Programmed cell death, known as apoptosis, is widely accepted as a key tumour suppression mechanism. The oncogene MYC promotes cell growth and proliferation but also sensitises cells to apoptosis, which limits its oncogenic potential. MYC-induced apoptosis requires the pro-apoptotic BCL2 family proteins BAX/BAK and can be blocked by anti-apoptotic family members such as BCL2 and BCL-xL. Previous studies have identified glutamine withdrawal as a trigger for MYC-induced apoptosis. Through untargeted metabolomic analyses of cells with perturbed BCL2 family member composition and of cells undergoing glutamine-dependent MYC-induced apoptosis, we found that nucleosides and nucleotides were altered in correlation with apoptotic status. Glutamine is an important biosynthetic substrate and energy source and we show global transcription and translation of the cells decreased upon glutamine withdrawal. However, MYC-activated cells promote transcription and translation even in the absence of glutamine and thus still drive huge demand for energy. Deregulated MYC promotes nucleotide catabolism and depletes cellular energy charge upon glutamine withdrawal, indicating energy shortage driven by MYC. Nucleotide conversion and remodeling by adenylate kinase 2 (AK2) protects cellular energy charge and inhibits MYC-induced apoptosis. These results indicate a homeostatic model for MYC-induced apoptosis based upon mitochondrial energy supply and demand. We propose that the transcriptional activity of MYC drives huge demand for energy to support global transcription and translation and thereby sensitises cells to apoptosis under conditions of limiting energy supply.

Futebol é um esporte de natureza intermitente, consistindo de séries repetidas (6-segundos) de trabalho rápido de alta intensidade e curta duração e de períodos de recuperação de intensidade baixa a moderada (60-segundos). O objetivo deste estudo foi caracterizar a antropometria, o consumo alimentar, o desempenho e as respostas bioquímicas, moleculares e celulares de jogadores de futebol no teste de esforço máximo e determinar se a reposição aguda de glutamina altera essas variáveis. Quatorze jogadores de futebol (idade: 18 e 21 anos), de time profissional, representativos dos atletas dessa modalidade e faixa etária, foram avaliados. A avaliação antropométrica mostrou que esses jogadores apresentavam altura, peso e percentual de gordura, compatíveis com a idade e modalidade. A avaliação do consumo alimentar mostrou um consumo inadequado de macronutrientes e cálcio segundo IDR e ACSM. O desempenho desses atletas no teste foi dentro do esperado para atletas desta modalidade. O exercício causou acidose metabólica; aumento dos níveis plasmáticos de produtos de peroxidação lipídica, glutamina e glutamato, e aumento dos leucócitos circulantes, sem alterações na glicemia, enzimas musculares, ou proteína do choque térmico 27. A suplementação com glutamina peptídeo não proporcionou melhora no desempenho nem alterou as variáveis analisadas, após o exercício, exceto os produtos da peroxidação lipídica, que diminuíram com a suplementação. Frente aos resultados obtidos, concluiu-se que sem uma alimentação adequada para faixa etária, nível de maturação sexual e modalidade esportiva, mesmo com a prescrição correta de treinamento, a suplementação com glutamina não será capaz de proporcionar efeitos positivos no desempenho físico. / Soccer is an intermittent sport in nature, consisting of repeated bouts of brief (6-second) maximal/near-maximal work interspersed with relatively short (60-second) moderate/low-intensity recovery periods. The aim of this study was to characterize anthropometry, food habit, performance, as well as biochemical, molecular and cellular responses of fourteen soccer players (age 18-21) to maximum exercise testing and to determine whether glutamine peptide supplementation changes these variables. The anthropometric and body composition assessment showed that the players presented height, weight and amount of body fat compatible with international sport criteria; however, body fat revealed a great variability. The dietary assessment identified an inadequate macronutrient and calcium consumption, compared to RDI e ACSM recommendations. Athlete\'s performance in maximum exercise testing was close to this modality average. The exercise testing induced a metabolic acidosis, with increased blood lactate levels; increased levels of glutamine and glutamate; increase in lipid peroxidation markers and in white blood cell count, while no change was detectable in blood glucose, muscle enzymes or heat shock protein 27 expression. Glutamine supplementation caused no change either in performance or in biochemical, molecular or cellular variables after aerobic exercise, except for lipid peroxidation markers, which decreased with supplementation. Therefore, it was concluded that without a healthy diet, ajusted to age and sport, glutamine supplement is not able to induce positive effects on performance.

O desenvolvimento de uma resposta imune adequada é um processo extremamente importante para a manutenção da homeostase do organismo. Uma série de processos são desencadeados a partir do primeiro contato com micro-organismos patógenos até a efetivação da resposta imune de memória. Todos esses processos envolvem a participação e a complexa atuação de mediadores como as citocinas inflamatórias e também citocinas regulatórias, que exercerão efeitos controlando o processo inflamatório. Diversos mecanismos moleculares, subjacentes à resposta inflamatória, ainda não estão totalmente compreendidos, como por exemplo o controle da expressão de genes inflamatórios exercido pela IL-10. Os processos envolvidos na resposta inflamatória são mantidos às custas do consumo de nutrientes, dentre eles podemos destacar o aminoácido glutamina, que atua em nível molecular, fornecendo nitrogênio para a formação do material genético e fonte energética para determinadas células do sistema imunológico como os macrófagos. Portanto, neste trabalho, investigamos os efeitos da IL-10 na modificação de nucleossomos, evidenciando o papel dessa citocina em regular a expressão de genes inflamatórios em macrófagos. Avaliamos também a função da glutamina, modulando a expressão de RNAm de citocinas inflamatórias e regulatórias dessas células. E por último, desenvolvemos um modelo de restrição alimentar em camundongos, nos quais avaliamos os efeitos desse modelo considerando-se alguns aspectos hematológicos e estudamos as alterações na resposta inflamatória em células esplênicas e do peritônio, bem como avaliamos a suplementação de glutamina in vitro na produção das citocinas (IL-12, TNF-alfa, IL-10) e a expressão do fator de transcrição NFkB. Os resultados compilados mostraram que a IL-10 leva a uma rápida redução da acetilação de nucleossomos, modulando a arquitetura da cromatina de genes inflamatórios como a IL-12. A glutamina modula a expressão de citocinas inflamatórias, regulando positivamente a expressão de IL-10 e Interferon beta. E a restrição alimentar induz a redução de citocinas proinflamatórias (IL-12 e TNF-α), influenciadas pelo aumento da produção de IL-10 e finalmente a suplementação com glutamina não interfere nesses parâmetros nas células peritoneiais e esplênicas do grupo submetido à restrição alimentar. Conclusão: a IL-10 modula a expressão gênica através da modificação de nucleossomos em macrófagos derivados da medula; a glutamina modula a expressão de IL-10 inibindo a resposta inflamatória, e a restrição alimentar modula alguns aspectos hematológicos e possui propriedades anti-inflamatórias. / The development of an appropriate immune response is an important process to the organism\'s homeostatic maintenance. A series of processes are triggered upon the very first contact with pathogens, up to the immunological memory establishment. These processes implicate in the participation of complex mediators, such as inflammatory and regulatory cytokines that will control the inflammatory process. Some mechanisms underlying the inflammatory response are not totally understood, the control of inflammatory genes exerted by IL-10 is an example. The processes involved in the inflammatory response are kept with nutrients expense, among these nutrients we can highlight the amino acid glutamine. It acts in a molecular level, supplying nitrogen to genetic material formation and as an energy supply for immune cells such as macrophages. Thus, we investigated the IL-10 effects on nucleosome modifications evidencing this cytokine role regulating inflammatory genes expression in macrophages. We also evaluated glutamine functions modulating inflammatory and regulatory cytokines mRNA expression on these cells. Ultimately, we developed a dietary restriction animal model where we evaluated it\'s effects on selected haematological aspects, analyzing the alteration in the inflammatory response of splenic and peritoneal cells. We also evaluated in vitro glutamine supplementation assessing cytokines production (IL-12, TNF-α, and IL-10) and the expression of NFkB transcription factor. The compiled results a expressive reduction in nucleosome acetylation modifying the chromatin architecture of inflammatory genes such as IL-12 and IL-6. Glutamine modulates inflammatory cytokines gene expression upregulating the expression of IL-10 and interferon beta. The dietary restriction reduces proinflammatory cytokines production (IL-12 and TNF-α), these results are influenced by the upregulated IL-10 production, glutamine supplementation have no effect on these parameters in the dietary restriction group. In conclusion, we can infer that IL-10 modulates gene expression trough nucleosome modification in bone marrow derived macrophages, glutamine has a potential effect on IL-10 expression, inhibiting the inflammatory response and dietary restriction modifies hematological parameters, presenting anti-inflammatory properties.

Dados da Literatura têm demonstrado que a CTX, toxina majoritária do veneno de serpente Crotalus durissus terrificus apresenta efeitos anti-inflamatório, imunomodulatório e antitumoral. A CTX modula, particularmente, as funções de macrófagos, células fundamentais para os mecanismos da defesa inata e de importância central na gênese e progressão tumoral. No início da progressão tumoral, os macrófagos apresentam fenótipo pró-inflamatório, caracterizado pela liberação de citocinas inflamatórias, tais como IL-1, IL-6 e TNF-<font face=\"Symbol\">a, reativos intermediários do nitrogênio (RNI) e do reativos intermediários do oxigênio (ROI). No contexto tumoral, os macrófagos inflamatórios inibem o crescimento tumoral, erradicando as células tumorais e estimulando a resposta imune. Por outro lado, os macrófagos anti-inflamatórios encontrados quando o tumor está estabelecido, ou seja, quando atinge o tamanho de 1 cm3, contribuem para a progressão do tumor por produzir mediadores pró-angiogênicos por expressarem baixos níveis das citocinas inflamatórias, perda da capacidade de liberação e de produção de ROI e RNI, importantes para ação tumoricida. Estudos realizados pelo nosso grupo demonstraram que a CTX, após 14 dias de uma única administração, estimula a liberação de peróxido de hidrogênio e produção de óxido nítrico, além de modular a secreção de citocinas por macrófagos obtidos da cavidade peritoneal de ratos normais. Desta forma, considerando que esses mediadores são fundamentais no controle de progressão tumoral, é relevante investigar se a CTX estimula a produção desses mediadores pelos macrófagos obtidos de animais portadores de tumor. Portanto, o objetivo deste trabalho foi investigar os efeitos da CTX sobre a função e o metabolismo de macrófagos peritoneais obtidos de ratos portadores de tumor de Walker 256. Para tanto, ratos machos da linhagem Wistar foram injetados, por via s.c., no flanco posterior direito, o volume de 1 mL de salina estéril contendo 2x107 de células tumorais. Numa primeira abordagem, a CTX (18<font face=\"Symbol\">mg/animal) foi administrada no subcutâneo dos animais no 5º dia após a inoculação das células tumorais. Após 14 dias da inoculação das células tumorais, macrófagos peritoneais foram obtidos e os seguintes parâmetros foram avaliados: a) liberação de H2O2 e, produção de NO e citocinas pró-inflamatórias (IL-1<font face=\"Symbol\">b, TNF-<font face=\"Symbol\">a e IL-6) e b) a atividade máxima da hexoquinase, glicose-6-fosfato desidrogenase, citrato sintase e a produção de 14CO2 de glicose [U-14C] e glutamina [U-14C]. O tratamento com a CTX estimulou a produção de H2O2 e de NO, a secreção das citocinas IL-1<font face=\"Symbol\">b e do TNF-<font face=\"Symbol\">a e a atividade do metabolismo de glicose e glutamina. Em outra abordagem, a administração da CTX foi concomitante à inoculação das células tumorais. Após 14 dias, os macrófagos apresentaram as atividades secretórias e metabólicas estimuladas. Esses dados colocam a CTX não apenas como ferramenta científica para investigar as funções e o metabolismo de macrófagos, como também para a melhor compreensão dos mecanismos envolvidos na progressão tumoral. / Crotoxin (CTX), the major toxin of Crotalus durissus terrificus venom induces an inhibitory effect on tumoral growth and modulates, particularly, the functions of macrophages, fundamental cells to provide a defense mechanism against tumor cell. In early tumor progression, macrophages are avidly phagocytic (inflammatory cell) releasing reactive nitrogen intermediates-RNI/ROI and cytokines TNF-<font face=\"Symbol\">a, IL-1<font face=\"Symbol\">b and IL-6. However, when the tumor is installed, tumor-associated macrophage (angiogenic cell) presents a decrease in these abilities. Our group demonstrates that CTX stimulates the H2O2 release and NO production and the secretion of the cytokines by peritoneal M<font face=\"Symbol\">&#198; obtained from non-tumor-bearing rats. Thus, considering that these are key mediators in the control of tumor progression, it is important to investigate whether CTX stimulates the production of these mediators by macrophages obtained from tumor-bearing animals. The aim of this work was to evaluate the CTX effect on macrophage functions and metabolism of peritoneal macrophage obtained of Walker 256 tumor-bearing rats. For this purpose, male Wistar rats were inoculated subcutaneously in the right flank with 1 ml of sterile suspension of 2×107 Walker 256 tumor cells. In a first approach, CTX (18<font face=\"Symbol\">mg/animal) was administered subcutaneously animals on day 5 after inoculation of tumor cells. After 14 days of tumor cell inoculation, peritoneal macrophages were obtained and the following parameters were evaluated: a) release of H2O2 and NO production and pro-inflammatory cytokines (IL-1<font face=\"Symbol\">b, TNF-<font face=\"Symbol\">a and IL-6) and b) maximal activity of hexokinase, glucose-6-phosphate dehydrogenase, citrate synthase and 14CO2 production from [U-14C]-glucose and [U-14C]-glutamine. The treatment with CTX stimulated NO or H2O2 production, cytokine secretion and glucose and glutamine metabolism. In another set of experiments, CTX injected concomitant with tumor cell inoculation. After 14 days macrophages presented metabolism and secretory activity stimulated. These data CTX not only as a scientific tool to investigate the functions and metabolism of macrophages, but also for better understanding of the mechanisms involved in tumor progression.

Os objetivos desse trabalho foram avaliar se provas de enduro de diferentes distâncias causam estresse em equinos treinados, avaliar os efeitos das provas de enduro de diferentes distâncias sobre a atividade dos linfócitos e a relação com a imunocompetência dos equinos atletas e investigar a suplementação alimentar com glutamina como possível atenuante desse efeito depressivo do estresse sobre o sistema imunológico. Foram utilizados 33 cavalos treinados para enduro, 13 em 80 km, 14 em 120 km e 6 em 160 km, avaliados em 4 provas. Metade dos cavalos de cada categoria recebeu suplementação com glutamina via oral 30 dias antes e 14 dias após as provas. Amostras de sangue venoso foram colhidas antes (M0), imediatamente após a última inspeção veterinária (M1), 3 horas depois (M2) e nos haras 3 (M3), 7 (M4), e 14 dias (M5). Houve aumento dos níveis de cortisol, amônia, neutrófilos, aumento da relação neutrófilos/linfócitos e diminuição da contagem de linfócitos em M1 e M2 em todos os cavalos. Houve diminuição da relação CD4/CD8 nos animais de 120 (M2, M3 e M4) e 160 km (M3) que não receberam suplementação e diminuição de IFN em todos os cavalos. Nos suplementados, houve diminuição da relação CD4/CD8 em 80 (M2), 120 (M2 e M3) e 160 km (M3 e M4) e aumento tardio de IFN (M4 e M5) nos cavalos de 80 e 120 km. As concentrações de IL-2, IL-4 e IL-10 aumentaram após a prova em todos os cavalos, porém, nos suplementados o aumento foi maior ou mais prolongado. Com base nesses resultados, não foi possível observar estresse nos animais, nem imunodepressão, embora a suplementação tenha exercido efeito sobre os linfócitos. / The objectives of this study were to assess whether a different distances endurance races cause stress in trained horses, assess the effects of different distances an endurance races on the lymphocytes and relationship with the immune competence of equine athletes and investigate dietary supplementation with glutamine as possible mitigating this depressive effect of stress on the immune system . 33 well trained endurance horses were used, 13 at 80 km, 14 and 120 km and 6 in 160 km in 4 endurance rides . Half of the horses in each category received oral supplementation with glutamine to 30 days before and 14 days after the race. Venous blood samples were collected before (M0), immediately after the last veterinary inspection (M1), 3 hours after (M2) and in their farms 3 (M3), 7 (M4) and 14 days (M5) after. There was an increase in cortisol levels, ammonia and neutrophils, increase in neutrophils / lymphocytes ratio and reduction in lymphocyte counts at M1 and M2 in all horses. There was a decrease in LT CD4/CD8 ratio in 120 (M2, M3 and M4) and 160 km (M3) without supplementation and either a decreased in IFN in all animals. Horses that had received glutamine supplementation showed a decreased in CD4/CD8 ratio in 80 (M2), 120 (M2 and M3) and 160 km (M3 and M4) and late increase of IFN (M4 and M5) in 80 and 120 km. INF production was increased later (7 and 14 days) in 80 and 120 km horses that received supplementation and decreased in all 160 km. The concentrations of IL-2 , IL-4 and IL-10 increased after the race on all horses , but the increase was greater or more prolonged in supplemented ones. Based on these results, it was not possible to observe stress in these animals, or immunosuppression either, although supplementation has exerted effects on lymphocytes.

A sepse é a principal causa de morte em unidades de terapia intensiva (UTIs) no mundo. A reduzida disponibilidade do aminoácido mais abundante do organismo, a glutamina contribui para o complicado estado catabólico da sepse. No presente estudo investigamos os efeitos da suplementação oral com L-glutamina e L-alanina (GLN+ALA), ambos na norma livre e como dipeptídeo, L-alanil-L-glutamina (DIP), sobre o eixo glutamina-glutationa (GSH), sistema imune, inflamação, proteínas de choque térmico (HSPs) e expressão de genes envolvidos com vias de sinalização proteica em animais endotoxêmicos. Camundongos C57/B6 foram submetidos à endotoxemia (Escherichia coli LPS, 5 mg.kg-1, grupo LPS) e suplementados por 48 horas com L-glutamina (1 g.kg-1) e L-alanina (0,61 g.kg-1, grupo GLN+ALA-LPS) ou 1,49 g.kg-1 de DIP (grupo DIP-LPS). A endotoxemia promoveu depleção da concentração de glutamina no plasma (71%), músculo esquelético (50%) e fígado (49%), quando comparado ao grupo CTRL, sendo restauradas nos grupos DIP-LPS e GLN+ALA-LPS (P<0,05), fato que atenuou a redução da GSH e o estado redox (taxa GSSG/GSH) em eritrócitos circulantes, musculo e fígado (P<0,05). A suplementação em animais endotoxêmicos resultou em uma upregulation dos genes GSR, GPX1 e GCLC no músculo e fígado. A concentração das citocinas plasmáticasTNF-&#945;, IL-6, IL-1&#946; e IL-10 foi atenuada pelas suplementações, bem como a expressão de mRNAs envolvidos com a resposta inflamatória, ativadas pela via do NF-&#954;B(P<0,05). Concomitantemente, verificou-se aumento da capacidade proliferativa de linfócitos T e B circulantes nos grupos GLN+ALA-LPS e DIP-LPS. A expressão de mRNAs e a concentração de HSPs no tecido muscular foi restabelecida pelas suplementações, contudo, a expressão mRNAs relacionados às vias de síntese e degradação proteica foi somente estimulada no tecido hepático(P<0,05). Os resultados do presente estudo demonstram que a suplementação por via oral com GLN+ALA ou DIP podem ser utilizados clinicamente como métodos nutricionais em reverter o quadro de depressão da disponibilidade de glutamina corporal da sepse induzida por LPS, tendo impacto no eixo glutamina-glutationa, sistema imune e inflamatório. / Sepsis is the leading cause of death inintensive care units (ICUs) in the world.The availability ofthe most abundant amino acid in the body, glutamine, is reduced in this situation, fact that contribute to the complicated catabolic state of sepsis. In the present study, we investigated the effects of oral supplementation with L-glutamine and L-alanine (GLN+ALA), both in their free form and as a dipeptide, L-alanyl-L-glutamine (DIP) on glutamine-glutathione axis (GSH), immune and inflammatory system, heat shock proteins (HSPs) expression and gene expressions involved in protein signaling pathways during endotoxemia. C57/B6 mice were subjected to endotoxemia (Escherichia coli LPS, 5 mg.kg-1, LPS group) and supplemented for 48 hours with L-glutamine (1 g.kg-1) plus L-alanine(0.61 g.kg-1, GLN+ALA-LPS group) or 1.49 g.kg-1of DIP (DIP-LPS group). Endotoxemia promoted depletion glutamine concentration in plasma (71%), skeletal muscle (50%) and liver (49%), when compared to the CTRL group, and was restored in the DIP-LPS e GLN+ALA-LPS (P<0.05), fact that attenuate the reduction of GSH and the redox state (GSSG/GSH rate) in circulating erythrocytes, liver and muscle (P<0.05). Supplementations in endotoxemic mice resulted in upregulation of GSR, GCLC and GPX1 genes in muscle and liver. Plasma concentration of TNF-&#945;, IL-6, IL-1&#946; and IL-10 were attenuated by supplementation as well as the expression of mRNAs involved in the inflammatory response, activated by NF&#954;-B pathway (P <0.05). At the same time, high proliferative capacity of circulating T and B lymphocytes GLN+ALA-LPS e DIP-LPS were observed. HSPs (protein and mRNAs) and in muscle were restored by the supplements, however, the mRNAs expression related to the synthesis and degradation of protein pathways was only stimulated in the liver (P <0.05). Our results demonstrate that oral supplementation with GLN+ALA or DIP can be used as clinically nutritional methods to reverse the depression of body glutamine availability during sepsis induced by LPS, impacting on the glutamine-glutathione axis, immune and inflammatory system.

An important cause of pulmonary arterial hypertension (PAH) in children with congenital heart disease (CHD) is increased pulmonary blood flow (PBF). To gain a better understanding of the disease process, the changes in biochemical pathways and metabolism of pulmonary arterial smooth muscle cells (PASMCs) were studied using a unique surgical ovine model of increased pulmonary blood flow. PASMCs isolated from 4-week-old lambs with increased PBF (shunt) showed lower oxygen consumption rates and lower extracellular acidification rates linked to glutamine metabolism when compared to controls. Shunt and control PASMCs both exhibited a switch into the reverse tricarboxylic acid (TCA) cycle, while only shunt cells showed a decrease of glucose being transformed into Acetyl CoA to enter the forward TCA cycle. Shunt PASMCs also demonstrated increased levels of yes-associated protein 1 (YAP1) expression in the nucleus. These results indicate changes in glutamine metabolism, glucose metabolism, and protein signaling cascades associated with increased mechanical forces in the setting of increased PBF, as seen in PAH in children with CHD.

Consumption of endophyte-infected tall fescue results in a syndrome of negatively altered physiological systems, collectively known as fescue toxicosis. Another challenge to endophyte-infected tall fescue -based beef cattle operations is that the soils often are selenium (Se) poor, necessitating the need to provide supplemental Se. To test the general hypothesis that different forms of supplemental Se would ameliorate the negative effects of fescue toxicosis, predominately-Angus steers (BW = 183 ± 34 kg) were randomly selected from herds of fall-calving cows grazing an endophyte-infected tall fescue pasture and consuming vitamin-mineral mixes that contained 35 ppm Se as sodium selenite (ISe), SELPLEX (OSe), or an 1:1 blend of ISe and OSe (MIX). Steers were commonly weaned and depleted of Se for 98 d. Steers were assigned (n = 8 per treatment) to the same Se-form treatments upon which they were raised and subjected to summer-long common grazing of an endophyte-infected tall fescue pasture (0.51 ppm ergot alkaloids: ergovaline plus ergovalinine; 10.1 ha). Selenium treatments were administered by daily top-dressing 85 g of vitamin-mineral mix onto 0.23 kg soyhulls, using in-pasture Calan gates. The first project objective was to determine the effect of forms of supplemental Se on whole blood Se, serum prolactin, liver glutamine synthetase (GS) activity, carcass parameters, and growth performance (Experiment 1). In Experiment 1, whole blood Se increased for all treatments from day 0 to 22 and then did not change. Across periods, MIX and OSe steers had greater whole blood Se than ISe steer. Compared to ISe steers, MIX and OSe steers had more serum prolactin. Liver GS mRNA, protein content, and activity were greater in MIX and OSe steers than ISe steers. However, the ADG and carcass parameters were not affected by Se treatments. The second project objective was to determine the effect of forms of supplemental Se on serum clinical parameters of Experiment 1 steers (Experiment 2). In Experiment 2, across periods, MIX steers had more serum albumin than OSe, and ISe steers, respectively. Serum alkaline phosphatase (ALP) activity was greater in MIX and OSe steers. In addition, blood urea nitrogen (BUN), serum sodium, phosphorus, and magnesium concentration were affected by Se treatments. Partial correlation analysis revealed that serum albumin, BUN, and ALP activity were correlated with whole blood Se concentration. The third project objective was to evaluate the hepatic transcriptome profiles of Experiment 1 steers using microarray and targeted RT-PCR analyses (Experiment 3). In Experiment 3, bioinformatic analysis of microarray data indicated that hepatic glutamate/glutamine, proline, arginine, and citrulline metabolism was affected by different forms of supplemental Se. The mRNA expression of critical proteins involved in glutamate/glutamine (GLS2, GLUD1, GLUL), proline (PYCR1, ALDH18A1), and urea (ARG1, ARG2, OAT, NAGS, OTC, ORNT1) metabolism were differentially expressed by Se treatments. Collectively, we conclude that consumption of 3 mg Se/d as OSe or MIX forms of Se in vitamin-mineral mixes 1) increased whole blood Se content, an indicator of greater whole-body Se assimilation; 2) increased serum prolactin, albumin, and ALP, the reduction of which are hallmarks of fescue toxicosis; and 3) altered hepatic nitrogen metabolism, as indicated by changes in key enzymes of glutamate/glutamine, proline, and urea metabolism. However, 4) these positive effects on metabolic parameters were not accompanied by increased growth performance.

As nitrogen (N) is a critical nutrient for plant growth, the development of synthetic N fertilisers dramatically changed agricultural production in the twentieth century. Improvement in N use efficiency (NUE) has been a focus of breeding for grain crop species, since protein is an important component of the harvested product. The study of NUE in sugarcane has lagged behind grain crops, mainly because N is not a component of sucrose, the primary product of the traditional sugarcane industry. Recently, improvement in NUE has become a focus of sugarcane breeding, due largely to environmental concerns regarding pollution from high N fertilisation, and the increasing cost of N fertilisers. This thesis aimed to gain an initial understanding of the genetic basis for variability in NUE in sugarcane. This was achieved through: (i) the screening of 168 sugarcane genotypes under limiting and non-limiting N supply in two glasshouse experiments; (ii) the mapping of marker-trait associations (MTA) for biomass and physiological traits under limiting and non-limiting N supply in a sugarcane mapping population; (iii) the analysis of expression of candidate genes encoding enzymes involved in the central processes of N assimilation and remobilisation in plants; and (iv) the mapping of candidate genes in a sugarcane genetic map. Genetic variation was identified for growth traits as well as physiological traits including %N, internal NUE (iNUE, g dry weight g-1 N) and leaf glutamine synthetase (GS) activity in a sugarcane mapping population. These traits were also analysed for linkage with genetic markers. Genetic variation in the screened genotypes was higher under limiting N supply, a finding that was reflected by the fact that marker-trait associations (MTA) for increases in iNUE were not identified under non-limiting N supply in the commercial parent of the mapping population. Contrary to findings in grain crop species, there was no link between GS activity and other traits, either through phenotypic correlations or co-location of MTA. The expression of candidate genes encoding GS, nitrate reductase (NR) and alanine amintotransferase (AlaAT) was quantified with Sequenom™ MassARRAY technology. Plants were grown under growth-limiting N supply, non-limiting N supply, or a N-pulse treatment, which consisted of growth-limiting N supply followed by non-limiting N supply 24 hours prior to sampling. Two genes, scAlaAT.d and scGS1.a, encoding AlaAT and GS respectively, were identified as non-responsive to changes in N supply, whereas scAlaAT.a, scGS1.b and scGS1.c had significantly (p<0.05) increased expression under a N-pulse, indicating an important role for these genes in the response of sugarcane to a sudden increase in N availability. The location of candidate genes associated with variation in NUE in a sugarcane genetic map were sought through restriction fragment length polymorphism (RFLP) markers. Twenty-two probes were screened, of which two generated single-dose markers, allowing the mapping of a single allele of scAspAT, encoding aspartate aminotransferase, and two alleles of scGS2, encoding plastidic GS. Because of the economic and environmental consequences of inefficient N fertiliser application, the development of sugarcane cultivars with improved NUE is essential. Since variation for NUE exists, especially in unimproved sugarcane varieties, this may be achieved through traditional breeding methods by screening existing breeding populations under limiting N supply. Additionally, an improved understanding of the genetic basis of variation for NUE in sugarcane should be pursued by further analysis of candidate gene response to changing N availability by screening widely varying cane species for differences in gene expression, enzyme activity and metabolite profiles. The further addition of candidate gene locations to sugarcane genetic maps will aid both future marker-assisted selection in breeding, and a fundamental understanding of genetic control of NUE variation. Through the development of sugarcane cultivars with improved NUE and an enhanced knowledge of the genetic control underpinning sugarcane N physiology, concerns regarding high N fertiliser applications may be mitigated and sustainability ensured.

Oxidative stress has been associated with many disease conditions in adults and neonates based on clinical and post mortem studies. Trauma is the commonest cause of oxidative stress. However a gap in knowledge of the natural history of oxidative stress in humans was identified as most studies have been post mortem or in animals. <p>The aim of this research is to understand treat and oxidative stress in trauma and surgical patients. The study involved three components including: the development and evaluation of the novel oxistress assay; study of clinical trauma and oxidative stress; and clinical trial of alanyl-glutamine supplementation following major surgery. The novel oxistress assay was used on urine samples in the normal population to determine reference values and subsequently on hospital patients to determine sensitivity and specificity. The study of clinical trauma and oxidative stress evaluated plasma antioxidants (FRAP assay), red cell glutathione (Asensis method), plasma and urine protein carbonyl (Levines method) and total oxidants in plasma and urine (oxistress assay) over 7 day period following trauma. The clinical trial was a double blind study of 69 major surgery patients evaluating biochemical and clinical parameters over 7 day period in comparison with pre-operative status. <p>The novel oxistress assay proves to be a sensitive and accurate bedside diagnostic tool for oxidative stress. It can also be used in the laboratory setting. Oxidative stress is associated with increased trauma severity resulting in antioxidant depletion, strong oxidant production and protein degradation. The presence of pre-morbid medical factors also increased oxidative stress in trauma patients. Oral alanyl-glutamine supplementation (0.3 g/kg) increased plasma glutamine and antioxidant levels while decreasing urine oxidant levels. It significantly reduced hospital stay in non-cancer and higher disease complexity patients. The intervention also reduced the resource intensity weighting (RIW) score. <p>Oxidative stress is a clinical problem in surgery and trauma patients that can now be easily diagnosed at the bedside using the novel oxistress assay. Treatment with alanyl-glutamine is effective in reducing oxidative stress and improving clinical outcome. It is highly recommended probably at a higher dose in order to achieve optimal results.