Several lines of evidence link Alzheimer's disease (AD) to atherosclerosis (CVD), including that elevated low density lipoprotein (LDL)-cholesterol is a common risk factor. Development of genomic instability could also link the two diseases. Previous fluorescence in situ hybridization (FISH) analyses revealed a clonal expansion of aneuploid smooth muscle cells underlying atherosclerotic plaques. Likewise, cellular and mouse models of AD revealed tau-dependent mitotic defects and subsequent aneuploidy partly resulting from amyloid-beta (A&beta) interference with microtubule (MT) stability, and specific MT motors function. Moreover, AD patients develop aneuploid/hyperploid cells in brain and peripheral tissues, implicating similar mechanism that may lead to apoptosis and neurodegeneration. This dissertation tested the hypothesis that elevated lipoproteins and cholesterol may contribute to genomic instability in AD and CVD and showed that: (1) treatment with oxidized LDL (OX-LDL), LDL and water soluble cholesterol, but not high density lipoprotein (HDL), induced chromosome mis-segregation, including trisomy and tetrasomy 12, 21, and 7 in human epithelial cells (hTERT-HME1), primary aortic smooth muscle cells, fibroblasts, mouse splenocytes and neural precursors; (2) LDL-induced aneuploidy may depend on a functional LDL receptor (LDLR), but not amyloid precursor protein (APP) gene; (3) fibroblasts and brain cells of patient with the mutation in the Niemann-Pick C1 gene (NPC1) characterized by impaired intracellular cholesterol trafficking and changed intracellular cholesterol distribution harbored trisomy 21 cells; (4) young wild-type mice fed high and low cholesterol diets developed aneuploidy in spleen but not in brain cells within 12 weeks; (5) like with the studies on A&beta-induced aneuploidy, calcium chelation reduced OX-LDL and LDL-mediated chromosomal instability; and (6) altering plasma membrane fluidity with ethanol attenuated OX-LDL and LDL-induced aneuploidy. These results suggest a novel biological mechanism by which disrupted cholesterol homeostasis may promote both atherosclerosis and AD by inducing chromosome mis-segregation and development of aneuploid cells. Understanding the cause and consequence of chromosomal instability as a common pathological trait in AD and CVD may be beneficial to designing therapies relevant for both diseases.

Diabetes mellitus is estimated to affect approximately 7% of the populations living a western lifestyle. Of the multiple etiologies associated with diabetes, heart failure is the most common cause of death. A specific type of heart disease called diabetic cardiomyopathy is thought to be partially responsible. At this time, no one specific treatment is available for diabetic cardiomyopathy due to the wide variety of possible complex molecular changes, including metabolic disturbances, myocardial fibrosis, LV hypertrophy, and increased ROS production. Abnormal copper metabolism in diabetes has been proposed to form part of the pathway that leads to diabetic cardiomyopathy. Our group have shown that treatment with the copper (CuII) chelator, triethylenetetramine, ameliorates the effects of diabetes on the heart at both the functional and molecular level. This thesis aimed to further these studies by increasing our understanding of the mechanism of triethylenetetramine action on the diabetic heart. This was primarily achieved through the use of microarray technology but included the use of a range of molecular experimental techniques. During this investigation it was determined that the most suitable microarray platform for our studies was the Affymetrix GeneChip® system. Using this system we identified more than 1600 gene changes associated with diabetes in the left ventricle wall. A disproportionate number of significant messenger RNA transcript changes were associated with the mitochondria and further investigation of these genes revealed changes associated with perturbed lipid metabolism and increased oxidative stress. A second study investigated the molecular mechanisms underpinning improved cardiac function in the left ventricle of the heart from diabetic and sham animals treated with triethylenetetramine. There was an observed decrease in diabetic cardiac tissue triglyceride towards normal, possibly through improvement of the structure and stability of the mitochondria. Only a small number of changes in gene expression were detected after triethylenetetramine treatment using microarray technology, and none were detected using real time-quantitative PCR. The final aim of this thesis was to understand the absorption and excretion of triethylenetetramine by both sham and diabetic animals. Our study found differences in the ability of diabetic animals to absorb and metabolise triethylenetetramine compared to sham animals. Also, the length of exposure was found to be an influencing factor in triethylenetetramine metabolism.

Diabetes mellitus is estimated to affect approximately 7% of the populations living a western lifestyle. Of the multiple etiologies associated with diabetes, heart failure is the most common cause of death. A specific type of heart disease called diabetic cardiomyopathy is thought to be partially responsible. At this time, no one specific treatment is available for diabetic cardiomyopathy due to the wide variety of possible complex molecular changes, including metabolic disturbances, myocardial fibrosis, LV hypertrophy, and increased ROS production. Abnormal copper metabolism in diabetes has been proposed to form part of the pathway that leads to diabetic cardiomyopathy. Our group have shown that treatment with the copper (CuII) chelator, triethylenetetramine, ameliorates the effects of diabetes on the heart at both the functional and molecular level. This thesis aimed to further these studies by increasing our understanding of the mechanism of triethylenetetramine action on the diabetic heart. This was primarily achieved through the use of microarray technology but included the use of a range of molecular experimental techniques. During this investigation it was determined that the most suitable microarray platform for our studies was the Affymetrix GeneChip® system. Using this system we identified more than 1600 gene changes associated with diabetes in the left ventricle wall. A disproportionate number of significant messenger RNA transcript changes were associated with the mitochondria and further investigation of these genes revealed changes associated with perturbed lipid metabolism and increased oxidative stress. A second study investigated the molecular mechanisms underpinning improved cardiac function in the left ventricle of the heart from diabetic and sham animals treated with triethylenetetramine. There was an observed decrease in diabetic cardiac tissue triglyceride towards normal, possibly through improvement of the structure and stability of the mitochondria. Only a small number of changes in gene expression were detected after triethylenetetramine treatment using microarray technology, and none were detected using real time-quantitative PCR. The final aim of this thesis was to understand the absorption and excretion of triethylenetetramine by both sham and diabetic animals. Our study found differences in the ability of diabetic animals to absorb and metabolise triethylenetetramine compared to sham animals. Also, the length of exposure was found to be an influencing factor in triethylenetetramine metabolism.

Diabetes mellitus is estimated to affect approximately 7% of the populations living a western lifestyle. Of the multiple etiologies associated with diabetes, heart failure is the most common cause of death. A specific type of heart disease called diabetic cardiomyopathy is thought to be partially responsible. At this time, no one specific treatment is available for diabetic cardiomyopathy due to the wide variety of possible complex molecular changes, including metabolic disturbances, myocardial fibrosis, LV hypertrophy, and increased ROS production. Abnormal copper metabolism in diabetes has been proposed to form part of the pathway that leads to diabetic cardiomyopathy. Our group have shown that treatment with the copper (CuII) chelator, triethylenetetramine, ameliorates the effects of diabetes on the heart at both the functional and molecular level. This thesis aimed to further these studies by increasing our understanding of the mechanism of triethylenetetramine action on the diabetic heart. This was primarily achieved through the use of microarray technology but included the use of a range of molecular experimental techniques. During this investigation it was determined that the most suitable microarray platform for our studies was the Affymetrix GeneChip® system. Using this system we identified more than 1600 gene changes associated with diabetes in the left ventricle wall. A disproportionate number of significant messenger RNA transcript changes were associated with the mitochondria and further investigation of these genes revealed changes associated with perturbed lipid metabolism and increased oxidative stress. A second study investigated the molecular mechanisms underpinning improved cardiac function in the left ventricle of the heart from diabetic and sham animals treated with triethylenetetramine. There was an observed decrease in diabetic cardiac tissue triglyceride towards normal, possibly through improvement of the structure and stability of the mitochondria. Only a small number of changes in gene expression were detected after triethylenetetramine treatment using microarray technology, and none were detected using real time-quantitative PCR. The final aim of this thesis was to understand the absorption and excretion of triethylenetetramine by both sham and diabetic animals. Our study found differences in the ability of diabetic animals to absorb and metabolise triethylenetetramine compared to sham animals. Also, the length of exposure was found to be an influencing factor in triethylenetetramine metabolism.

Diabetes mellitus is estimated to affect approximately 7% of the populations living a western lifestyle. Of the multiple etiologies associated with diabetes, heart failure is the most common cause of death. A specific type of heart disease called diabetic cardiomyopathy is thought to be partially responsible. At this time, no one specific treatment is available for diabetic cardiomyopathy due to the wide variety of possible complex molecular changes, including metabolic disturbances, myocardial fibrosis, LV hypertrophy, and increased ROS production. Abnormal copper metabolism in diabetes has been proposed to form part of the pathway that leads to diabetic cardiomyopathy. Our group have shown that treatment with the copper (CuII) chelator, triethylenetetramine, ameliorates the effects of diabetes on the heart at both the functional and molecular level. This thesis aimed to further these studies by increasing our understanding of the mechanism of triethylenetetramine action on the diabetic heart. This was primarily achieved through the use of microarray technology but included the use of a range of molecular experimental techniques. During this investigation it was determined that the most suitable microarray platform for our studies was the Affymetrix GeneChip® system. Using this system we identified more than 1600 gene changes associated with diabetes in the left ventricle wall. A disproportionate number of significant messenger RNA transcript changes were associated with the mitochondria and further investigation of these genes revealed changes associated with perturbed lipid metabolism and increased oxidative stress. A second study investigated the molecular mechanisms underpinning improved cardiac function in the left ventricle of the heart from diabetic and sham animals treated with triethylenetetramine. There was an observed decrease in diabetic cardiac tissue triglyceride towards normal, possibly through improvement of the structure and stability of the mitochondria. Only a small number of changes in gene expression were detected after triethylenetetramine treatment using microarray technology, and none were detected using real time-quantitative PCR. The final aim of this thesis was to understand the absorption and excretion of triethylenetetramine by both sham and diabetic animals. Our study found differences in the ability of diabetic animals to absorb and metabolise triethylenetetramine compared to sham animals. Also, the length of exposure was found to be an influencing factor in triethylenetetramine metabolism.

Heavy metals are non-degradable and their persistence constitutes ecotoxicological ramifications. Their presence in wastewater is inhibitory to microbial digestion processes and can lead to biomagnification in the environment. Clay minerals have been widely studied as inexpensive sorbent materials of aqueous-phase contaminants. Particularly in the case of kaolinite, a non-swelling clay with a low cation-exchange capacity, chemically enhanced derivatives exposing its OH-rich interlayer space to interactions with its external environment are of exceptional interest. The objective of this study was to prepare a novel functionalised kaolinite with the aim of significantly improving its heavy-metal sorption capacity. Due to the robustness of sulfur-metal linkages, the compound 3,6-dithia-1,8-octanediol (HO(CH2)2S(CH2)2S(CH2)2OH) was grafted onto both urea- and DMSO precursors of two kaolinitic clays: the Source clay KGa-1b and a Portuguese clay sample collected from the Clays of Taveiro formation. All samples were characterised by X-ray diffraction and 13C nuclear magnetic resonance spectroscopy (NMR). Initial materials and final samples were also characterised by thermal gravimetric analysis and infrared spectroscopy. The results describe nanohybrid materials that are resistant to hydrolysis and exhibit different grafting fashions. Application as a heavy metal sorbent was tested in ZnCl2 solutions as well as in zinc-spiked raw wastewater. Zinc concentrations were analysed concomitant with other metal species by inductively coupled plasma – mass spectrometry, demonstrating variable sorption capacities of the different clay samples tested and a consistently greater zinc removal in the wastewater system compared to simple zinc solutions. Measured concentrations of concomitantly analysed metals reveals a potentially alarming release of certain heavy metal species from some clay samples as a result of zinc sorption.

La nicotianamine est une molécule naturelle qui joue un rôle essentiel dans la régulation des concentrations en métaux de transition dans les plantes. Sa structure comprend trois acides carboxyliques et trois fonctions amines. L'objectif du travail présenté dans ce manuscrit est d'établir un protocole permettant de reproduire les propriétés de chélation de cette molécule grâce à des calculs de chimie quantique. L'intérêt est d'appliquer ce protocole à des analogues synthétiques de la nicotianamine, afin de prédire leurs propriétés.Le protocole que nous avons mis au point s'articule autour de quatre axes : le calcul des pKa, l'étude des propriétés de chélation, le calcul de tenseur g de résonance paramagnétique électronique et l'analyse des propriétés vibrationnelles. Ces différents points ont nécessité le développement de méthodes de calcul et d'outils innovants. Les calculs de chimie quantique qui ont été effectués sont basés sur la théorie de la fonctionnelle de la densité. On a utilisé des fonctionnelles hybrides, associées à un modèle de solvant implicite ou semi-explicite qui permet de prendre en compte l'effet du milieu.Nous avons proposé une méthode graphique originale adaptée à la nicotianamine permettant le calcul de ses six pKa ainsi que celui des pka des complexes formés avec les métaux de transition. Cette méthode conduit à une bonne précision sur les calcul de pKa dans l'intervalle de pH physiologique. Ces pKa renseignent sur le comportement de la nicotianamine dans différents milieux végétaux. Nous avons aussi envisagé plusieurs protocoles pour l'étude des propriétés de chélation de la nicotianamine.Pour compléter l'étude des complexes de la nicotianamine, nous avons paramétré une méthode simple et rapide de calcul du tenseur g de systèmes moléculaires contenant du cuivre paramagnétique. Enfin, nous avons développé un programme graphique permettant l'analyse des modes de vibration infrarouges de systèmes moléculaires comme les complexes métal-nicotianamine étudiés. / Nicotianamine is a triamino acid that plays an essential role in regulation and transportation of transition metals in plants by a chelation process. Functional analogues of this molecule have already been synthesised that we hope to use for selective soil decontamination thanks to their property changes with respect to nicotianamine. Therefore, the aim of the present work is to find a predictive protocol for the chelating properties of these analogues, using the know example of nicotianamine. We worked out a predictive protocol focusing on the four following points : pKas, chelation, EPR tensors and vibrational properties. It uses quantum chemistry methods such as hybrid DFT functionals and implicit solvent models.The pKa calculations for nicotianamine and metal-nicotianamine complexes were performed using a novel graphical method well suited to these systems. The pKas are useful to understand the effect of the various plants pHs on the complexes. Then we computed the chelation constants and the structures of metal-nicotianamine complexes. As a complement to the study of these complexes, we set up protocols to calculate the EPR tensors of the copper-nicotianamine complex and to analyse the infra-red vibrations of the complexes, using respectively a parametrised method and a local code.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Botany Candidate: Ingrid Salanciová Supervisor: PharmDr. Jana Karlíčková, Ph.D. Title of Thesis: Interactions of reduced and oxidized glutathione with copper, iron and zinc Copper (Cu), iron (Fe) and zinc (Zn) are important trace elements that are necessary for proper function of the body. Excess but also the lack of these metals may lead to pathological conditions. Glutathione is the main antioxidant in the human body so it is expected to protect the organism against concequences of metal excess. Glutathione occurs in the reduced (GSH) and oxidized state as glutathione disulfide (GSSG) in the organism, both states forming an important redox system. On the other hand, glutathione can reduce these metals so that could participate in the formation of free radicals (metal based Fenton reaction). In this diploma thesis, was tested the ability of reduced and oxidized glutathione to interact with Fe, Cu and Zn in various (patho) physiological pH conditions was tested by using in vitro spectrophotometric competitive methods. Interactions include not only the chelation of Cu, Fe and Zn ions, but also their reductive activity toward Cu2+ and Fe3+ cations. Hematoxylin, bathocuproinedisulfonic acid disodium salt, ferrozine...

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