Thesis (Ph.D.) -- University of Adelaide, Department of Agricultural Biochemistry, 1979.

xxi, 215 leaves : photos, graphs, tables ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Agricultural Biochemistry, 1979

Oral creatine supplementation has been shown to increase skeletal muscle total creatine (TCr) content, and in some cases improve performance in high-intensity short duration exercise. A variety of factors related to an enhanced efficacy of adenine nucleotide metabolism have been demonstrated as partly responsible for this ergogenic effect. Also, there is evidence that high-intensity sprint training results in a decrease in muscle total adenine nucleotide (TAN) and/or ATP stores. This placebo controlled double-blind study examined whether an oral creatine supplementation regimen would 1) increase muscle TCr content, 2) attenuate any loss in TAN or ATP during intermittent sprint training, and 3) have an ergogenic effect on performance after sprint training. Thirteen male endurance trained cyclists ingested 20 g of creatine monohydrate supplement or placebo per day for 7 days, after which they ingested a maintenance dose of 2 g creatine or placebo per day for the remainder of the trial (15d). While on the maintenance dose, subjects performed intermittent sprint training (ST) on a cycle ergometer (10 x 10 s sprints with 140 s active recovery) for 6 consecutive days and a 7th day after one day of rest. Performance tests were performed before and after ST, and metabolic tests were performed on the 1st and 7th day of ST. TCr increased significantly with creatine supplementation (creatine group pre: 121 ± 4, post: 147 ± 9; vs. placebo group pre: 122 ± 4, post: 125 ± 4 mmol/kg dm; mean± SEM; p<0.05). The increase in TCr correlated with the percentage Type IIB fibres (r=0.95, p<0.005). By day 7 of ST, TCr content was no longer significantly higher than pre-supplementation levels despite the maintenance dose of creatine. ST resulted in a significant decrease in resting muscle TAN and ATP content in both groups (ATP content in creatine group pre: 24.1 ± 0.8, post: 17.2 ± 0.5; and placebo group pre: 26.5 ± 1.1, post: 18.0 ± 0.6 mmol/kg dm; p<0.001). During and in recovery from ST on day 7, both groups had lower plasma ammonia (p<0.05), hypoxanthine (p<0.001) and urate (p<0.001) accumulation than on day 1 of ST. There was no improvement in 1-hr cycle distance performance after ST, but peak sustained power output increased in the creatine group and not in the placebo group after ST (p<0.05). Peak and mean power during a 30 s Wingate test increased significantly (p<0.05) after ST but there was no additional ergogenic effect of creatine supplementation. In conclusion, this study shows that 1) the efficacy of muscle creatine uptake was dependent on the percentage of Type IIB fibres, 2) creatine supplementation and maintenance (2 g/d) did not attenuate ATP or TAN loss during 7 days of ST, 3) ST decreased the accumulation of plasma products of adenine nucleotide degradation and improved 30 s sprint performance, and 4) creatine supplementation and ST did not improve I-hr cycle distance performance.

Heart failure (HP) is a common, disabling and deadly condition that causes high rates of morbidity and mortality worldwide. It is widely recognised that the failing heart is energy-starved, and that restoring energy homeostasis is a promising approach towards improving cardiac output. This thesis aims to address the role of energetics in the failing heart, by focussing on modulation of the creatine transporter (CrT). Creatine (Cr), together with the phosphocreatine shuttle, plays a vital role in maintaining energy supplies via ATP in times of high energy demand. Key to the regulation of intracellular [Cr] is the CrT, a Na+ and Cl - dependent membrane transporter. Previous CrT genetic mouse models include a knockout model, found to still express cardiac CrT, and a cardiac-specific CrT overexpressing (OE) model with large variations in myocardial [Cr] between animals and Cr levels high enough to cause spontaneous hypertrophy. To overcome the shortfalls of this CrT-OE model, a novel in vivo model of temporal inducible expression of CrT is described, using a cardiac-specific tetracycline inducible (Tet-On) system . ..,. .A' Ten transgenic lines (RCT) were created with a construct containing . zhe CrT-HA (CrT cDNA with an haemagglutinin epitope tag), following successful doxycyline-inducibility in vitro. Eight lines showed germline transmission, with LV CrT OE achieved in an individual mouse that displayed double LV [Cr] compared to WT. Issues with the inducer line (rtTA) were ruled out by its use in the creation of a luciferase overexpressing mouse line; all mice tested demonstrated LV luciferase expression in response to doxycycline feeding. The failure to overexpress CrT could be attributed to position or copy number dependent suppression, or to position effect variegation in the case of the single OE mouse obtained. Subsequent work focus sed on regulatory pathways in vitro in a cell line of mouse fibroblasts stably overexpressing CrT·HA. Post-translational modifications (PTMs) had been previously suggested to regulate CrT activity. Two N-linked glycosylation sites exist, in addition to the putative phosphorylation sites. Inhibition of glycosylation by tunicamycin led to decreased CrT activity, reflected by decreased Cr uptake capacity. Strategies to confirm the presence of phosphorylation were employed, including isolation of CrT -HA by immunoprecipitation and subsequent LC-MS / MS analysis to identify PTMs. Although the presence of CrT was confirmed in 5 different sized species- one previously unreported- inadequate sequence coverage prevented identification of any PTM sites. Tyrosine phosphorylation was not detected using a phosphospecific antibody on immunopurified CrT -HA. Candidate signalling pathways in vitro were then investigated to elucidate CrT regulation, namely the IGF-IR signalling pathway. This study included a cardiomyocyte-like mouse cell line (HL-l) in addition to 3T3-CrT -HA. Exposure of cells to extracellular insulin, growth hormone and IGF-1 led to increased Cr uptake of 125% - 300% of normal. Pharmacological inhibition of the downstream kinases PKA and PKC reduced the effect of insulin and GH, while PMA, sapintoxin (STX) and Go 6976 induced CrT activity. The mammalian target of rapamycin (mTOR) is also a candidate regulator of CrT, as incubation with rapamycin decreased Cr uptake in 3T3-CrT -HA. Finally, a targeted approach on transcription factors in the 5'UTR region of mouse CrT identified HEYl as a highly conserved site. In siRNA experiments, HEYl was found to exert a mild effect on CrT activity, suggesting that regulation at the transcriptional level merits further investigation. Together, this work has provided novel insights into the modulation of CrT in vitro, identifying molecular and pharmacological targets in a known therapeutic signalling pathway. Further work could potentially develop these findings by identifying candidate compounds that would increase CrT activity, potentially in a tissue-specific manner. 3

A lesão de isquemia e reperfusão (IR) é um evento que pode elevar o risco de morte após o transplante pulmonar, por ativar o sistema imune inato a induzir a inflamação. Em situação de isquemia, a oferta de oxigênio se encontra abaixo das necessidades metabólicas, resultando na depleção das reservas celulares de ATP e no aumento da produção de espécies reativas de oxigênio (EROs) e nitrogênio (ERNs). Adicionalmente, a IR desencadeia um processo inflamatório intenso, caracterizado principalmente pela presença de neutrófilos e macrófagos ativados, os quais liberam inúmeros mediadores inflamatórios, perpetuando a inflamação. Nossa hipótese inicial era que a suplementação com creatina (Cr) poderia atenuar a lesão de IR pelo aumento dos níveis de fosfocreatina (PCr) nas células, o que facilitaria a formação de adenosina trifosfato (ATP), promovendo a manutenção dos níveis de Ca2+ intracelular, desestimulando assim a formação de EROs e, consequentemente, diminuindo o processo inflamatório. Portanto, o objetivo do presente estudo foi avaliar o papel da suplementação com creatina na atenuação da lesão de IR em ratos submetidos ao transplante pulmonar, segundo aspectos inflamatórios, estruturais e funcionais do tecido pulmonar. Foram utilizados 64 ratos machos da raça Sprague Dawley distribuídos em quatro grupos: A90, controle/água + 90 minutos de isquemia; Cr90, creatina + 90 minutos de isquemia; A180, controle/água + 180 minutos de isquemia; Cr180, creatina + 180 minutos de isquemia. Os animais doadores receberam creatina (0,5g/kg/dia) diariamente durante cinco dias antes do transplante pulmonar. Os animais do grupo controle receberam apenas o veículo. Após a extração, os pulmões permaneceram em isquemia fria por 90 ou 180 minutos sendo, a seguir, implantados e reperfundidos por 120 minutos. Ao final da reperfusão, foram coletados os dados de mecânica respiratória, além de amostras de ar exalado, sangue arterial e periférico, lavado broncoalveolar e tecido pulmonar. Os parâmetros avaliados foram: resistência das vias aéreas, resistência e elastância do tecido pulmonar, óxido nítrico exalado, pressão parcial de oxigênio e de dióxido de carbono, creatinina sérica, células inflamatórias, índice de edema, PCNA, Caspase-3, TLR 4 e 7, IL1-beta, IL6, TNF-alfa, IL10 e CINC1. Os animais tratados com creatina apresentaram melhora da mecânica pulmonar, dos níveis de creatinina sérica, da gasometria arterial, além da diminuição da fração exalada de óxido nítrico e da inflamação verificada no sangue periférico, no lavado broncoalveolar e no parênquima pulmonar. Estes animais também apresentaram diminuição da proliferação e da apoptose de células inflamatórias, de TLR4, dos níveis de IL6 e CINC1, além de aumento de IL10. Concluímos que o prétratamento com creatina tem efeito protetor na lesão de IR após transplante pulmonar unilateral em ratos / Ischemia and reperfusion injury (IRI) is an event that can increase the risk of death after lung transplantation (LTx) by activating the innate immune system to induce inflammation. In ischemia events, oxygen supply is below metabolic requirements, resulting in depletion of ATP cellular reserves and increased production of reactive oxygen (ROS) and nitrogen species (RNS). In addition, IRI triggers an intense inflammatory process characterized mainly by the presence of activated neutrophils and macrophages, which release innumerable inflammatory mediators, perpetuating the inflammation. Our initial hypothesis was that creatine supplementation (Cr) could attenuate IRI by increasing phosphocreatine (PCr) levels in cells, which would facilitate the formation of adenosine triphosphate (ATP), promoting the maintenance of intracellular Ca2+ levels, thus discouraging the formation of ROS and, consequently, decreasing the inflammatory process. Therefore, the objective of this study was to evaluate the role of Cr supplementation in the attenuation of IRI in rats underwent to LTx in according to inflammatory, structural and functional aspects of the lung tissue. Sixty Sprague Dawley male rats were distributed into four groups: A90, control / water + 90 minutes of ischemia; Cr90, creatine + 90 minutes of ischemia; A180, control / water + 180 minutes of ischemia; Cr180, creatine + 180 minutes of ischemia. Donor animals received creatine (0.5g/kg/day) daily for five days prior to LTx. Animals in the control group received only the vehicle. The donor`s lung remained in cold ischemia for 90 or 180 minutes and then, were implanted and reperfused during 120 minutes. After reperfusion, respiratory mechanics data were performed and collected samples of exhaled air, arterial and peripheral blood, bronchoalveolar lavage fluid and pulmonary tissue. The parameters evaluated were: airway resistance, resistance and elastance of the pulmonary tissue, exhaled nitric oxide, partial pressure of oxygen and carbon dioxide, serum creatinine, inflammatory cells, edema index, PCNA, Caspase-3, TLR 4 and 7, IL1-beta, IL6, TNF-alpha, IL10, and CINC1. The animals treated with Cr showed an improvement in pulmonary mechanics, serum creatinine levels, and arterial blood gases. In addition, there was a decrease in the exhaled fraction of nitric oxide and in the inflammation in the peripheral blood, BALF, and pulmonary parenchyma in creatine-treated animals. These rats also had a decrease in the proliferation and apoptosis of inflammatory cells, TLR4, IL6, and CINC1. Moreover, there was an increase in the IL10 levels after Cr treatment. We conclude that pre-treatment with Cr has a protective effect on IRI after LTx in rats

A lesão de isquemia e reperfusão (IR) é um evento que pode elevar o risco de morte após o transplante pulmonar, por ativar o sistema imune inato a induzir a inflamação. Em situação de isquemia, a oferta de oxigênio se encontra abaixo das necessidades metabólicas, resultando na depleção das reservas celulares de ATP e no aumento da produção de espécies reativas de oxigênio (EROs) e nitrogênio (ERNs). Adicionalmente, a IR desencadeia um processo inflamatório intenso, caracterizado principalmente pela presença de neutrófilos e macrófagos ativados, os quais liberam inúmeros mediadores inflamatórios, perpetuando a inflamação. Nossa hipótese inicial era que a suplementação com creatina (Cr) poderia atenuar a lesão de IR pelo aumento dos níveis de fosfocreatina (PCr) nas células, o que facilitaria a formação de adenosina trifosfato (ATP), promovendo a manutenção dos níveis de Ca2+ intracelular, desestimulando assim a formação de EROs e, consequentemente, diminuindo o processo inflamatório. Portanto, o objetivo do presente estudo foi avaliar o papel da suplementação com creatina na atenuação da lesão de IR em ratos submetidos ao transplante pulmonar, segundo aspectos inflamatórios, estruturais e funcionais do tecido pulmonar. Foram utilizados 64 ratos machos da raça Sprague Dawley distribuídos em quatro grupos: A90, controle/água + 90 minutos de isquemia; Cr90, creatina + 90 minutos de isquemia; A180, controle/água + 180 minutos de isquemia; Cr180, creatina + 180 minutos de isquemia. Os animais doadores receberam creatina (0,5g/kg/dia) diariamente durante cinco dias antes do transplante pulmonar. Os animais do grupo controle receberam apenas o veículo. Após a extração, os pulmões permaneceram em isquemia fria por 90 ou 180 minutos sendo, a seguir, implantados e reperfundidos por 120 minutos. Ao final da reperfusão, foram coletados os dados de mecânica respiratória, além de amostras de ar exalado, sangue arterial e periférico, lavado broncoalveolar e tecido pulmonar. Os parâmetros avaliados foram: resistência das vias aéreas, resistência e elastância do tecido pulmonar, óxido nítrico exalado, pressão parcial de oxigênio e de dióxido de carbono, creatinina sérica, células inflamatórias, índice de edema, PCNA, Caspase-3, TLR 4 e 7, IL1-beta, IL6, TNF-alfa, IL10 e CINC1. Os animais tratados com creatina apresentaram melhora da mecânica pulmonar, dos níveis de creatinina sérica, da gasometria arterial, além da diminuição da fração exalada de óxido nítrico e da inflamação verificada no sangue periférico, no lavado broncoalveolar e no parênquima pulmonar. Estes animais também apresentaram diminuição da proliferação e da apoptose de células inflamatórias, de TLR4, dos níveis de IL6 e CINC1, além de aumento de IL10. Concluímos que o prétratamento com creatina tem efeito protetor na lesão de IR após transplante pulmonar unilateral em ratos / Ischemia and reperfusion injury (IRI) is an event that can increase the risk of death after lung transplantation (LTx) by activating the innate immune system to induce inflammation. In ischemia events, oxygen supply is below metabolic requirements, resulting in depletion of ATP cellular reserves and increased production of reactive oxygen (ROS) and nitrogen species (RNS). In addition, IRI triggers an intense inflammatory process characterized mainly by the presence of activated neutrophils and macrophages, which release innumerable inflammatory mediators, perpetuating the inflammation. Our initial hypothesis was that creatine supplementation (Cr) could attenuate IRI by increasing phosphocreatine (PCr) levels in cells, which would facilitate the formation of adenosine triphosphate (ATP), promoting the maintenance of intracellular Ca2+ levels, thus discouraging the formation of ROS and, consequently, decreasing the inflammatory process. Therefore, the objective of this study was to evaluate the role of Cr supplementation in the attenuation of IRI in rats underwent to LTx in according to inflammatory, structural and functional aspects of the lung tissue. Sixty Sprague Dawley male rats were distributed into four groups: A90, control / water + 90 minutes of ischemia; Cr90, creatine + 90 minutes of ischemia; A180, control / water + 180 minutes of ischemia; Cr180, creatine + 180 minutes of ischemia. Donor animals received creatine (0.5g/kg/day) daily for five days prior to LTx. Animals in the control group received only the vehicle. The donor`s lung remained in cold ischemia for 90 or 180 minutes and then, were implanted and reperfused during 120 minutes. After reperfusion, respiratory mechanics data were performed and collected samples of exhaled air, arterial and peripheral blood, bronchoalveolar lavage fluid and pulmonary tissue. The parameters evaluated were: airway resistance, resistance and elastance of the pulmonary tissue, exhaled nitric oxide, partial pressure of oxygen and carbon dioxide, serum creatinine, inflammatory cells, edema index, PCNA, Caspase-3, TLR 4 and 7, IL1-beta, IL6, TNF-alpha, IL10, and CINC1. The animals treated with Cr showed an improvement in pulmonary mechanics, serum creatinine levels, and arterial blood gases. In addition, there was a decrease in the exhaled fraction of nitric oxide and in the inflammation in the peripheral blood, BALF, and pulmonary parenchyma in creatine-treated animals. These rats also had a decrease in the proliferation and apoptosis of inflammatory cells, TLR4, IL6, and CINC1. Moreover, there was an increase in the IL10 levels after Cr treatment. We conclude that pre-treatment with Cr has a protective effect on IRI after LTx in rats