Prospecting major genes in dairy buffaloes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Sociología y ética del deporte: Desde la perspectiva relacional

Esta tesis presenta un breve estado de la cuestión de la sociología y la ética del deporte como nueva disciplina académica, desde su origen hasta el presente. En segundo lugar pretende ver que aporta la perspectiva de la sociologia relacional al estudio del deporte. La primera contribución es la “trans-discipliniariedad” entre disciplinas como la sociología y la ética, evitando caer en una ética “sociologizzata” o en una sociología “eticizzata”. Gracias a la relacionalidad se obtiene la des-mercantilización de bienestar. La lógica relacional busca el desarrollo económico sin olvida que la sociedad está formada por personas, que son lo realmente importantes por encima de los interés económicos. Otra contribución es la distinción entre la “sociedad humana” y la “sociedad de lo humano” que evita muchos de los actuales problemas que trae consigo las nuevas tecnologías. Y finalmente aporta el esquema “AGIL” como “brújula relacional” que ayuda a relaizar los objetivos, medios y reglas éticas dentro de la práctica deportiva.

SNPs for parentage testing and traceability in globally diverse breeds of sheep

DNA-based parentage determination accelerates genetic improvement in sheep by increasing pedigree accuracy. Single nucleotide polymorphism (SNP) markers can be used for determining parentage and to provide unique molecular identifiers for tracing sheep products to their source. However, the utility of a particular "parentage SNP" varies by breed depending on its minor allele frequency (MAF) and its sequence context. Our aims were to identify parentage SNPs with exceptional qualities for use in globally diverse breeds and to develop a subset for use in North American sheep. Starting with genotypes from 2,915 sheep and 74 breed groups provided by the International Sheep Genomics Consortium (ISGC), we analyzed 47,693 autosomal SNPs by multiple criteria and selected 163 with desirable properties for parentage testing. On average, each of the 163 SNPs was highly informative (MAF≥0.3) in 48±5 breed groups. Nearby polymorphisms that could otherwise confound genetic testing were identified by whole genome and Sanger sequencing of 166 sheep from 54 breed groups. A genetic test with 109 of the 163 parentage SNPs was developed for matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The scoring rates and accuracies for these 109 SNPs were greater than 99% in a panel of North American sheep. In a blinded set of 96 families (sire, dam, and non-identical twin lambs), each parent of every lamb was identified without using the other parent's genotype. In 74 ISGC breed groups, the median estimates for probability of a coincidental match between two animals (PI), and the fraction of potential adults excluded from parentage (PE) were 1.1×10(-39) and 0.999987, respectively, for the 109 SNPs combined. The availability of a well-characterized set of 163 parentage SNPs facilitates the development of high-throughput genetic technologies for implementing accurate and economical parentage testing and traceability in many of the world's sheep breeds.

Disease-modifying genes and monogenic disorders: experience in cystic fibrosis

The mechanisms responsible for the determination of phenotypes are still not well understood; however, it has become apparent that modifier genes must play a considerable role in the phenotypic heterogeneity of Mendelian disorders. Significant advances in genetic technologies and molecular medicine allow huge amounts of information to be generated from individual samples within a reasonable time frame. This review focuses on the role of modifier genes using the example of cystic fibrosis, the most common lethal autosomal recessive disorder in the white population, and discusses the advantages and limitations of candidate gene approaches versus genome-wide association studies. Moreover, the implications of modifier gene research for other monogenic disorders, as well as its significance for diagnostic, prognostic, and therapeutic approaches are summarized. Increasing insight into modifying mechanisms opens up new perspectives, dispelling the idea of genetic disorders being caused by one single gene.

GFP-tagged CFTR transgene is functional in the G551D cystic fibrosis mouse colon

Trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is central to its function, with the most common mutation, DeltaF508, resulting in abnormal processing and trafficking. Therefore, there is a significant need to develop tools, which enable the trafficking of CFTR to be studied in vitro and in vivo. In previous studies it has been demonstrated that fusion of the green fluorescent protein (GFP) to the N-terminus of CFTR does lead to functional expression of CFTR chloride channels in epithelial cell lines. The aim of the present study was to examine whether it is possible to express GFP-tagged CFTR as a transgene in colonic and airway epithelial cells of cystic fibrosis (CF) mice and to correct the CF defect. Using the epithelial-specific human cytokeratin promoter K18, we generated bitransgenic mice cftr(G551D/G551D) K18-GFP-CFTR+/-, designated GFP mice. Transcripts for GFP-CFTR could be detected in bitransgenic mice by use of RT-PCR techniques. Expression of GFP-CFTR protein was detected specifically in the colonic epithelium by both direct GFP fluorescence and the use of an anti-GFP antibody. Ussing chamber studies showed that the ion transport defect in colon and airways observed in cftr(G551D/G551D) mice was partially corrected in the bitransgenic animals. Thus, K18-GFP-CFTR is functionally expressed in transgenic mice, which will be a valuable tool in studies on CFTR synthesis, processing and ion transport in native epithelial tissues.

On systems thinking, systems biology and the in Silico Plant

The recent summary report of a Department of Energy Workshop on Plant Systems Biology (P.V. Minorsky [2003] Plant Physiol 132: 404-409) offered a welcomed advocacy for systems analysis as essential in understanding plant development, growth, and production. The goal of the Workshop was to consider methods for relating the results of molecular research to real-world challenges in plant production for increased food supplies, alternative energy sources, and environmental improvement. The rather surprising feature of this report, however, was that the Workshop largely overlooked the rich history of plant systems analysis extending over nearly 40 years (Sinclair and Seligman, 1996) that has considered exactly those challenges targeted by the Workshop. Past systems research has explored and incorporated biochemical and physiological knowledge into plant simulation models from a number of perspectives. The research has resulted in considerable understanding and insight about how to simulate plant systems and the relative contribution of various factors in influencing plant production. These past activities have contributed directly to research focused on solving the problems of increasing biomass production and crop yields. These modeling approaches are also now providing an avenue to enhance integration of molecular genetic technologies in plant improvement (Hammer et al., 2002).

Functional promoter analysis using an approach based on an in vitro evolution strategy

In vitro evolution imitates the natural evolution of genes and has been very successfully applied to the modification of coding sequences, but it has not yet been applied to promoter sequences. We propose an alternative method for functional promoter analysis by applying an in vitro evolution scheme consisting of rounds of error-prone PCR, followed by DNA shuffling and selection of mutant promoter activities. We modified the activity in embryogenic sugarcane cells of the promoter region of the Goldfinger isolate of banana streak virus and obtained mutant promoter sequences that showed an average mutation rate of 2.5% after applying one round of error-prone PCR and DNA shuffling. Selection and sequencing of promoter sequences with decreased or unaltered activity allowed us to rapidly map the position of one cis-acting element that influenced promoter activity in embryogenic sugarcane cells and to discover neutral mutations that did not affect promoter Junction. The selective-shotgun approach of this promoter analysis method immediately after the promoter boundaries have been defined by 5' deletion analysis dramatically reduces the labor associated with traditional linker-scanning deletion analysis to reveal the position of functional promoter domains. Furthermore, this method allows the entire promoter to be investigated at once, rather than selected domains or nucleotides, increasing the, prospect of identifying interacting promoter regions.

Roles of transmembrane domain 2 and the first intracellular loop in human noradrenaline transporter function: pharmacological and SCAM analysis

The aim was to investigate the roles of transmembrane domain 2 and the adjacent region of the first intracellular loop in determining human noradrenaline transporter (hNET) function by pharmacological and substituted-cysteine accessibility method (SCAM) analyses. It was first necessary to establish a suitable background NET for SCAM. Alanine mutants of endogenous hNET cysteines, hC86A, hC131A and hC339A, were examined and showed no marked effects on expression or function. hNET and the mutants were also resistant to methanethiosulfonate (MTS), ethylammonium (MTSEA) and MTStrimethylammonium (MTSET). Hence, wild-type hNET is an appropriate background for production of cysteine mutants for SCAM. Pharmacological investigation showed that all mutants except hT99C and hL109C showed reduced cell-surface expression, while all except hM107C showed a reduction in functional activity. The mutations did not markedly affect the apparent affinities of substrates, but apparent affinities of cocaine were decreased 7-fold for hP97C and 10-fold for hF101C and increased 12-fold for hY98C. [H-3]Nisoxetine binding affinities were decreased 13-fold for hP97C and 5-fold for hF101C. SCAM analysis revealed that only hL102C was sensitive to 1.25 mM MTSEA, and this sensitivity was protected by noradrenaline, nisoxetine and cocaine. The results suggest that this region of hNET is important for interactions with antidepressants and cocaine, but it is probably not involved in substrate translocation mechanisms.

Human tartrate-resistant acid phosphatase becomes an effective ATPase upon proteolytic activation

Proteolytic, cleavage in an exposed loop of human tartrate-resistant acid phosphatase (TRAcP) with trypsin leads to a significant increase in activity. At each pH value between 3.25 and 8.0 the cleaved enzyme is more active. Substrate specificity is also influenced by proteolysis. Only the cleaved form is able to hydrolyze unactivated substrates efficiently, and at pH > 6 cleaved TRAcP acquires a marked preference for ATP. The cleaved enzyme also has altered sensitivity to inhibitors. Interestingly, the magnitude and mode of inhibition by fluoride depends not only on the proteolytic state but also pH. The combined kinetic data imply a role of the loop residue D158 in catalysis in the cleaved enzyme. Notably, at low pH this residue may act as a proton donor for the leaving group. In this respect the mechanism of cleaved TRAcP resembles that of sweet potato purple acid phosphatase. (c) 2005 Elsevier Inc. Ail rights reserved.

Morfometria craniana do boto (Phocoena phocoena) na costa portuguesa

Eventos climáticos extremos poderão ter conduzido à instabilidade de habitats e em última instância à sua fragmentação, causando repercussões ainda visíveis nos predadores de topo, tais como o boto (Phocoena phocoena). Consequentemente, os indivíduos que não se adaptaram, tiveram de procurar novos habitats com as respetivas condições ideais, nomeadamente os indivíduos da população de botos do Atlântico Este (norte e sul da Baía de Biscaia). O presente estudo focou-se em indivíduos da costa portuguesa (representando uma amostra da população residente a sul da Baía de Biscaia) e, recorrendo a informação proveniente de estudos previamente realizados, procurou salientar diferenças entre os botos que habitam ao longo do Atlântico Este. Foi aplicado um método de limpeza e branqueamento em crânios de boto, para posterior medição. Foram medidos 65 crânios de botos (programa Image J®), providenciados pela rede de arrojamentos de animais marinhos e obtidos no âmbito do projeto LIFE+ MarPro. As comparações das medidas dos crânios entre géneros foram realizadas a partir de modelos alométricos (programa GraphPad Prism ®) e as comparações entre géneros de cada estágio de desenvolvimento foram efetuadas com recurso a uma análise de PERMANOVA unifatorial (usando distâncias euclidianas), através do programa PAST® v. 2.12. Concluiu-se que na população em estudo, tanto as fêmeas como os machos apresentaram um modelo de alometria negativa. Reportou-se que as fêmeas apresentam comprimentos corporais superiores aos machos, assim como comprimentos e larguras cranianas. Comparativamente aos indivíduos da população a norte da Baía de Biscaia, a amostra de estudo da população do sul da Baía de Biscaia (representada pelos indivíduos da costa portuguesa) apresentou maiores comprimentos corporais e cranianos e maior largura craniana. Estes resultados corroboram observações prévias sobre a população de botos do Atlântico, que detetaram já diferenças genéticas e partilha limitada de genes, diferenças nos hábitos alimentares e habitats distintos nos botos de norte e sul da Baía de Biscaia.

Revisiting the question of limited genetic variation within Schistosoma japonicum

Recent electrophoretic data have indicated that Schistosoma japonicum in mainland China may be a species complex, with the existence of a cryptic species being predicted from the analysis of schistosome populations from Sichuan province. To investigate the Sichuan form of S. japonicum, 4.9 kbp of mitochondrial DNA from each of three samples of the parasite from China (two from Sichuan and one from Hunan) and one from Sorsogon in the Philippines were amplified, sequenced and characterized. The sequence data were compared with those from the related South-east Asian species of S. mekongi (Khong Island, Laos) and S. malayensis (Baling, Malaysia) and that from S. japonicum from Anhui (China). At both the nucleotide and amino-acid levels, the variation among the five S. japonicum samples was limited ( < 1%). This was consistent with the conclusions drawn from previous molecular studies, in which minimal variation among S. japonicum populations was also detected. In contrast, S. mekongi and S. malayensis, species recognized as separate but closely related, differ from each other by about 10%, and each differs by 25%-26% from S. japonicum. Phylogenetic trees provided a graphic representation of these differences, showing all S. japonicum sequences to be very tightly clustered and distant from S. mekongi and S. malayensis, the last two being clearly distinct from each other. The results thus indicate no significant intraspecific genetic variation among S. japonicum samples collected from different geographical areas and do not support the idea of a distinct form in Sichuan.

Dried blood spots collected on filter paper: an international resource for the diagnosis and genetic characterization of human immunodeficiency virus Type-1

The collection of dried blood spots (DBS) on filter paper provides a powerful approach for the development of large-scale, population-based screening programs. DBS methods are particularly valuable in developing countries and isolated rural regions where resources are limited. Large numbers of field specimens can be economically collected and shipped to centralized reference laboratories for genetic and (or) serological analysis. Alternatively, the dried blood can be stored and used as an archival resource to rapidly establish the frequency and distribution of newly recognized mutations, confirm patient identity or track the origins and emergence of newly identified pathogens. In this report, we describe how PCR-based technologies are beginning to interface with international screening programmes for the diagnosis and genetic characterization of human immunodeficiency virus type 1 (HIV-1). In particular, we review recent progress using DBS specimens to resolve the HIV-1 infection status of neonates, monitor the genetic evolution of HIV-1 during early infancy and establish a sentinel surveillance system for the systematic monitoring of HIV-1 genetic variation in Asia.

Limited genetic diversity and high differentiation among the remnant adder (Vipera berus) populations in the Swiss and French Jura Mountains

Although the adder (Vipera berus) has a large distribution area, this species is particularly threatened in Western Europe due to high habitat fragmentation and human persecution. We developed 13 new microsatellite markers in order to evaluate population structure and genetic diversity in the Swiss and French Jura Mountains, where the species is limited to only a few scattered populations. We found that V. berus exhibits a considerable genetic differentiation among populations (global F-ST = 0.269), even if these are not geographically isolated. Moreover, the genetic diversity within populations in the Jura Mountains and in the less perturbed Swiss Alps is significantly lower than in other French populations, possibly due to post-glacial recolonisation processes. Finally, in order to minimize losses of genetic diversities within isolated populations, suggestions for the conservation of this species in fragmented habitats are proposed.