An acetonitrile based protein extraction method was developed that demonstrated high efficient and effective removal of high abundant proteins from both human and murine serum. The protein content of the extract was characterised using gel electrophoresis, the Bradford assay and liquid chromatography tandem mass spectrometry (LC-MS/MS) with database searching. Selected reaction monitoring (SRM) analysis was used to quantify the levels of high abundant serum proteins to further validate the extraction methodology. The ACN depletion method, in combination with artificial neural networks (ANNs) data mining software, was applied to a murine growth hormone (GH) gene doping study with the aim of identifying biomarker ions capable of detecting gene doping. The LC-MS and ANNs analysis approach failed to conclusively identify a biomarker to gene doping in the mouse model. However, the application of the same technique to serum from a rhGH administration study in humans, returned models capable of discriminating between rhGH treated placebo states. The ion identified as being the most discriminatory was characterised using mass spectrometry, and was derived from the protein leucine-rich a-2-glycoprotein (LRG). Multiple LRG related tryptic peptides were identified as being up-regulated upon dosing with recombinant human GH (rhGH). A high throughput LC-MS/MS and SRM approach was developed to quantify proteins in human serum. The approach was validated by comparison of LC-MS/MS derived APO A1 concentrations with those obtained using established clinical analyser technologies. The LC-MS/MS methodology was applied to a large cohort of 257 serum samples from two rhGH administration studies performed at Royal Free Hospital . The two administrations included serum samples from 15 individuals who had been dosed daily with rhGH. Serum concentrations of the established rhGH biomarker insulin-like growth factor-I (IGF-I) were quantified by LC-MS/MS and compared well with those determined using two different immunoassay-based methodologies. Serum concentrations of the LRG protein were measured simultaneously with IGF-I and appeared to increase in 14 of the 15 rhGH dosed individuals. Combining the LRG and IGF-I data further increased the separation of rhGH treated and placebo states within each individual, and the application of ANNs analysis showed that the combination of the two proteins increased the discrimination characteristics over using IGF-I alone. The murine equivalent of the LRG protein was identified and SRM transitions for a tryptically derived peptide were developed, along with transitions for monitoring a peptide from the murine IGF-I protein. These transitions were used to quantify the two proteins in the remaining aliquots from a murine GH gene doping experiment, however neither protein appeared to increase in the GH +ve plasmid samples that were analysed.

Made available in DSpace on 2014-08-20T13:32:56Z (GMT). No. of bitstreams: 1 dissertacao_thais_farias_collares.pdf: 421000 bytes, checksum: d922362fc9456c8a75d15a49744bf882 (MD5) Previous issue date: 2010-05-28 / The administration of recombinant proteins is being used in sport as gene doping. In medicine, a recent therapeutic technique is the genetic therapy, which, up to this moment, shows results that indicate its efficiency in the treatment of some diseases. Recently, the potential for misuse of gene therapy among athletes has called the attention of scientists and sports regulating organs. The transfer of genes that could enhance athletic performance was named gene doping. The most important candidate genes for gene doping are Erythropoietin (EPO), vascular endothelial growth factor (VEGF), insulin-like growth factor I (IGF-1) and myostatin blockers. Nevertheless, gene therapy presents adverse indicators, such as inflammatory response and lack of control of gene activation. It is probable that in healthy individuals such problems would be aggravated. There are still no conclusive tests capable of detecting gene doping. However, recent researches have studied promising strategies. The reflection of the use of EPO on reproductive parameters in vivo has not yet been described. On the other hand, in vitro studies with cultured cells have shown that erythropoietin stimulates steroidogenesis in Leydig cells, triggering an increase in testosterone production. The objective of this study is to evaluate the effects of the administration of recombinant erythropoietin (rHuEpo) and erythropoietin gene transfer in reproductive parameters of rabbits. Fifteen rabbits were divided in 3 groups: group I (rHuEpo) received subcutaneously 25UI/kg of recombinant human erythropoietin, three times a week for 5 weeks; group II (pTarget/Epo) received a single dose of recombinant vector with the gene of the rabbit erythropoietin; group III (pTarget) received a single dose of empty pTarget vector (control). Throughout the experiment, reproductive and blood parameters were monitored, such as: sperm motility, spermatic vigor, sperm concentration, sperm viability, sperm morphology, erythrocytes and hematocrit level. Erythropoietin gene transfer and rHuEpo administration caused a significant increase in the number of erythrocytes. The animals which received rHuEpo showed an increase in the hematocrit level, reaching numbers between 41,34 and 52,32. The statistical analysis proved that the treatment and the time did not interfer on sperm motility, sperm concentration and spermatic vigor (P<0.05). The percentage of morphologically normal cells in group I as well as in group II decreased over time, however, there was no statistical difference between the treatments (P<0.05). This study is the first to show the answers to the use of gene doping with the erythropoietin gene and the rHuEpo administration in reproductive and blood parameters. / A administração de proteínas recombinantes vem sendo utilizada no esporte como um meio de doping. Na medicina, um método terapêutico bastante recente é a terapia gênica, que até o momento, possui resultados indicando sua eficiência no tratamento de algumas doenças. Recentemente, o potencial para uso indevido desta terapia entre atletas tem despertado a atenção de cientistas e órgãos reguladores do esporte. A transferência de genes que poderiam melhorar o desempenho esportivo de atletas saudáveis foi denominada de doping genético. Os principais genes candidatos são eritropoetina (EPO), fator de crescimento endotelial vascular (VEGF), fator de crescimento semelhante à insulina tipo 1 (IGF-1) e bloqueadores da miostatina. Porém a terapia gênica apresenta indicadores adversos, como resposta inflamatória e falta de controle da ativação do gene. Em indivíduos saudáveis, é provável que essa situação seja agravada. Ainda não existem testes conclusivos para a detecção do doping genético, no entanto alguns estudos recentes têm o intuito de investigar algumas estratégias que apontam como promissoras. O reflexo do uso da EPO sobre parâmetros reprodutivos in vivo ainda não tem sido descritos, por outro lado, estudos in vitro com cultivo de células têm demonstrado que a eritropoetina estimula a esteroidogênese nas células de Leydig desencadeando um aumento na produção de testosterona. O objetivo deste estudo foi avaliar os efeitos da administração de rHuEpo (eritropoetina recombinante) e da transferência gênica com eritropoetina em parâmetros reprodutivos de coelhos. Quinze coelhos foram divididos em 3 grupos: grupo I (rHuEpo) receberam por via subcutânea 25UI/kg de eritropoetina humana recombinante, três vezes por semana durante cinco semanas, grupo II (pTarget/Epo) receberam dose única de vetor recombinante com o gene da eritropoetina de coelho; grupo III (pTarget) receberam dose única de vetor pTarget vazio (controle). Parâmetros sanguíneos e reprodutivos foram monitorados durante o experimento, tais como: motilidade, vigor espermático, concentração espermática, viabilidade espermática, morfologia espermática, hemácias e hematócrito. A transferência gênica com eritropoietina e a administração de rHuEpo causaram um aumento significativo no número de eritrócitos. Os animais que receberam rHuEpo obtiveram um aumento no hematócrito, alcançando valores entre 41,34 e 52,32. A análise estatística mostrou que os tratamentos e o tempo não interferiram na motilidade, concentração espermática e vigor espermático (P <0,05). A porcentagem de células com morfologia normal, tanto do grupo I como do grupo II diminuiu no decorrer do tempo, mas não houve diferença estatística entre os tratamentos (P<0,05). Este é o primeiro estudo que relata as respostas do uso do doping genético com o gene da eritropoetina e da administração de rHuEpo em parâmetros sanguíneos e reprodutivos.