This study combined concentration-discharge analyses (filtration at 0.45 m), cascade filtrations (at 1.2, 0.4, and 0.025 m) and asymmetrical flow field flow fractionation (AF4) to probe the influence of colloidal carriers (dissolved organic matter and inorganic nanoparticles) on observed concentration-discharge relationships for trace metals in a 155 ha forested catchment of the Santa Catalina Mountains Critical Zone Observatory (SCM CZO), Arizona. Many major elements (Na, Mg, Si, K, Ca) show no colloidal influence, and concentration-discharge relationships for these species are explained by previous work. However, the majority of trace metals (Al, Ti, V, Mn, Fe, Cu, Y, REE, U) show at least some influence of colloids on chemistry when filtered at the standard 0.45 m cutoff. Concentration-discharge slopes of trace metals with modest colloidal influence are shallow (approximate to 0.3) similar to that measured for dissolved organic carbon (DOC, 0.24), whereas elements with greater colloidal influence have steeper concentration-discharge slopes approaching that of Al (0.76), the element with the largest colloidal influence in this study (on average 68%). These findings are further supported by AF4 measurements that show distinct and resolvable pools of low hydrodynamic diameter DOC-sized material coexistent with larger diameter inorganic colloids, and the ratio of these carriers changes systematically with discharge because the DOC pool has a concentration-discharge relationship with shallower slope than the inorganic colloidal pool. Together these data sets illustrate that positive concentration-discharge slopes of trace metals in stream waters may be explained as the relative partitioning of trace metals between DOC and inorganic colloids, with contributions of the latter likely increasing as a result of increased prevalence of macropore flow.

Despite the importance of coalbed methane (CBM) as a natural gas resource, little is known about the microbial communities responsible for production of biogenic CBM (~20% of all CBM gas). It is thought that coal-associated microbial communities are limited by nutrients, such as nitrogen or phosphate and a of suite trace elements, but it's not clear whether these nutrients are sourced from in-situ biodegradation of the coal or transported in with groundwater recharge. To address this knowledge gap, we examined the nitrogen and phosphorous species and trace metal geochemistry of the solid coal and overlying siliciclastic sediment and associated groundwater from monitoring wells across the Powder River Basin (PRB). Four Groups were identified that represent the geochemical evolution of groundwater from shallow unconsolidated siliciclastic aquifers, to recharge-associated coal waters, to sulfate-reducing coal waters, and finally, methanogenic coal waters, along a hydrologic gradient within single coal seams. The highest nutrient concentrations were found in the first two water types, and the lowest in the last two, suggesting that essential nutrients are mobilized at the surface and transported downgradient into the coal and overlying unconsolidated siliciclastic sediment. However, by the time groundwater reaches sulfate-reducing or methanogenic conditions, the nutrients are either utilized in reactions or precipitated under changing redox conditions. Sequential chemical extraction experiments of coal and siliciclastic core materials revealed that all essential nutrients for microbial methanogenesis are present and easily leachable into groundwater. Therefore, under anoxic conditions, microbial communities are unlikely to be limited by the presence of essential elements; rather, they are likely limited by the slow rates of coal biodegradation and liberation of essential nutrients.

A contaminação dos ecossistemas aquáticos por metais-traço demanda preocupação já que estes contaminantes podem exercer efeitos tóxicos sobre a biota e aumentar os custos para tratamento da água. Em geral, tais problemas de degradação são decorrentes de uma gestão de recursos hídricos deficiente. Tendo como área de estudo a represa Paiva Casto, reservatório integrante do Sistema Cantareira, maior sistema de abastecimento público da região metropolitana de São Paulo, este trabalho teve o intuito de: investigar o histórico da contaminação por metais-traço (Cd, Cr, Cu, Ni, Pb, Zn, Mn, Al) por meio da geocronologia por 210Pb e indicar um valor de referência para estes; identificar a heterogeneidade espacial destes contaminantes; analisar a biodisponibilidade e com base na possibilidade de adequar etapas do sistema de gestão de recursos hídricos europeu, a Diretiva Quadro da Água (DQA) discutir a qualidade química dos sedimentos da represa em estudo. Foram efetuadas duas coletas em campo, destinadas à avaliação do histórico da contaminação ambiental por metais-traço e à análise da biodisponibilidade por meio da técnica de sulfetos volatilizáveis por acidificação (SVA) e metais simultaneamente extraídos (MES). O histórico da contaminação por metais-traço abrangeu um período de 100 anos e indicou que os maiores impactos na região, em relação a estes contaminantes, ocorreu em período anterior ao início de operação da represa Paiva Castro, com exceção do Cu. Para o Cu, em sedimentos superficiais, os teores excederam até três vezes e meia o VR. Este resultado é provável consequência da aplicação de sulfato de cobre para o controle de florações de algas. Os dados para os VR divergiram daqueles estabelecidos para a crosta terrestre, porém foram semelhantes aos encontrados para a bacia do Alto Tietê, com exceção dos metais Cr e Pb. A análise de (SVA/MES) indicou que os metais não estavam biodisponíveis. Dentre as áreas amostradas, a região da barragem apresentou ao longo do tempo as maiores taxas de acumulação para metais-traço em contraposição à área de captação das águas para abastecimento público. Em relação a DQA, constatou-se que existe capacidade científica para avançar na incorporação de novos instrumentos na gestão, principalmente no Estado de São Paulo, entretanto, é preciso que haja sistemas de saneamento e monitoramento adequados e especialmente decisão política e rigor na aplicação destes novos instrumentos, caso contrário a incorporação da DQA é inviável. Aplicando algumas das normas da DQA na avaliação da qualidade química dos sedimentos da represa Paiva Castro constatou-se que a qualidade geral para este manancial é boa. Apesar disto é necessário que as políticas públicas sejam de fato aplicadas contra a degradação deste importante manancial, caso contrário a boa qualidade deste reservatório estará em risco / Contamination of aquatic ecosystems by trace metals demand concern since these contaminants may cause toxic effects on biota and increase costs for water treatment. In general, such degradation problems are due to a poor water management. A study was conducted at Paiva Castro reservoir, one of the reservoirs of the Cantareira System, largest public water supply system in the metropolitan region of São Paulo, this study aimed to: investigate the history of the accumulation of trace-metals (Cd, Cr, Cu, Ni, Pb, Zn, Mn, Al), through geochronology by 210Pb; establish background values (BV); analyze spatial heterogeneity; bioavailability and based on the possibility of adjusting some instruments of the European water resources management system, the Water Framework Directive (WFD), discuss the chemical quality of Paiva Castro\'s sediments. Two collections were performed, to assess the historical environmental contamination by trace-metals and to analyze the bioavailability using the technique of sulfides volatilizable by acidification (AVS) and simultaneously extracted metals (SEM). The history of contamination by trace- metals covered a period of 100 years and indicated that the greatest impacts in the region occurred in periods prior to the reservoir operation, with the exception of Cu. For Cu, in surface sediments, the levels exceeded three times and a half the BV. This result is probable consequence of copper sulphate application for controlling algal blooms. Data for BV were different from those found in the earth\'s crust, but similar to those found for the Alto Tietê basin, except for Cr and Pb metals. Analysis (AVS/ SEM) indicated that metals were not bioavailable. Among the sampled areas, the region of the dam, showed the highest rates of accumulation for trace- metals in contrast to the catchment area for public water supply. Regarding WFD, it was observed that there is scientific capacity to incorporate new instruments in the management, mainly in São Paulo, however, there must be adequate sanitation and monitoring systems and especially political decision and rigor in the application of these new tools, otherwise the incorporation of the WFD is unviable. Applying some of the WFD instruments in assessing the chemical quality of Paiva Castro\'s sediments, it was found that the quality of this ecosystem is good. Nevertheless, it is necessary that public policies are actually applied against the degradation of this important ecosystem; otherwise, reservoir\'s \'good quality\' will be at risk

The atmospheric transport of various substances from the continents to the oceans plays an important role in biogeochemical processes. Trace metals, iron in particular is of great interest as its availability regulates the growth of phytoplankton over large areas of the ocean. This dissertation focuses on examining and characterizing the factors that affect the solubility of trace metals in Miami and Barbados aerosols and precipitation, in particular species that could play a role in surface seawater biogeochemistry (Fe and trace metals such as Al, V, Cr, Mn, Cu, Co, Ni, Zn, As, Tl, Ba, Cd, Pb, Th, Ti, Zr, and REE's). To enable this study existing methods of colorimetric spectroscopic and inductively coupled plasma mass spectrometry analysis were improved and modified. This dissertation examines several issues related to source inputs: 1.) Are single spot sources within the North African dust source distinguishable after long transport by their bulk metal composition and thus important in the characteristics of individual mineral dust samples? 2.) What is the temporal variability and controlling factors in trace metal solubility in trade wind aerosols collected over Barbados? 3.) Which factors control the observed trend of speciation and increasing iron solubility in decreasing aerosol loading? Additionally a kinetic model of species specific iron (II) to iron(III) oxidation kinetics in NaCl Brines was conducted at nano-molar levels. This study greatly expands the ability to predict rates of iron oxidation at concentrations closer to those observed in natural systems.

Water samples were collected along salinity gradients during different seasons from three estuaries (Tseng-Wen, Gao-Ping, Er-Ren) and coasts in/off southwestern Taiwan. In order to assess the partitioning of trace metals between solution and particle, the concentrations of dissolve and particulate trace metals (Cd, Cu, Ni, Pb) along with their chemical affinity fractions, were determined. This study investigated the variations in distribution and partitioning of the different metals in estuarine waters, and examined the effects of oxides and particulate organic matter on the partitioning of trace metals in waters from different estuaries. Fractionation of dissolved trace metal species was based on ion exchange (Chelex-100 and AG MP-1 resins) separation techniques. The fractions obtained were operationally defined as labile (Chelex), organic (AG MP-1) and inert. Particles were extracted to three phases (surface adsorbed phases, Fe¡VMn oxide/organic phases and refractory phases) using sequential extraction techniques. Seasonally variable distributions of dissolved trace metals were found in the Tseng-Wen estuary. The behavior of trace metals was mainly influenced by anthropogenic input during the dry season in the upper Tseng-Wen estuary, while mixing processes controlled the distribution of trace metals during the wet season. The dilution effect was a major factor in the metal distribution in the Gao-Ping estuary due to high river discharge. The higher concentration of metals in the Er-Ren estuary, in contrast to other estuaries indicated that the Er-Ren estuary has serious pollution concerns. According to the results of particulate metal fractions obtained, Cd and Pb existed predominantly in the surface adsorbed phase. The speciation and spatial distribution of Cd were similar to those of Mn, indicating that the formation of authigenic Mn oxides affected the distribution of Cd in estuaries. The percentage of oxide /organic phase for Cu accounted for 25% of total particulate Cu, but dose not correlate well with particulate organic carbon, implying that organic carbon is not the only factor controlling particulate Cu distribution. Ni was present mainly in lattice phase, except in the Er-Ren estuary where anthropogenic Ni loading was high. In the Tseng-Wen and Gao-Ping estuaries, the percentages of lattice phase of all metals determined in this study during the wet season were higher than those during the dry season. These seasonal variations are probably resulted from different flushing times in dry/wet seasons, which control the extent of geochemical processes for trace elements.

Diabetic cardiomyopathy is one of the causes of mortality and morbidity associated with diabetes. Diabetes is a disorder characterised by chronic hyperglycaemia and cardiovascular complications. The relationship between these integrally linked conditions has long been recognised, and for a significant portion of individuals the two conditions co-exist as part of metabolic syndrome. The presence of diabetes increases the risk of heart failure up to fivefold and three-fold in women and men, respectively, when compared to individuals without diabetes. While there has been a significant declining trend in cardiovascular mortality and morbidity in the general population over the past two decades, unfortunately such trends have not been seen among diabetic patients. As a result, this has persuaded many health professionals to re-evaluate their current treatment and pharmacological regimens. It is a well established fact that oxidative stress is a contributory mechanism in many agerelated disorders including T2DM, especially in those with poor glycaemic control. Thus far, clinical trials with antioxidant or carbonyl-trapping agents have produced mixed results, suggesting that the mechanisms underlying this disorder may be more complex than previously thought. Although altered systemic regulation of trace metals in diabetes has been previously investigated, it is still unclear whether changed trace metal metabolism would cause heart disease in common forms of diabetes and whether metal chelation can reverse this condition. Our hypothesis is that the accumulation of redox-active trace metals including Cu and Fe in cardiac muscle may, at least in part, result in cardiomyopathy through the generation of excess reactive oxygen species. We believed that the administration of a specific metal chelator should ameliorate this process by increasing the excretion of free systemic Cu and Fe, consequently limiting the production of superoxide oxygen free radicals and arresting the process of diabetic cardiomyopathy. Data from pre-clinical studies conducted in our laboratory using diabetic animal model with diabetes-induced abnormal Cu metabolism have been remarkably consistent in demonstrating that oral dosing with triethylenetetramine (TETA) can effectively remove systemic Cu via increased urinary Cu excretion, improve cardiomyocyte structure, reverse elevations in left ventricular collagen and β1-integrin, and alleviate heart failure, all in the presence of a consistently high circulating blood glucose profile. Taken together, these findings support the beneficial role of TETA in diabetic animal model and lay the foundation for its potential therapeutic effect in humans with diabetes. This thesis describes a series of randomised, placebo-controlled clinical trials that have investigated the metabolism of Cu and Fe and seven other trace metals in patients with chronic T2DM compared with non-diabetic control subjects. This thesis also examines the mechanism of action of TETA and addresses the hypothesis that a decrease in body systemic Cu pool through chelation therapy may improve cardiac complication in diabetic subjects. Trial 1 is a randomised, double-blind, placebo- and diet-controlled study which measured the 6d balance of Cu and Fe and seven other essential trace metals, in twenty male T2DM and twenty age-matched control subjects in whom we later probed systemic metal balance with oral TETA. Basal urinary output and balance of Cu and Fe was significantly elevated in diabetes, and the two output values correlated strongly (p<0.05). 6d treatment with 2400mg/d dose of TETA (maximum Wilson’s disease dose) has increased the urinary excretion of Cu, which was predicted by basal urinary Cu excretion, thereby causing a positive Cu balance to become negative in diabetes. Regulation of Cu metabolism was shown to be abnormal in diabetes and was selectively modified by TETA, which did not concomitantly modify Fe metabolism. Moreover, TETA did not cause a negative balance in any of the other seven trace metals monitored. These findings are consistent with TETA reversing the accumulation of free systemic Cu in diabetes, which may help to explain its potential therapeutic effects in some diabetic complications. Trial 2 investigated the acute response effect of a single 2400mg dose of TETA on urinary and serum trace metals in the first 10hr and 10~24hr post-dose. The results showed that TETA markedly increased the urinary Cu and Zn excretion in diabetes for the duration of 10hrs with the maximum excretion phase between 4~6hr post-drug (p<0.05). TETA did not change the metabolism of Mg and six other essential trace metals monitored. Trial 3 examined the dose-response effect of TETA, at and below the dose given to patients with the Wilson’s disease over a 7d period, on Cu and eight other trace metals in a subgroup of seven T2DM and seven control subjects who had completed trial 1. The results of this i i trial showed that there was a linear dose-response relationship over the dose range 300~2400 mg/d on urinary Cu excretion in both T2DM and control subjects. However, there was no significant difference between the two subject groups at any of the four doses tested. In addition, 300mg/d of TETA was effective in mobilizing Cu in both T2DM and healthy control subjects. Trial 4 described the full work-up of a sensitive LC-MS methodology to identify and quantify TETA and its metabolite(s) in human urine. Using the LC-MS, TETA metabolism and excretion was investigated by analysing the urine of seven T2DM and seven control subjects who received escalating doses of TETA (samples obtained from trial 3). I have successfully identified and characterised two major metabolites of TETA in the urine of both T2DM and control subjects, N1-monoacetytriethylenetetramine (MAT) and diacetytriethylenetetramine (DAT), the latter which has not been previously reported. The results from urinary TETA excretion analyses also showed that T2DM may metabolise TETA more extensively than control subjects, which in turn is associated with its higher uptake or bioavailability. Urinary Zn excretion was mainly linked with urinary TETA and MAT in T2DM and healthy controls, respectively, whereas urinary Cu excretion was associated with urinary TETA excretion in healthy controls and urinary TETA+MAT excretion in T2DM subjects. These results suggest that MAT may also be involved in the mechanism by which TETA extracts systemic free Cu in diabetes. The identification of the two major metabolites of TETA and the development of a robust analytical LC-MS methodology reported in this study is an important step to further investigate the pharmacological actions of TETA in diabetic individuals. Collectively, the results presented in this thesis and in association with previous animal and clinical studies from our laboratory have provided consistent supporting evidences for the use of TETA clinically as a safe and effective therapy to prevent the genesis of some diabetic complications, in conjunction with conventional complication modifying therapies.

Diabetic cardiomyopathy is one of the causes of mortality and morbidity associated with diabetes. Diabetes is a disorder characterised by chronic hyperglycaemia and cardiovascular complications. The relationship between these integrally linked conditions has long been recognised, and for a significant portion of individuals the two conditions co-exist as part of metabolic syndrome. The presence of diabetes increases the risk of heart failure up to fivefold and three-fold in women and men, respectively, when compared to individuals without diabetes. While there has been a significant declining trend in cardiovascular mortality and morbidity in the general population over the past two decades, unfortunately such trends have not been seen among diabetic patients. As a result, this has persuaded many health professionals to re-evaluate their current treatment and pharmacological regimens. It is a well established fact that oxidative stress is a contributory mechanism in many agerelated disorders including T2DM, especially in those with poor glycaemic control. Thus far, clinical trials with antioxidant or carbonyl-trapping agents have produced mixed results, suggesting that the mechanisms underlying this disorder may be more complex than previously thought. Although altered systemic regulation of trace metals in diabetes has been previously investigated, it is still unclear whether changed trace metal metabolism would cause heart disease in common forms of diabetes and whether metal chelation can reverse this condition. Our hypothesis is that the accumulation of redox-active trace metals including Cu and Fe in cardiac muscle may, at least in part, result in cardiomyopathy through the generation of excess reactive oxygen species. We believed that the administration of a specific metal chelator should ameliorate this process by increasing the excretion of free systemic Cu and Fe, consequently limiting the production of superoxide oxygen free radicals and arresting the process of diabetic cardiomyopathy. Data from pre-clinical studies conducted in our laboratory using diabetic animal model with diabetes-induced abnormal Cu metabolism have been remarkably consistent in demonstrating that oral dosing with triethylenetetramine (TETA) can effectively remove systemic Cu via increased urinary Cu excretion, improve cardiomyocyte structure, reverse elevations in left ventricular collagen and β1-integrin, and alleviate heart failure, all in the presence of a consistently high circulating blood glucose profile. Taken together, these findings support the beneficial role of TETA in diabetic animal model and lay the foundation for its potential therapeutic effect in humans with diabetes. This thesis describes a series of randomised, placebo-controlled clinical trials that have investigated the metabolism of Cu and Fe and seven other trace metals in patients with chronic T2DM compared with non-diabetic control subjects. This thesis also examines the mechanism of action of TETA and addresses the hypothesis that a decrease in body systemic Cu pool through chelation therapy may improve cardiac complication in diabetic subjects. Trial 1 is a randomised, double-blind, placebo- and diet-controlled study which measured the 6d balance of Cu and Fe and seven other essential trace metals, in twenty male T2DM and twenty age-matched control subjects in whom we later probed systemic metal balance with oral TETA. Basal urinary output and balance of Cu and Fe was significantly elevated in diabetes, and the two output values correlated strongly (p<0.05). 6d treatment with 2400mg/d dose of TETA (maximum Wilson’s disease dose) has increased the urinary excretion of Cu, which was predicted by basal urinary Cu excretion, thereby causing a positive Cu balance to become negative in diabetes. Regulation of Cu metabolism was shown to be abnormal in diabetes and was selectively modified by TETA, which did not concomitantly modify Fe metabolism. Moreover, TETA did not cause a negative balance in any of the other seven trace metals monitored. These findings are consistent with TETA reversing the accumulation of free systemic Cu in diabetes, which may help to explain its potential therapeutic effects in some diabetic complications. Trial 2 investigated the acute response effect of a single 2400mg dose of TETA on urinary and serum trace metals in the first 10hr and 10~24hr post-dose. The results showed that TETA markedly increased the urinary Cu and Zn excretion in diabetes for the duration of 10hrs with the maximum excretion phase between 4~6hr post-drug (p<0.05). TETA did not change the metabolism of Mg and six other essential trace metals monitored. Trial 3 examined the dose-response effect of TETA, at and below the dose given to patients with the Wilson’s disease over a 7d period, on Cu and eight other trace metals in a subgroup of seven T2DM and seven control subjects who had completed trial 1. The results of this i i trial showed that there was a linear dose-response relationship over the dose range 300~2400 mg/d on urinary Cu excretion in both T2DM and control subjects. However, there was no significant difference between the two subject groups at any of the four doses tested. In addition, 300mg/d of TETA was effective in mobilizing Cu in both T2DM and healthy control subjects. Trial 4 described the full work-up of a sensitive LC-MS methodology to identify and quantify TETA and its metabolite(s) in human urine. Using the LC-MS, TETA metabolism and excretion was investigated by analysing the urine of seven T2DM and seven control subjects who received escalating doses of TETA (samples obtained from trial 3). I have successfully identified and characterised two major metabolites of TETA in the urine of both T2DM and control subjects, N1-monoacetytriethylenetetramine (MAT) and diacetytriethylenetetramine (DAT), the latter which has not been previously reported. The results from urinary TETA excretion analyses also showed that T2DM may metabolise TETA more extensively than control subjects, which in turn is associated with its higher uptake or bioavailability. Urinary Zn excretion was mainly linked with urinary TETA and MAT in T2DM and healthy controls, respectively, whereas urinary Cu excretion was associated with urinary TETA excretion in healthy controls and urinary TETA+MAT excretion in T2DM subjects. These results suggest that MAT may also be involved in the mechanism by which TETA extracts systemic free Cu in diabetes. The identification of the two major metabolites of TETA and the development of a robust analytical LC-MS methodology reported in this study is an important step to further investigate the pharmacological actions of TETA in diabetic individuals. Collectively, the results presented in this thesis and in association with previous animal and clinical studies from our laboratory have provided consistent supporting evidences for the use of TETA clinically as a safe and effective therapy to prevent the genesis of some diabetic complications, in conjunction with conventional complication modifying therapies.

Diabetic cardiomyopathy is one of the causes of mortality and morbidity associated with diabetes. Diabetes is a disorder characterised by chronic hyperglycaemia and cardiovascular complications. The relationship between these integrally linked conditions has long been recognised, and for a significant portion of individuals the two conditions co-exist as part of metabolic syndrome. The presence of diabetes increases the risk of heart failure up to fivefold and three-fold in women and men, respectively, when compared to individuals without diabetes. While there has been a significant declining trend in cardiovascular mortality and morbidity in the general population over the past two decades, unfortunately such trends have not been seen among diabetic patients. As a result, this has persuaded many health professionals to re-evaluate their current treatment and pharmacological regimens. It is a well established fact that oxidative stress is a contributory mechanism in many agerelated disorders including T2DM, especially in those with poor glycaemic control. Thus far, clinical trials with antioxidant or carbonyl-trapping agents have produced mixed results, suggesting that the mechanisms underlying this disorder may be more complex than previously thought. Although altered systemic regulation of trace metals in diabetes has been previously investigated, it is still unclear whether changed trace metal metabolism would cause heart disease in common forms of diabetes and whether metal chelation can reverse this condition. Our hypothesis is that the accumulation of redox-active trace metals including Cu and Fe in cardiac muscle may, at least in part, result in cardiomyopathy through the generation of excess reactive oxygen species. We believed that the administration of a specific metal chelator should ameliorate this process by increasing the excretion of free systemic Cu and Fe, consequently limiting the production of superoxide oxygen free radicals and arresting the process of diabetic cardiomyopathy. Data from pre-clinical studies conducted in our laboratory using diabetic animal model with diabetes-induced abnormal Cu metabolism have been remarkably consistent in demonstrating that oral dosing with triethylenetetramine (TETA) can effectively remove systemic Cu via increased urinary Cu excretion, improve cardiomyocyte structure, reverse elevations in left ventricular collagen and β1-integrin, and alleviate heart failure, all in the presence of a consistently high circulating blood glucose profile. Taken together, these findings support the beneficial role of TETA in diabetic animal model and lay the foundation for its potential therapeutic effect in humans with diabetes. This thesis describes a series of randomised, placebo-controlled clinical trials that have investigated the metabolism of Cu and Fe and seven other trace metals in patients with chronic T2DM compared with non-diabetic control subjects. This thesis also examines the mechanism of action of TETA and addresses the hypothesis that a decrease in body systemic Cu pool through chelation therapy may improve cardiac complication in diabetic subjects. Trial 1 is a randomised, double-blind, placebo- and diet-controlled study which measured the 6d balance of Cu and Fe and seven other essential trace metals, in twenty male T2DM and twenty age-matched control subjects in whom we later probed systemic metal balance with oral TETA. Basal urinary output and balance of Cu and Fe was significantly elevated in diabetes, and the two output values correlated strongly (p<0.05). 6d treatment with 2400mg/d dose of TETA (maximum Wilson’s disease dose) has increased the urinary excretion of Cu, which was predicted by basal urinary Cu excretion, thereby causing a positive Cu balance to become negative in diabetes. Regulation of Cu metabolism was shown to be abnormal in diabetes and was selectively modified by TETA, which did not concomitantly modify Fe metabolism. Moreover, TETA did not cause a negative balance in any of the other seven trace metals monitored. These findings are consistent with TETA reversing the accumulation of free systemic Cu in diabetes, which may help to explain its potential therapeutic effects in some diabetic complications. Trial 2 investigated the acute response effect of a single 2400mg dose of TETA on urinary and serum trace metals in the first 10hr and 10~24hr post-dose. The results showed that TETA markedly increased the urinary Cu and Zn excretion in diabetes for the duration of 10hrs with the maximum excretion phase between 4~6hr post-drug (p<0.05). TETA did not change the metabolism of Mg and six other essential trace metals monitored. Trial 3 examined the dose-response effect of TETA, at and below the dose given to patients with the Wilson’s disease over a 7d period, on Cu and eight other trace metals in a subgroup of seven T2DM and seven control subjects who had completed trial 1. The results of this i i trial showed that there was a linear dose-response relationship over the dose range 300~2400 mg/d on urinary Cu excretion in both T2DM and control subjects. However, there was no significant difference between the two subject groups at any of the four doses tested. In addition, 300mg/d of TETA was effective in mobilizing Cu in both T2DM and healthy control subjects. Trial 4 described the full work-up of a sensitive LC-MS methodology to identify and quantify TETA and its metabolite(s) in human urine. Using the LC-MS, TETA metabolism and excretion was investigated by analysing the urine of seven T2DM and seven control subjects who received escalating doses of TETA (samples obtained from trial 3). I have successfully identified and characterised two major metabolites of TETA in the urine of both T2DM and control subjects, N1-monoacetytriethylenetetramine (MAT) and diacetytriethylenetetramine (DAT), the latter which has not been previously reported. The results from urinary TETA excretion analyses also showed that T2DM may metabolise TETA more extensively than control subjects, which in turn is associated with its higher uptake or bioavailability. Urinary Zn excretion was mainly linked with urinary TETA and MAT in T2DM and healthy controls, respectively, whereas urinary Cu excretion was associated with urinary TETA excretion in healthy controls and urinary TETA+MAT excretion in T2DM subjects. These results suggest that MAT may also be involved in the mechanism by which TETA extracts systemic free Cu in diabetes. The identification of the two major metabolites of TETA and the development of a robust analytical LC-MS methodology reported in this study is an important step to further investigate the pharmacological actions of TETA in diabetic individuals. Collectively, the results presented in this thesis and in association with previous animal and clinical studies from our laboratory have provided consistent supporting evidences for the use of TETA clinically as a safe and effective therapy to prevent the genesis of some diabetic complications, in conjunction with conventional complication modifying therapies.

Diabetic cardiomyopathy is one of the causes of mortality and morbidity associated with diabetes. Diabetes is a disorder characterised by chronic hyperglycaemia and cardiovascular complications. The relationship between these integrally linked conditions has long been recognised, and for a significant portion of individuals the two conditions co-exist as part of metabolic syndrome. The presence of diabetes increases the risk of heart failure up to fivefold and three-fold in women and men, respectively, when compared to individuals without diabetes. While there has been a significant declining trend in cardiovascular mortality and morbidity in the general population over the past two decades, unfortunately such trends have not been seen among diabetic patients. As a result, this has persuaded many health professionals to re-evaluate their current treatment and pharmacological regimens. It is a well established fact that oxidative stress is a contributory mechanism in many agerelated disorders including T2DM, especially in those with poor glycaemic control. Thus far, clinical trials with antioxidant or carbonyl-trapping agents have produced mixed results, suggesting that the mechanisms underlying this disorder may be more complex than previously thought. Although altered systemic regulation of trace metals in diabetes has been previously investigated, it is still unclear whether changed trace metal metabolism would cause heart disease in common forms of diabetes and whether metal chelation can reverse this condition. Our hypothesis is that the accumulation of redox-active trace metals including Cu and Fe in cardiac muscle may, at least in part, result in cardiomyopathy through the generation of excess reactive oxygen species. We believed that the administration of a specific metal chelator should ameliorate this process by increasing the excretion of free systemic Cu and Fe, consequently limiting the production of superoxide oxygen free radicals and arresting the process of diabetic cardiomyopathy. Data from pre-clinical studies conducted in our laboratory using diabetic animal model with diabetes-induced abnormal Cu metabolism have been remarkably consistent in demonstrating that oral dosing with triethylenetetramine (TETA) can effectively remove systemic Cu via increased urinary Cu excretion, improve cardiomyocyte structure, reverse elevations in left ventricular collagen and β1-integrin, and alleviate heart failure, all in the presence of a consistently high circulating blood glucose profile. Taken together, these findings support the beneficial role of TETA in diabetic animal model and lay the foundation for its potential therapeutic effect in humans with diabetes. This thesis describes a series of randomised, placebo-controlled clinical trials that have investigated the metabolism of Cu and Fe and seven other trace metals in patients with chronic T2DM compared with non-diabetic control subjects. This thesis also examines the mechanism of action of TETA and addresses the hypothesis that a decrease in body systemic Cu pool through chelation therapy may improve cardiac complication in diabetic subjects. Trial 1 is a randomised, double-blind, placebo- and diet-controlled study which measured the 6d balance of Cu and Fe and seven other essential trace metals, in twenty male T2DM and twenty age-matched control subjects in whom we later probed systemic metal balance with oral TETA. Basal urinary output and balance of Cu and Fe was significantly elevated in diabetes, and the two output values correlated strongly (p<0.05). 6d treatment with 2400mg/d dose of TETA (maximum Wilson’s disease dose) has increased the urinary excretion of Cu, which was predicted by basal urinary Cu excretion, thereby causing a positive Cu balance to become negative in diabetes. Regulation of Cu metabolism was shown to be abnormal in diabetes and was selectively modified by TETA, which did not concomitantly modify Fe metabolism. Moreover, TETA did not cause a negative balance in any of the other seven trace metals monitored. These findings are consistent with TETA reversing the accumulation of free systemic Cu in diabetes, which may help to explain its potential therapeutic effects in some diabetic complications. Trial 2 investigated the acute response effect of a single 2400mg dose of TETA on urinary and serum trace metals in the first 10hr and 10~24hr post-dose. The results showed that TETA markedly increased the urinary Cu and Zn excretion in diabetes for the duration of 10hrs with the maximum excretion phase between 4~6hr post-drug (p<0.05). TETA did not change the metabolism of Mg and six other essential trace metals monitored. Trial 3 examined the dose-response effect of TETA, at and below the dose given to patients with the Wilson’s disease over a 7d period, on Cu and eight other trace metals in a subgroup of seven T2DM and seven control subjects who had completed trial 1. The results of this i i trial showed that there was a linear dose-response relationship over the dose range 300~2400 mg/d on urinary Cu excretion in both T2DM and control subjects. However, there was no significant difference between the two subject groups at any of the four doses tested. In addition, 300mg/d of TETA was effective in mobilizing Cu in both T2DM and healthy control subjects. Trial 4 described the full work-up of a sensitive LC-MS methodology to identify and quantify TETA and its metabolite(s) in human urine. Using the LC-MS, TETA metabolism and excretion was investigated by analysing the urine of seven T2DM and seven control subjects who received escalating doses of TETA (samples obtained from trial 3). I have successfully identified and characterised two major metabolites of TETA in the urine of both T2DM and control subjects, N1-monoacetytriethylenetetramine (MAT) and diacetytriethylenetetramine (DAT), the latter which has not been previously reported. The results from urinary TETA excretion analyses also showed that T2DM may metabolise TETA more extensively than control subjects, which in turn is associated with its higher uptake or bioavailability. Urinary Zn excretion was mainly linked with urinary TETA and MAT in T2DM and healthy controls, respectively, whereas urinary Cu excretion was associated with urinary TETA excretion in healthy controls and urinary TETA+MAT excretion in T2DM subjects. These results suggest that MAT may also be involved in the mechanism by which TETA extracts systemic free Cu in diabetes. The identification of the two major metabolites of TETA and the development of a robust analytical LC-MS methodology reported in this study is an important step to further investigate the pharmacological actions of TETA in diabetic individuals. Collectively, the results presented in this thesis and in association with previous animal and clinical studies from our laboratory have provided consistent supporting evidences for the use of TETA clinically as a safe and effective therapy to prevent the genesis of some diabetic complications, in conjunction with conventional complication modifying therapies.

Les sédiments de surface du Golfe du Morbihan et de la Baie de Quiberon ont été analysés afin d’évaluer les éléments traces métalliques (ETMs), leur comportement, les processus qui contrôlent leur distribution spatiale, leur spéciation, leur réactivité et leur biodisponibilité. Les potentielles sources métalliques ont été recherchées à partir de l’analyse des sédiments des ports à proximité et des sédiments du bassin versant. Les analyses ont été faites sur la fraction inférieure à 63 µm des sédiments collectés en Avril 2013. Les concentrations totales et la spéciation des ETMs ont été obtenues à partir des attaques totales et sélectives, respectivement. L’enrichissement en Zn, Cu, Cd et Pb a été mis en évidence dans les sédiments de surface du Golfe du Morbihan. De même, la labilité et la biodisponibilité du Pb ont été montrées dans les sédiments de la Baie de Quiberon. Le comportement des ETMs dans les sédiments de la côte morbihannaise est principalement contrôlé par le gradient terre/mer. Cependant, les ports et les rivières constituent des sources d’ETMs, principalement de Cu et Zn. / Surface sediments in the Gulf of Morbihan and the Bay of Quiberon were analyzed in order to characterize the trace metals elements, their behavior, the processes that control their spatial distribution, their speciation, reactivity and bioavailability. Potential sources of metals were looked for, based on the analysis of nearby harbors and river basin’s sediments. The analyses were done on the fraction below 63 µm of the sediments collected in April 2013. MTEs total concentration and speciation were obtained from total and selective attacks, respectively.The enrichment of Zn, Cu, Cd et Pb was identified in the surface sediments of the Gulf of Morbihan. Pb lability and bioavailability were shown in the sediments of the Bay of Quiberon. The MTEs behavior of the Morbihan’s coast is mostly controlled by the land-sea gradient. However, harbors and rivers form sources of ETMs, principally of Cu and Zn.