Selenium is a required trace element which was originally discovered by the Swedish chemist Jons Jacob Berzelius in 1817. It was initially believed to be a toxin as it was identified as being the cause of hoof maladies and excessive hair loss in horses that feed upon plants with high selenium content. It wasn’t until 1957 that the potential contributions of selenium to physiology were first demonstrated. Selenium is now known to play a critical role in the maintenance of human health. Interestingly, unlike other trace metals/semi-metals, selenium is directly incorporated into proteins in the form of the amino acid selenocysteine (Sec) in a very complicated and energetically costly fashion. Though rare, being found in only 25 human proteins, Sec proteins are involved in numerous vital biological processes including maintenance of redox homeostasis and anti-oxidant defense. Even though Sec is essential, the reason that Sec replaces its structural analog cysteine (Cys) in only 25 proteins is not widely agreed upon. A previous model suggests that the replacement of Cys with Sec provides enzymes with a type of catalytic advantage. The presence of Cys-containing orthologs of mammalian Sec-enzymes in other eukaryotes argues against this model. A newer model to explain the use of Sec is that the gain of function imparted to an enzyme by replacing Cys with Sec is the ability of Sec to impart chemical reversibility. Building on previous results from our lab demonstrating the ability of Sec to confer proteins with the ability to resist over oxidation we have elucidated the mechanism by which Sec containing thioredoxin reductase (TrxR) resists over oxidation. The ability of Sec-TrxR to resist oxidative inactivation is due to the greater electrophilicity of Sec relative to Cys. This allows for quicker resolution and prevents over oxidation. Based on these findings we also investigate the utility of the alkylating agent dimedone to probe the oxidation state of Sec. Interestingly, it was discovered that dimedone will react with seleneninic acid with the resulting adduct being labile. Additonally it was discovered that dimedone will also react with seleninic acid, resulting in the formation of a dimedone dimer. These results call into question the usefulness of dimedone in deteremining the oxidation state of Sec. Finally, we provide evidence that Sec-TrxR enzymes are able to catalyze single electron reductions. This is most likely due to the formation of a stable Sec radical intermediate. As a whole this project provides support for the theory that Sec was selected for due to its ability to convey chemical reversiablity to proteins.

No presente trabalho, estudaram-se os efeitos da l-alanil glutamina (Ala-Gln), do precondicionamento isquÃmico (PCI) e das duas tÃcnicas concomitantemente sobre a lesÃo pulmonar provocada por isquemia e reperfusÃo (I/R) causada por pinÃamento da aorta infra-renal em ratos. Foram utilizados 60 ratos machos Wistar, randomizados em cinco grupos (n = 12) divididos em dois tempos (n = 6): Grupo Simulado, Grupo I/R, Grupo PCI + I/R, Grupo Ala-Gln + I/R, Grupo Ala-Gln + PCI + I/R. Tempos: T1 (4h de isquemia) e T2 (4 horas de isquemia e 1h de reperfusÃo). Todos os grupos receberam soluÃÃo salina previamente, menos os grupos prÃ-tratados com Ala-Gln que receberam o dipeptÃdeo e soluÃÃo salina em igual volume. Foi utilizado o modelo de pinÃamento da aorta infra-renal com 4 horas de isquemia e 1 hora de reperfusÃo. Determinaram-se as concentraÃÃes de mieloperoxidase (MPO) pulmonar, substÃncias reativas ao Ãcido tiobarbitÃrico (TBARS) e glutationa reduzida (GSH) no sangue e pulmÃo para avaliar os grupos em estudo. O teste de Kolmogorov-Smirnoff mostrou distribuiÃÃo normal dos dados. Dados expressos como mÃdia acompanhada pelo seu desvio padrÃo (MÃdia  DPM) sendo realizado teste t de Student. Para anÃlise comparativa simultÃnea de trÃs grupos utilizou-se o teste Anova com pÃs-teste de Tukey. Em todos os casos foi adotado o nÃvel de significÃncia de p<0,05. Houve elevaÃÃo das concentraÃÃes de MPO pulmonar tanto no grupo submetido à isquemia quanto no grupo que realizou a I/R. Ocorreu reduÃÃo significante das concentraÃÃes de MPO pulmonar nos grupos submetidos à isquemia prÃ-tratados com Ala-Gln e com PCI. Na avaliaÃÃo dos grupos que sofreram I/R nÃo foi observada alteraÃÃo nas concentraÃÃes de MPO nos grupos prÃ-tratados Ala-Gln ou PCI. O grupo prÃ-tratado com as duas tÃcnicas apresentou aumento significante da MPO nos tempos estudados. A Ala-Gln como prÃ-tratamento isolado reduziu TBARS plasmÃtico na isquemia e o aumentou no pulmÃo na I/R. Jà no pulmÃo durante isquemia e no plasma na I/R houve reduÃÃo da GSH. O PCI como prÃ-tratamento isolado elevou o TBARS pulmonar na I/R e reduziu a GSH pulmonar na I/R. A associaÃÃo da Ala-Gln e PCI acresceu o TBARS plasmÃtico na isquemia, tambÃm o elevando no pulmÃo e mÃsculo na I/R. Jà a GSH, com os dois prÃ-tratamentos, sofre reduÃÃo plasmÃtica na isquemia e pulmonar na I/R, com elevaÃÃo plasmÃtica na I/R. O presente estudo demonstra que tanto o prÃ-tratamento com Ala-Gln como o PCI protegem contra a lesÃo isquÃmica à distÃncia, em modelo murino de pinÃamento da aorta infra-renal quando avaliado MPO pulmonar. O mesmo nÃo ocorre na lesÃo por I/R. NÃo hà benefÃcio, e sim agravamento de lesÃo à distÃncia pulmonar, na associaÃÃo dos dois prÃ-tratamentos ao mensurar a MPO pulmonar. / The present work determined the effects of pre-treatment with L-alanyl glutamine (Ala-Gln) and ischemic preconditioning (IPC), alone and in combination, against lesions caused by I/R by clamping the infrarenal aorta in rats. Sixty Wistar rats were distributed into five groups (n = 12) divided into two times (n = 6): Control, Group I/R, Group IPC + I/R, Group Ala-Gln + I/R, Group Ala-Gln + IPC + I/R. Times: T1 (infrarenal-aorta clamping ischemia-4h); T2 (ischemia-4h plus reperfusion-1h). Pulmonary myeloperoxidase (MPO) and plasma TBARS concentrations were measured. Data expressed as mean  standard-deviation, analyzed by Studentâs t-test and ANOVA/Tukeyâs post-test. P-values < 0,05 were considered significant. Increased MPO concentrations in ischemic group and in I/R group occurred as compared to control. Reduction in MPO concentrations happened in ischemic groups pre-treated with either Ala-Gln or IPC. I/R induced no change in MPO concentrations in groups pre-treated with either Ala-Gln or IPC. Pre-treating with the two procedures showed increased MPO at both times studied. Reduction in TBARS concentrations occurred in Ala-Gln pre-treated group, whereas significant elevation was observed when Ala-Gln and IPC were associated in ischemic animals. Ischemia/reperfusion induced elevation of plasma TBARS. Pre-treatment with either Ala-Gln or IPC protects against distant pulmonary lesion due to ischemia. The same did not occur in I/R lesion. Combining the two procedures aggravated inflammation indicated by increased MPO concentrations. Elevated TBARS concentrations in ischemic animals pre-treated with the two procedures indicate increased lipid peroxidation, whereas pre-treatment with Ala-Gln induced decreased TBARS concentrations.