Šiame darbe tirta TetA ir AcrAB siurblių veikla Escherichia coli ląstelėse, panaudojant lipofilinius tetrafenilfosfonio (TPP+) jonus bei mikrobiologiniais ląstelių gyvybingumo tyrimo metodais įvertintas pasirinktų E. coli padermių jautrumas tetraciklinui. Buvo nustatyta, kad atspariausios tetraciklinui yra IQ86 ląstelės, transpozone Tn10 turinčios TetA siurblį koduojančius genus. Registruodami TPP+ pasiskirstymą tarp ląstelių ir jų inkubacinės terpės, nustatėme, kad tetraciklinas didina E. coli ląstelių išorinės membranos (IM) laidumą lipofiliniams junginiams. EDTA pagalba pralaidinus E. coli ląstelių IM, tetraciklinas lengviau patenka į ląstelę. Tai rodo, kad šis antibiotikas į ląstelę patenka ne tik per porinus, bet ir prasiskverbdamas per IM lipidinį dvisluoksnį. TPP+ jonus iš E.coli ląstelių išmetinėjantys DVA siurbliai taip pat sąveikauja ir su tetraciklinu bei chloramfenikoliu. Inkubuojant ląsteles su tetraciklinu, indukuojama šį antibiotiką išstuminėjančio TetA siurblio genų raiška. Vienok, inkubacija su tetraciklinu gali sutrikdyti kitų E. coli ląstelėse esančių DVA siurblių veiklą. Rezerpinas ir chlorpromazinas efektyviai slopina tetracikliną iš ląstelių išstuminėjančių siurblių veiklą, o fenilalanin-arginin- naftilamidas E. coli ląstelių atveju nepasižymi DVA siurblius slopinančiu poveikiu. Nustatyta, kad DVA siurblių slopiklių poveikis stipresnis, kai jie sąveikauja su ląstelėmis terpėje be antibiotikų. / We used lipophilic tetraphenilphosphonium (TPP+) ions for functional analysis of multidrug resistance (MDR) pumps TetA and AcrAB in Escherichia coli cells. We have found that tetracycline increases the cell outer membrane permeability to lipophilic compounds. Tetracycline entered the cells faster when the bacterial outer membrane was permeabilized using EDTA. These results indicate that tetracycline enters the cell not only through the porins, but also it penetrates the lipid bilayer of the outer membrane. MDR pumps extruding TPP+ ions from E. coli cells, also interact with tetracycline and chloramphenicol. Genes of TetA pump are induced, when the cells are incubated with low concentrations of tetracycline. On the other hand, incubation of the cells with tetracycline disturbs the activity of others MDR pumps in E. coli. Reserpine and chlorpromazine effectively inhibit activity of tetracycline-extruding MDR pumps, but Phe-Arg-β-naphtylamide does not show any inhibitory activity. Finally, it has been shown that action of the MDR pump inhibitors is stronger, when they are added to the bacterial suspension in the absence of antibiotics.

Ce travail porte sur la prévision du risque de fissuration en fretting fatigue des contacts Teta/Frette des conduites flexibles servant à l’acheminement du pétrole et du gaz. Les mouvements de la houle produisent des micros déplacements typiques du fretting entre les fils de Teta et de Frette. Le problème des conduites flexibles est particulièrement complexe puisqu’il implique de prendre en compte des forts niveaux de pressions induisant de la plasticité et des sollicitations variables. Pour répondre à ce problème, des études expérimentales et numériques ont été menés en parallèle. L’étude de fissuration est divisée entre prévision du risque d’amorçage des fissures et étude des conditions de propagation. L’amorçage est étudié avec le critère de fatigue multiaxial de Crossland appliqué à distance critique pour prendre en compte les gradients de contrainte spécifiques au fretting. Cette distance critique est optimisée pour être représentative du large spectre de gradient des contraintes étudié. Il est montré que si la distance critique est constante quelle que soit le gradient des contraintes, elle est associée à une longueur de fissure amorcée dépendante du gradient (couple ℓopt-bopt). Cette approche optimisée, combinée à une loi élastoplastique représentative permet des estimations très précises des conditions d’amorçage des fissures. [...] / This work concerns the prediction of fretting fatigue cracking risk in the Teta/Frette contacts of flexible pipes used for oil&gaz transportation. Swell movements produce micro-displacements between Teta wires and Frette wires, which are typical of fretting. Fretting problem in flexible pipes is particularly complex as it involves taking account of high pressure levels inducing plasticity and variable loadings. To answer this problem, experimental and numerical studies have been carried out. Cracking study is divided between crack nucleation risk prediction and the study of crack propagation conditions. Crack nucleation is studied using Crossland multiaxial fatigue criterion applied at a critical distance to address fretting stress gradient effect. This critical distance is optimized to be representative of the wide spectrum of stress gradient studied. It is shown that if the critical distance is constant whatever the stress gradient, it is combined with a crack nucleation length which depends on the stress gradient (ℓopt-bopt couple). This optimized approach, combined with a representative elastic-plastic material law, allows very precise estimates of crack nucleation conditions. [...]

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.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2015-09-17T21:10:57Z No. of bitstreams: 1 AnnieDaCostaSouza_DISSERT.pdf: 4581835 bytes, checksum: 75b58eb36554ceea210d1f40fcbb7f5e (MD5) / Approved for entry into archive by clediane guedes (clediane@bczm.ufrn.br) on 2015-10-27T15:25:21Z (GMT) No. of bitstreams: 1 AnnieDaCostaSouza_DISSERT.pdf: 4581835 bytes, checksum: 75b58eb36554ceea210d1f40fcbb7f5e (MD5) / Made available in DSpace on 2015-10-27T15:25:21Z (GMT). No. of bitstreams: 1 AnnieDaCostaSouza_DISSERT.pdf: 4581835 bytes, checksum: 75b58eb36554ceea210d1f40fcbb7f5e (MD5) Previous issue date: 2014-10-15 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / O ritmo teta consiste em uma oscila??o eletrofisiol?gica hipocampal presente em v?rias esp?cies de mam?feros (4-12 Hz, com varia??es entre esp?cies). Essa oscila??o est? presente durante a vig?lia ativa de ratos e tamb?m ? predominante no PCL desta esp?cie durante o sono de movimento r?pido dos olhos (sono REM). V?rios trabalhos demonstraram que o ritmo teta ? importante em tarefas cognitivas. O septo medial ? uma regi?o importante na gera??o do ritmo teta hipocampal. Possui proje??es colin?rgicas, GABA?rgicas e glutamat?rgicas para o hipocampo, que por sua vez, possui proje??es de feedback para o septo. Al?m do septo, outras regi?es est?o envolvidas na regula??o do teta, formando uma rede complexa de intera??o e coordena??o entre ?reas que resultam no ritmo. A optogen?tica ? uma ferramenta desenvolvida recentemente que tem sido amplamente utilizada em pesquisas de diversas ?reas. Ela nos permite manipular a atividade el?trica de neur?nios atrav?s de estimula??o luminosa. A t?cnica consiste em, atrav?s de um vetor viral, induzir a express?o neuronal de canais i?nicos associados a opsinas (ex.: ChR2), que uma vez infectados passam a ser sens?veis a luz de determinado comprimento de onda. O presente trabalho de pesquisa de mestrado teve como objetivo implantar a optogen?tica em animais em livre comportamento pioneiramente no Brasil, atrav?s de experimentos com implantes cr?nicos de eletrodos e fibras ?ptica em animais infectados com vetor viral para express?o de ChR2. Foram realizadas cirurgias de inje??es de v?rus no septo medial; resultados histol?gicos confirmaram a express?o de ChR2 atrav?s da marca??o da prote?na rep?rter eYFP no septo e tamb?m em processos hipocampais. Al?m disso, foram realizados experimentos agudos com estimula??o luminosa do septo medial e registro de potenciais de campo local (PCL) no pr?prio septo e hipocampo em animais anestesiados. Ainda nesses experimentos foi poss?vel registrar potenciais de a??o no septo. Nesses experimentos observamos aumento da taxa de disparo dos neur?nios septais durante estimula??o luminosa (n=300 est?mulos). Al?m disso, encontramos uma resposta evocada no PCL do hipocampo no in?cio do pulso luminoso. Tamb?m foram realizados experimentos cr?nicos com estimula??o luminosa do septo medial e registro de PCL do hipocampo em animais em livre comportamento. Atrav?s de an?lise do PCL, verificamos se a estimula??o luminosa do septo ? capaz de induzir ritmo teta no hipocampo. / Theta rhythm consists of an electrophysiological hippocampal oscillation present in mammalian species (4-12 Hz with variations across species). This oscillation is present during active waking and is also prevalent in local field potentials (LFP) during rapid eye movement sleep (REM sleep). Several studies have shown that theta rhythm is important in cognitive tasks and that the medial septum is a key region for its occurrence. The septum sends cholinergic, GABAergic and glutamatergic projections to the hippocampus, which in turn projects axons to the septum. Besides the septum, other regions are involved in regulating theta rhythm, forming a complex network of interactions among brain areas that result in theta rhythm. Optogenetics is a recently developed method that has been widely used in various research areas. It allows us to manipulate the electrical activity of neurons through light stimulation. One of the existing techniques consists in using a viral vector to induce the neuronal expression of ion channels associated with the light-sensitive molecule rhodopsin (e.g. ChR2). Once infected, the neurons become sensitive to light of a particular wavelength. The present M. Sc. research aimed to perform luminous stimulation of the brain in anesthetized and freely behaving animals using chronically implanted electrodes and optical fibers in animals infected with a viral vector for ChR2 expression. Surgical viral injections were performed in the medial septum; histological results confirmed the expression of ChR2 by way of the presence of the eYFP reporter protein in the septum and also in hippocampal processes. Moreover, we performed acute experiments with luminous stimulation of the medial septum and LFP recordings of the septum and hippocampus of anesthetized animals. Action potentials were recorded in the septum. In these experiments we observed a significant increase in the firing rates of septal neurons during luminous stimulation (n = 300 trials). Furthermore, we found an early light-evoked response in the hippocampal LFP. Chronic experiments with luminous stimulation of the medial septum and hippocampus in freely behaving animals were also performed in combination with LFP recordings. We found that the luminous stimulation of the septum is able to induce theta rhythm in the hippocampus. Together, the results demonstrate that the luminous stimulation of the medial septum in optogenetically-modified animals causes relevant electrophysiological changes in the septum and the hippocampus.

Bacterial antibiotic resistance is a globally recognized problem that has prompted extensive research into novel antimicrobial compounds. This dissertation describes research focusing on two types of potential antimicrobial molecules, organometallic compounds (OMC) and peptide nucleic acids (PNA). Organometallic compounds show promise as antimicrobial drugs because of their structural difference from conventional antibiotics and antimicrobials, and because of the ability to "tune" their chemical and biological properties by varying ligand attachments. Peptide nucleic acids, when linked to a cell-penetrating peptide (CPP), can suppress bacterial gene expression by an antisense mechanism and are attractive candidates for antimicrobial drugs because they bind strongly to target nucleic acids and are resistant to nucleases. Chapters 1 and 2 of the dissertation provide an introduction and broad literature review to frame the experimental questions addressed. Chapter 3 describes work to test the cytotoxicity and cellular penetration capabilities of novel OMCs by evaluating their effects on J774A.1 murine macrophage-like cells that were either uninfected or were infected with Mycobacterium bovis BCG. Results indicate that OMCs with an iridium (Ir) metal center and an amino acid ligand show minimal cytotoxicity against eukaryotic cells but likely do not penetrate the intracellular compartment in significant amounts. Chapter 4 presents research into in vitro effects of CPP-PNAs targeting the tetA and tetR antibiotic resistance genes (CPP-anti-tetA PNA and CPP-anti-tetR PNA, respectively) in tetracycline-resistant Salmonella enterica ssp. enterica serovar Typhimurium DT104 (DT104). Through the use of modified minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays it was shown that both the CPP-anti-tetA PNA and CPP-anti-tetR PNA increase tetracycline susceptibility in DT104. Chapter 5 explores the molecular mechanism of the CPP-anti-tetA PNA and CPP-anti-tetR PNA through the use of reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Results indicate good specificity of the CPP-anti-tetA PNA for its nucleic acid target as evidenced by suppression of tetA mRNA expression in DT104 cultures treated with a combination of tetracycline and the PNA. Chapter 6 describes the development of a mouse model of DT104 infection using BALB/c mice, followed by implementation of that model to test in vivo antimicrobial effects of the CPP-anti-tetA PNA and the CPP-Sal-tsf PNA, which targets expression of the essential tsf gene. An optimal dose of DT104 was identified that causes clinical illness within 2-4 days. At the doses tested, concurrent treatment of infected mice with tetracycline and the CPP-anti-tetA PNA or with the CPP-Sal-tsf PNA alone did not have a protective effect. Final conclusions are 1) that further research with the OMCs should focus on compounds with an Ir center and an amino acid ligand, and should explore ways to enhance intracellular penetration, 2) that the in vitro results of the PNA studies suggest that PNAs targeting expression of antibiotic resistance genes could allow for repurposing of antibiotics to which bacteria are resistant, and 3) additional study of the behavior of PNAs in vivo is advised. / Doctor of Philosophy / Antibiotic-resistant bacteria are increasingly recognized as a threat to global health, and new antibacterial drugs are urgently needed. Before a chemical compound can advance far in the journey to becoming a new drug it must be tested for toxicity against mammalian cells. A portion of this dissertation research involved testing the toxicity of several organometallic compounds (OMCs) previously shown to have antibacterial potential. Mouse-derived mammalian cells were treated with several of the OMCs, and initial results indicated that one of the OMCs is non-toxic and is likely a safe option for additional analysis. This OMC was further tested to see if it could inhibit mycobacterial growth inside of the mammalian cells. It did not effectively clear bacteria from inside of the mammalian cells, likely because of poor penetration of the cell membrane. Further research with this compound should focus on ways to effectively transport the OMC inside infected mammalian cells so that it can reach the bacteria it is meant to target. A second portion of this research involved using a peptide nucleic acid (PNA) to try and reverse tetracycline antibiotic resistance in the bacterial strain Salmonella enterica ssp. enterica serovar Typhimurium DT104 (DT104). Peptide nucleic acids are short linear molecules that can bind strongly to complementary DNA and RNA sequences and thus be used to interfere with expression of specific genes. A PNA was designed to inhibit expression of the bacterial tetA gene that codes for a protein called the TetA tetracycline efflux pump, which imparts resistances to tetracycline. Treating the bacteria with the PNA resulted in a lower dose of tetracycline needed to inhibit bacterial growth, indicating a successful increase in tetracycline susceptibility. By using a molecular analysis technique called reversetranscriptase quantitative polymerase chain reaction (RT-qPCR), it was possible to measure the amount of tetA messenger RNA (mRNA) in cultures of DT104 treated only with tetracycline or with a combination of tetracycline and the PNA. As expected, bacteria treated with both the antibiotic and the PNA had less tetA mRNA than the cultures treated only with tetracycline, supporting the hypothesis that the PNA prevents the bacteria from effectively expressing the tetA gene. The PNA was next used in conjunction with tetracycline as an experimental treatment for mice infected with DT104. The PNA did not provide the expected protective effect under these circumstances. The overall conclusion for this part of the research is that PNAs offer an exciting potential avenue for counteracting antibiotic resistance, but additional experimentation is needed. Future research should focus on investigating more effective ways to get the PNAs inside the bacteria and on understanding more about how the PNAs behave in live animals. Several other PNAs targeting different genes involved in antibiotic resistance or essential bacterial functions were also tested against DT104 with variable success.

Polymer/clay nanocomposites consisting of an epoxy resin matrix filled with organoclays have been investigated. The main objective of this study was to determine which combination of components led to the greatest enhancement in properties of the epoxy resin. Exfoliation of the clay was desired, as exfoliated nanocomposites are known to exhibit great improvements in mechanical properties [1]. The epoxy resins studied were di-functional DGEBA and tetra-functional TGDDM. The epoxy resin was cured with three different hardeners, these included: the high functionality amine hardener, TETA, and two anhydride hardeners, accelerated MTHPA and pure HHPA. The three organoclays used, contained alkylammonium cations and were also compared to the unmodified clay. Morphology was investigated by XRD and TEM, and the flexural properties of the resulting nanocomposites were studied. The effect that the addition of an organoclay has on the cure of the epoxy resin was investigated using MDSC. Both the temperatures required to cure the resin with, and without, the clay, and any changes in the total heat flow that occurred were studied. The Tg++ of the cured nanocomposites was also measured using MDSC. The heat flow results indicated that the clays added to the epoxy resins act as a physical barrier, which prevents the resin from reaching full cure. In the higher functional resin, the addition of clay resulted in a significant decrease in the total heat flow, suggesting that a large amount of epoxy remains uncured, and, as a result, there should be a reduction in the amount of cross-linking. The lower cross-link density led to a significantly lower Tg and the mechanical properties were also poorer. The reactivity of the hardener towards the resin was found to have the greatest impact on the cured nanocomposite morphology. Intragallery polymerisation occurring at a faster rate than the extragallery polymerisation causes exfoliation. In order to achieve a balance that favours intragallery polymerisation, it was found that the curing reaction was required to be catalysed by the alkylammonium cation of the organoclay, and not catalysed by other means. The DGEBA cured with HHPA provided the largest layer expansion in the clay structure due to the alkylammonium cation catalysing the anhydride ring-opening reaction. The effect was not seen with TGDDM due to the tertiary amine in its structure. The accelerator within the MTHPA assisted extragallery polymerisation of the resin and the TETA cured readily without additional catalysis.

Made available in DSpace on 2014-12-17T15:36:42Z (GMT). No. of bitstreams: 1 HindiaelAB_TESE.pdf: 3415388 bytes, checksum: d9664bb45093eee642d43a4ac9712227 (MD5) Previous issue date: 2013-08-28 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The processing of spatial and episodic information during memory tasks depends on hippocampal theta oscillations. In the present study, I investigated the relationship between theta power and choice selection during spatial decision-making. I recorded local field potentials from the CA1 region of rats retrieving reward locations in a 4-arm maze. In trained but not in na?ve animals, I observed a significant increase in theta power during decision-making, which could not be explained by changes in locomotion speed. Furthermore, a Bayesian decoder based on theta power predicted choice outcomes in speed-matched trials. The decoding time course revealed that performance increased above chance before the decision moment exclusively for theta power, remaining flat for other frequency bands. These results occurred for trained animals, but no significant prediction could be made for na?ve animals. Altogether, the data support a mnemonic function of theta rhythm during spatial decision-making, indicating that these oscillations correlate with the retrieval of memories required for successful decisions / O hipocampo ? uma estrutura essencial para o processamento de mem?rias espaciais e epis?dicas. Evid?ncias recentes t?m sugerido que aumentos de amplitude nas oscila??es Teta (5-12 Hz) refletem a participa??o do hipocampo na execu??o de tarefas de tomada de decis?o. Com o objetivo de investigar a fun??o da oscila??o Teta hipocampal em processos de decis?o espacial, registrei o potencial de campo da regi?o CA1 dorsal do hipocampo do rato durante a execu??o de uma tarefa de escolha em labirinto radial de quatro bra?os. Observei que animais treinados apresentam aumento significativo das oscila??es Teta durante a decis?o, diferentemente de animais n?o treinados. Os resultados mostram que o aumento da pot?ncia de Teta durante a decis?o est? relacionado ao desempenho dos animais na tarefa, e sugerem que o ritmo Teta hipocampal reflete a evoca??o de mem?rias associada ? decis?o. Al?m disso, a pot?ncia de Teta foi significativamente maior durante decis?es corretas que nas decis?es incorretas. Esses resultados n?o est?o associados a diferen?as de atividade locomotora. Finalmente, verifiquei que ? poss?vel utilizar a pot?ncia das oscila??es Teta para predizer decis?es corretas e incorretas em cada tentativa. Em conjunto, os resultados apoiam uma fun??o mnem?nica das oscila??es Teta em tarefas de tomada de decis?o espacial