Santa Maria region. These results are consistent with the few studies reported in the literature on this species, showing that it is possible to increase the sample size N and reduce geographic representation, and thereby improve the performance of local management strategies from the results of phylogenetic studies for analysis of the structure population of the species

Development of a mouse computational model for MCNPx based on Digimouse (r) images and dosimetric assays

The aim of this study was to create and test a new mice 3D-voxel phantom named DM_BRA for mice and human first-estimation radiopharmaceutical dosimetry. Previously, the article reviews the state-of-art in animal model development. Images from Digimouse CT database were used in the segmentation and on the generation of the voxelized phantom. Simulations for validation of the DM_BRA model was performed at 0.015, 0.1, 0.5, 1 and 4 MeV photons with heart-source. Specific Absorbed Fractions (SAF) data were compared with literature data. The organ masses of DM_BRA correlated well with existing models based on the same dataset; however, few small organ masses hold significant variations. The SAF data in most simulated cases were statistically equal to a significant level of 0.01 to the reference data

Gd-GLU toward NMR imaging: synthesis, characterization and breast cell uptake assay

Breast cancer cell uptake of Gd-metal is investigated based on the formation of coordinate compounds of gadolinium and glucose (Glu) molecules in solution. The hypothesis is that glucose helps Gdinternalization by complex formations constituted of Gd3+ coordinate to m-glucose molecules, whose valence was complemented by Cl- anions. Such a proposal is an insight toward a metabolic-dependent contrast-agent for cancer and inflammation in magnetic resonance image. A solution was prepared based on anhydrous d-glucose and gadolinium chloride (Gd-Glu). Uptake assays for MDA-MB-231(c231) cells were elaborated collecting incubated c231-cells with Gd-Glu and measuring metal-uptake and their concentrations by Nuclear Activation Analysis (NAA). The ionic solution was studied using Direct-Infusion Electrospray Ionization Mass-Spectrometry (ESI-MS) to identify Gd-Glu interactions. Means and standard deviations of Gd-masses were 13.3±0.8 and 12.5±0.7μg, at 361.5 μg of Gd in 3mL Gd-Glu/PBS solution, in times of 30-50 min, equivalent to the concentrations of 13404±2104 and 11347±2742 μg.g-1 in dried cells. Such values were statistically higher than the control with metal presence. ESI-MS demonstrated the m/z-signals at 516, 552, 696, 923, attributed to positively loaded-species containing Glu, Gd+3 and Cl- . In conclusion, Gd-internalization was increased in aqueous solution due to the gadolinium-glucose coordination. Such findings drive the research to MRI with Gd-Glu complexes

Validation of the multiplex PCR for identification of Brucella spp.

ABSTRACT: A multiplex PCR technique for detection of Brucella spp. in samples of bacterial suspension was validated as a complementary tool in the diagnosis of the disease. This technique allows the characterization of the agent without performing biochemical tests, which greatly reduces the time for a final diagnosis, and provides more security for the analyst by reducing the time of exposure to microorganisms. The validation was performed in accordance with the Manual of Diagnostic Tests from OIE (2008) and following the requirements present in the ABNT NBR ISO/IEC 17025:2005. The mPCR validated in this study identified the different species of Brucella ( Brucella abortus , B. suis , B. ovis e B. melitensis ) of bacterial suspension obtained from the slaughterhouse samples, as well as distinguished the biovars (1, 2 e 4; 3b, 5, 6 e 9) of B. abortus in grouped form and differentiated the field strains from vaccine strains, as a quick, useful and less expensive technique in diagnosis of brucellosis in Brazil