Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-07-20T19:32:21Z No. of bitstreams: 1 virginiafernandesbraga.pdf: 2075207 bytes, checksum: 720e5dba0516d172aec1843372dcecc2 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-07-22T15:25:58Z (GMT) No. of bitstreams: 1 virginiafernandesbraga.pdf: 2075207 bytes, checksum: 720e5dba0516d172aec1843372dcecc2 (MD5) / Made available in DSpace on 2016-07-22T15:25:58Z (GMT). No. of bitstreams: 1 virginiafernandesbraga.pdf: 2075207 bytes, checksum: 720e5dba0516d172aec1843372dcecc2 (MD5) Previous issue date: 2011-04-20 / Pitcairnia albiflos Herb. (Bromeliaceae) atualmente se encontra na lista de espécies ameaçadas de extinção. Essa espécie é endêmica dos afloramentos rochosos do município do Rio de Janeiro, RJ, e vem sofrendo com o pisoteio de alpinistas, queimadas, invasão de gramíneas exóticas e extrativismo vegetal. A micropropagação pode ser utilizada como alternativa às condições de risco em que as populações dessa espécie se encontram submetidas, visando à recomposição de populações ameaçadas em ambiente natural, assim como o abastecimento do mercado consumidor. A etapa final da micropropagação é a aclimatização, período em que as plantas ficam mais susceptíveis e sofrem com o estresse oxidativo devido às mudanças nas condições ambientais. No presente trabalho foram avaliadas as atividades enzimáticas antioxidativas da CAT, SOD, POD, PPO e o conteúdo de prolina, além dos teores de pigmentos fotossintéticos em plantas de Pitcairnia albiflos cultivados in vitro, em meios de cultura contendo duas concentrações de sacarose (15 ou 30 g L-1), tampas não vedadas que permitiam trocas gasosas e frascos vedados com tampas e filme plástico de PVC, que impediam a ventilação. Sob essas condições, as plantas foram cultivadas em meios contendo GA3 ou ANA. Após o período de crescimento in vitro, as plantas foram transferidas para condições ex vitro em casa de vegetação. As análises supra-citadas e a determinação dos teores de carboidratos solúveis totais, sacarose, amido, açúcares redutores, conteúdo relativo de água e suculência também foram realizadas nas plantas previamente cultivadas in vitro com 15 ou 30 g L-1 de sacarose e GA3 em tubos com tampas vedadas, após submissão das mesmas a estresse hídrico durante 24, 38 ou 52 dias. Após o período de estresse hídrico, as plantas foram reidratadas durante 34 dias sob irrigação periódica em casa de vegetação. Nos tecidos cultivados in vitro percebeu-se o surgimento de características de hiperidricidade nas plantas cultivadas com 15 g L-1 de sacarose, GA3 e tubos com tampas vedadas, o que foi evidenciado pelo menor acúmulo de prolina, aumento das atividades das enzimas antioxidantes e menor acúmulo de pigmentos fotossintetizantes. Na condição ex vitro, as plantas cultivadas anteriormente em meio de cultura contendo 15 g L-1 de sacarose apresentaram maior atividade das enzimas antioxidantes não havendo, em alguns casos, diferenças significativas em comparação com a concentração mais elevada de sacarose. Nessa condição o acúmulo de prolina foi menor, o que é indicativo de maior estresse oxidativo nessas plantas durante a aclimatização. Durante o estresse hídrico houve queda na atividade de todas as enzimas estudadas, embora essa queda tenha sido mais acentuada para as plantas que inicialmente foram cultivadas com 15 g L-1 de vi sacarose. O acúmulo de prolina aumentou com o prolongamento do estresse hídrico, sendo maior nas plantas que foram cultivadas in vitro com 30 g L-1 de sacarose. Não houve diferenças significativas no conteúdo de pigmentos fotossintetizantes e nas suas relações para nenhuma das concentrações de sacarose, exceto para os carotenóides totais, que apresentaram aumento significativo ao longo do período de estresse hídrico para as plantas previamente cultivadas com a menor concentração de sacarose. Os conteúdos de carboidratos solúveis totais e sacarose aumentaram com o prolongamento do estresse, sendo mais acentuados na concentração de 30 g L-1 de sacarose. Os conteúdos de amido, o conteúdo relativo de água e a suculência apresentaram redução com o aumento do estresse hídrico. Após a reidratação, todas as plantas mostraram capacidade de recuperação, apresentando valores próximos aos dos controles para todas as variáveis analisadas. Ressalta-se, todavia, que as plantas tratadas com 30 g L-1 de sacarose tiveram melhor recuperação quando comparadas com aquelas que foram tratadas com 15 g L-1 de sacarose. Em função dos resultados obtidos, é possível concluir que a concentração de sacarose utilizada in vitro apresenta influência no processo de aclimatização ex vitro e também, posteriormente, no campo, na capacidade de recuperação das plantas à seca quando elas são submetidas a estresse hídrico. As plantas cultivadas in vitro com 15 g L-1 de sacarose se mostraram mais sensíveis à seca e, possivelmente, não sobreviveriam caso fossem transferidas dos tubos de ensaio diretamente para o campo. As plantas cultivadas in vitro com 30 g L-1 de sacarose aparentemente eram mais resistentes ao processo de aclimatização ex vitro, apresentando maiores chances de sobrevivência em campo, maior tolerância à seca e maior capacidade de recuperação após períodos prolongados de estresse hídrico. / Pitcairnia albiflos Herb. (Bromeliaceae) is currently in the list of endangered species. This species is endemic of the inselbergs of the city of Rio de Janeiro, RJ, and has been suffering with the mountaineer’s trampling, wildfires, invasion of exotic grasses and plant extraction. The micropropagation can be used as an alternative to the risk conditions under which populations of this species are submitted, aiming at the recomposition of endangered populations in the natural environment, as well as the supply of the consumer market. The final stage of the micropropagation is the acclimatization, period in which the plants become more susceptible and suffer from oxidative stress due to the changes in the environmental conditions. In the present study it were evaluated the antioxidative enzymatic activities of CAT, SOD, POD, PPO and proline content, besides the levels of photosynthetic pigments in plants of Pitcairnia albiflos grown in vitro in culture mediums containing two sucrose concentrations (15 or 30 g L-1). Part was covered with unsealed lids that allowed gas exchanges and part was kept in sealed flasks with lids and PVC plastic film, that didn’t allow the ventilation. Under these conditions, the plants were cultivated in culture mediums containing GA3 or NAA. After the in vitro growth period, the plants were transferred to ex vitro conditions at a greenhouse. The above mentioned analyses and the determination of total soluble carbohydrate levels, sucrose, starch, reducer sugars, relative water content and succulence were also performed on the plants previously grown in vitro with 15 or 30 g L-1 of sucrose and GA3 in tubes with sealed lids, after the submission of these to water stress during 24, 38 or 52 days. After the water stress period, the plants were rehydrated for 34 days under regular irrigation at the greenhouse. In the in vitro cultivated tissues it was noted the emergence of hyperhydricity characteristics in the plants grown with 15 g L-1 of sucrose, GA3 and tubes with sealed lids, which was evidenced by the lowest proline accumulation, the increased in the antioxidative enzymatic activities and the lowest accumulation of photosynthetic pigments. In the ex vitro condition, the plants previously grown in culture mediums containing 15 g L-1 of sucrose presented larger antioxidative enzymatic activities, which did not show, in some cases, significant differences compared with the largest concentration of sucrose. In this condition, the proline accumulation was lower, which is an indicative of larger oxidative stress in these plants during acclimatization. During the water stress, there was a fall in the activity of all studied enzymes, although that fall had been more evident in the plants that were initially cultivated with 15 g L-1 of sucrose. The proline accumulation increased with the extension of the water stress, being larger in the plants grown in vitro with 30 g L-1 of sucrose. There were no viii significant differences in the content of photosynthetic pigments and in their relation with any sucrose concentrations, except for total carotenoids, which significantly increased over the period of water stress for the plants previously grown with the lowest concentration of sucrose. The contents of total soluble carbohydrates and sucrose increased with the extension of the stress, being more accentuated in the 30 g L-1 of sucrose concentration. The contents of starch, the relative content of water and succulence presented a reduction with the increase of water stress. After the rehydration, all plants showed recovery capacity, presenting values close to those from the control groups for all the analyzed variables. It should be noted, however, that the plants treated with 30 g L-1 of sucrose had better recovery compared with those that were treated with 15 g L-1 of sucrose. According to the obtained results, it is possible to conclude that the concentration of sucrose used in vitro presents influence on the process of ex vitro acclimatization and also, later in the field, in the recovery capacity of the plants to drought when they are submitted to water stress. The plants grown in vitro with 15 g L-1 of sucrose were more sensitive to drought and, possibly, would not survive if they were transferred from the test tubes directly to the field. The plants grown in vitro with 30 g L-1 of sucrose were apparently more resistant to the ex vitro acclimatization process, presenting greater survival chances in the field, larger drought tolerance and larger recovery capacity after extended periods of water stress.

L’objectif de cette thèse est la synthèse de composés de manganèse et de matériaux hybrides qui soient intéressants du point de vue bioinorganique et magnétique. Pour accomplir ce but, nous avons découpé la stratégie en trois étapes constituant les différents chapitres de ce manuscrit :(a) Synthèse et caractérisation des composés moléculaires de manganèse et l’étude de leurs propriétés magnétiques.Nous avons réussi à obtenir la structure cristalline des vingt-trois nouveaux composés de Mn de différentes nucléarités, d’état d’oxydation II, III ou IV. Nous avons étudié les propriétés magnétiques de ces composés et nous avons établi des corrélations magnéto-structurales. Les composés de MnII ont été aussi étudiés par spectroscopie RPE.(b) Synthèse et caractérisation des matériaux hybrides basés sur des composés moléculaires de manganèse insérés dans de la silice mésoporeuse. Les composés moléculaires sélectionnés ont été insérés dans de la silice mésoporeuse (du type MCM-41). Les complexes de Mn dans les supports ont été caractérisés par ATG, XPS, ICP-OES, spectroscopie IR et mesures magnétiques. (c) Étude des propriétés catalytiques des composés moléculaires et des matériaux hybrides.Une famille de composés moléculaires obtenus dans cette thèse sont des modèles structuraux et fonctionnels de la catalase à Mn, une enzyme présente dans certaines bactéries, ayant des propriétés antioxydantes (H2O2 « scavenger »). L’activité catalase pour ces composés et les matériaux hybrides dérivés a été étudiée dans l’acétonitrile et dans l’eau. / The main objective of this work is the synthesis of manganese compounds and hybrid materials that may be relevant from a bioinorganic and magnetic point of view. The developed strategy comprises three main steps that form different sections in this thesis:(a) Synthesis and characterization of molecular manganese compounds and study of the magnetic propertiesThe crystal structure of twenty-three new Mn compounds of different nucleartities were obtained in which the Mn oxidation state is II, III, or IV. The magnetic properties of all these compounds were profoundly studied and they have been rationalized with their structural and electronic parameters. The MnII compounds were also studied with EPR spectroscopy. (b) Synthesis and characterization of hybrid materials based on molecular manganese compounds inside mesoporous silica.Selected molecular compounds were inserted inside mesoporous silica (MCM-41 type). The Mn complexes inside the supports were characterized with TGA, XPS, ICP-OES, IR spectroscopy, and magnetic measurements.(c) Study of the catalytic properties of both molecular compounds and hybrid materials.A family of the molecular compounds obtained in this work are structural and functional models of the Mn catalase, an enzyme found in some bacteria with antioxidant properties (H2O2 scavenger). The catalase activity for these compounds and the hybrid materials was studied in acetonitrile and water.

This project focuses on two important topics which may pose hazards to human health. Firstly, drinking water disinfection by-products (DBPs), which are generated by the chemical disinfection of water have been investigated. What has not been shown is the effect of DBPs in human germ cells as well as somatic cells and whether oxidative stress is involved in the mechanism of genotoxic action. Three different DBPs (halo acetic acids: HAAs), together with the antioxidants – catalase and butylated hydroxyanisole (BHA), were investigated in peripheral blood cells and sperm from healthy individuals using the Comet assay and lymphocytes only using the micronucleus assay. Secondly, nanoparticles of the non-steroidal anti-inflammatory drugs (NSAIDs), aspirin and ibuprofen, have been investigated in patients with respiratory diseases, in the micronucleus assay and the Comet repair assay. NSAIDs inhibit cyclooxygenase enzyme activity, which plays part in tumour progression. In the Comet assay, BHA and catalase were able to reduce DNA damage in both cell types compared to HAAs alone. Similarly, in the micronucleus assay, micronuclei were reduced with the antioxidants, suggesting oxygen radical involvement in both assays. With the NSAIDs, reductions were seen for DNA damage in the micronucleus assay with aspirin and ibuprofen nanoparticles compared to their bulk forms. Using the Comet repair assay, aspirin and ibuprofen nanoparticles aided repair of DNA to a greater extent than their bulk counterparts, which in turn showed better repair compared to samples repaired without NSAIDs. These observations show the importance of DBPs and NSAIDs in genotoxic public health issues.

Die Induktion antioxidativer Enzyme gilt als eine Möglichkeit, die antioxidative Kapazität von Zellen zu steigern und dadurch mit oxidativem Stress assoziierten Erkrankungen (z. B. Herz-Kreislauf-Erkrankungen, Neurodegeneration, Atherosklerose) vorzubeugen. Ausgehend davon wurde in der vorliegenden Arbeit der Dual-Luciferase-Reportergen-(DLR)-Assay zum Nachweis der Induktion der antioxidativen Enzyme Katalase (CAT), zytosolische Glutathion-Peroxidase (GPX1) und Kupfer-Zink-Superoxid-Dismutase (SOD1) entwickelt. Im Zuge dessen wurden drei Säugetierzelllinien (CaCo2, IEC-18, V79) auf ihre Eignung zur Modellzelllinie untersucht. Aufgrund der Transfektionseffizienz wurde die Fibroblastenzelllinie V79 ausgewählt. Zur Gewährleistung eines hohen Substanzdurchsatzes des DLR-Assays wurden bei der Etablierung Parameter wie Kulturplattenformat, DNA-Menge, Luciferasen-Kinetik berücksichtigt. Nach erfolgreicher Etablierung des Versuchs im 96-Well-Format wurden L-Carnitin, Catechin, Epigallocatechingallat, Genistein, Wasserstoffperoxid (H2O2), Natrium-Ascorbat, Paraquat, Quercetin, 12-O-Tetradecanoylphorbol-13-Acetat (TPA) und Trolox in nicht-zytotoxischen Konzentrationen hinsichtlich der Aktivierung des Ratten-CAT-, des humanen GPX1- und des humanen SOD1-Promotors untersucht. Die Bestimmung der maximal tolerierbaren Behandlungskonzentration erfolgte im Vorfeld mittels Resazurintest. Von den zehn Verbindungen zeichneten sich drei Substanzen als potente Induktoren für die SOD1 und die GPX1 aus. Die 24-stündige Behandlung von mit Reportergenkonstrukten transient transfizierten V79-Zellen mit 100 µM Paraquat resultierte in einer Verdopplung der relativen SOD1-Promotor-Aktivität und einer Erhöhung der relativen GPX1-Promotor-Aktivität auf 1,6 bzw. 1,7. Die Stimulation mit 20 µM Genistein oder 10 µM Quercetin führte wiederum zu einer Verdopplung bis Verdreifachung der relativen SOD1- und GPX1-Promotor-Aktivität. Der Promotor der Rattenkatalase konnte demgegenüber nur durch 50 µM H2O2 aktiviert werden (1,5fach). Für diesen DLR-Assays bieten sich folglich Genistein, Quercetin wie auch H2O2 als Referenzsubstanzen an. Um aber eine qualitative Charakterisierung der einzelnen Verbindungen hinsichtlich ihres Induktionspotentials zu gewährleisten, sollten von allen getesteten Substanzen Dosis-Wirkungskurven aufgenommen werden. Zudem wird für den routinemäßigen Einsatz die Verwendung stabil transfizierter Zellen zur Vermeidung von mit der Transfektion verbundenen experimentellen Schwankungen empfohlen. / The induction of antioxidative enzymes might be an opportunity to elevate the cellular antioxidative capacity and, thus, to prevent oxidative stress associated diseases (e. g. cardio-vascular disease, neurodegenerative disease, atherosclerosis). Based on this idea the dual luciferase reporter gene (DLR) assay was developed to demonstrate the induction of three antioxidative enzymes: catalase (CAT), cytosolic glutathione peroxidase (GPX1), and copper-zinc superoxide dismutase (SOD1). In the course of the development three mammalian cell lines (CaCo2, IEC-18, V79) were tested for their ability to serve as a model cell line. The line V79 was chosen due to the transfection efficiency. To give consideration to a high-throughput several parameters were studied (e. g. format of the cultural plates, amount of DNA, kinetics of the luciferases) and the DLR assay was successfully established in 96 well plates. Subsequently, L-carnitine, catechin, epigallocatechin gallate, genistein, hydrogen peroxide (H2O2), sodium ascorbate, paraquat, quercetin, 12-O-tetradecanoylphorbol-13-acetate (TPA) and trolox were tested in non-cytotoxic concentrations for the activation of the rat CAT, human GPX1 and human SOD1 promoter. The maximally tolerable concentrations were determined by resazurin test in advance. Three out of these ten compounds were identified as potent inducers of GPX1 and SOD1. Stimulation of reporter gene construct transient transfected V79 cells for 24 hours with 100 µM paraquat caused a duplication of the relative GPX1 promoter activity and a 1.6-/1.7-fold increase of the relative SOD1 promoter activity. The incubation with 20 µM gen-istein or 10 µM quercetin resulted in duplication to triplication of both, the relative GPX1 and SOD1 promoter activity. In contrast, the rat CAT promoter was activated by 50 µM H2O2 (1.5-fold). Consequently, genistein, quercetin, and H2O2 are considered to be suitable reference substances for this DLR assay. To further characterize the inducing potential of the tested compounds all of them should be tested in different concentrations. Furthermore, for the routinely performed DLR assay it is recommended to use stably transfected cells to eliminate transfection caused variations.

The vitamin D metabolite 1,25(OH)2D3 has long been known to inhibit growth of prostate cancer cells and this has been attributed to a VDR-mediated pathway controlling target gene expression, resulting in cell cycle arrest, apoptosis and differentiation. New research has shown that another vitamin D metabolite, 24,25(OH)2D3, inhibits proliferation of prostate cancer cells as well, more specifically, cells of the line LNCaP. It is not clear exactly how 24,25(OH)2D3 exerts this cancer growth inhibition but it has been shown that it is to some extent regulated via G protein coupled signalling pathways. Catalase is a haem-containing redox enzyme found in the majority of animal cells, plant cells and aerobic microorganisms. This enzyme is very important because it prevents excessive accumulation of the strongly oxidizing agent H2O2 which otherwise can do damage to the cells. Because of this preventive effect of catalase, important cellular processes which generate H2O2 as by-product can proceed safely. Biochemical analysis of catalase has shown that it binds endogenously to 24,25(OH)2D3. The fact that 24,25(OH)2D3 has anti-proliferative effects on prostate cancer cells combined with the fact that it binds to catalase generates the hypothesis that this binding interferes with the essential task of catalase to keep the cell free from accumulation of destructive H2O2, and by means of this interference induces apoptosis. Finding out about the cancer growth inhibiting mechanism behind each vitamin D metabolite is important and may be a lead in the search for a new, better treatment of prostate cancer. The specific aim of this project was to study if and in what way 24,25(OH)2D3 affects the enzymatic activity of catalase in LNCaP cells and to do this with dose and time responses in focus. In this experiment LNCaP cells were incubated for 48 hours together with 24,25(OH)2D3 in five different concentrations, then a catalase assay was performed on the cells including fluorescence-mediated measuring of catalase activity in both treated and untreated cells. The analysis of the result values showed that regardless of dose or time, 24,25(OH)2D3 has no statistically significant effect on catalase activity in cells of the line LNCaP.

The vitamin D metabolite 1,25(OH)2D3 has long been known to inhibit growth of prostate cancer cells and this has been attributed to a VDR-mediated pathway controlling target gene expression, resulting in cell cycle arrest, apoptosis and differentiation. New research has shown that another vitamin D metabolite, 24,25(OH)2D3, inhibits proliferation of prostate cancer cells as well, more specifically, cells of the line LNCaP. It is not clear exactly how 24,25(OH)2D3 exerts this cancer growth inhibition but it has been shown that it is to some extent regulated via G protein coupled signalling pathways. Catalase is a haem-containing redox enzyme found in the majority of animal cells, plant cells and aerobic microorganisms. This enzyme is very important because it prevents excessive accumulation of the strongly oxidizing agent H2O2 which otherwise can do damage to the cells. Because of this preventive effect of catalase, important cellular processes which generate H2O2 as by-product can proceed safely. Biochemical analysis of catalase has shown that it binds endogenously to 24,25(OH)2D3. The fact that 24,25(OH)2D3 has anti-proliferative effects on prostate cancer cells combined with the fact that it binds to catalase generates the hypothesis that this binding interferes with the essential task of catalase to keep the cell free from accumulation of destructive H2O2, and by means of this interference induces apoptosis. Finding out about the cancer growth inhibiting mechanism behind each vitamin D metabolite is important and may be a lead in the search for a new, better treatment of prostate cancer. This is a follow-up to an earlier study, and the specific aim of this project was to find out if and in what way 24,25(OH)2D3 affects the enzymatic activity of catalase in LNCaP cells during long-term treatment (up to 48 hours). In this experiment LNCaP cells were incubated for 48 hours together with 24,25(OH)2D3 of the concentration 10-8 M, then a catalase assay was performed on the cells including fluorescence-mediated measuring of catalase activity in both treated and untreated cells. The analysis of the result values showed that despite of the rather high dose used, 24,25(OH)2D3 has no statistically significant effect on catalase activity in cells of the line LNCaP, regardless of time.

<p>The vitamin D metabolite 1,25(OH)2D3 has long been known to inhibit growth of prostate cancer cells and this has been attributed to a VDR-mediated pathway controlling target gene expression, resulting in cell cycle arrest, apoptosis and differentiation. New research has shown that another vitamin D metabolite, 24,25(OH)2D3, inhibits proliferation of prostate cancer cells as well, more specifically, cells of the line LNCaP. It is not clear exactly how 24,25(OH)2D3 exerts this cancer growth inhibition but it has been shown that it is to some extent regulated via G protein coupled signalling pathways. Catalase is a haem-containing redox enzyme found in the majority of animal cells, plant cells and aerobic microorganisms. This enzyme is very important because it prevents excessive accumulation of the strongly oxidizing agent H2O2 which otherwise can do damage to the cells. Because of this preventive effect of catalase, important cellular processes which generate H2O2 as by-product can proceed safely. Biochemical analysis of catalase has shown that it binds endogenously to 24,25(OH)2D3. The fact that 24,25(OH)2D3 has anti-proliferative effects on prostate cancer cells combined with the fact that it binds to catalase generates the hypothesis that this binding interferes with the essential task of catalase to keep the cell free from accumulation of destructive H2O2, and by means of this interference induces apoptosis. Finding out about the cancer growth inhibiting mechanism behind each vitamin D metabolite is important and may be a lead in the search for a new, better treatment of prostate cancer. The specific aim of this project was to study if and in what way 24,25(OH)2D3 affects the enzymatic activity of catalase in LNCaP cells and to do this with dose and time responses in focus. In this experiment LNCaP cells were incubated for 48 hours together with 24,25(OH)2D3 in five different concentrations, then a catalase assay was performed on the cells including fluorescence-mediated measuring of catalase activity in both treated and untreated cells. The analysis of the result values showed that regardless of dose or time, 24,25(OH)2D3 has no statistically significant effect on catalase activity in cells of the line LNCaP.</p>

<p>The vitamin D metabolite 1,25(OH)2D3 has long been known to inhibit growth of prostate cancer cells and this has been attributed to a VDR-mediated pathway controlling target gene expression, resulting in cell cycle arrest, apoptosis and differentiation. New research has shown that another vitamin D metabolite, 24,25(OH)2D3, inhibits proliferation of prostate cancer cells as well, more specifically, cells of the line LNCaP. It is not clear exactly how 24,25(OH)2D3 exerts this cancer growth inhibition but it has been shown that it is to some extent regulated via G protein coupled signalling pathways. Catalase is a haem-containing redox enzyme found in the majority of animal cells, plant cells and aerobic microorganisms. This enzyme is very important because it prevents excessive accumulation of the strongly oxidizing agent H2O2 which otherwise can do damage to the cells. Because of this preventive effect of catalase, important cellular processes which generate H2O2 as by-product can proceed safely. Biochemical analysis of catalase has shown that it binds endogenously to 24,25(OH)2D3. The fact that 24,25(OH)2D3 has anti-proliferative effects on prostate cancer cells combined with the fact that it binds to catalase generates the hypothesis that this binding interferes with the essential task of catalase to keep the cell free from accumulation of destructive H2O2, and by means of this interference induces apoptosis. Finding out about the cancer growth inhibiting mechanism behind each vitamin D metabolite is important and may be a lead in the search for a new, better treatment of prostate cancer. This is a follow-up to an earlier study, and the specific aim of this project was to find out if and in what way 24,25(OH)2D3 affects the enzymatic activity of catalase in LNCaP cells during long-term treatment (up to 48 hours). In this experiment LNCaP cells were incubated for 48 hours together with 24,25(OH)2D3 of the concentration 10-8 M, then a catalase assay was performed on the cells including fluorescence-mediated measuring of catalase activity in both treated and untreated cells. The analysis of the result values showed that despite of the rather high dose used, 24,25(OH)2D3 has no statistically significant effect on catalase activity in cells of the line LNCaP, regardless of time.</p>

The etiology of abdominal aortic aneurysms (AAA) is characterized by localized extracellular matrix remodeling and vessel dilation. Population-based studies have shown that AAA account for nearly 1% of all deaths. This thesis seeks to identify the earliest molecular and biomechanical determinants of aneurysm formation. Our initial motivator was the lack of information defining the underlying mechanisms of AAA formation. We used isolated vessel testing and histological analysis to study the mechanical and morphological evolution of AAA. These factors were measured in murine models of reproducible AAA formation. From this study, we determined 1) that molecular events precede mechanical events in AAA progression and 2) aortic circumferential mechanics are well conserved during AAA pathogenesis. Next we sought to explore the mechanistic link between oxidative stress and AAA development. To determine this relationship we used isolated vessel testing as well as measurement of aortic residual circumferential strain. To isolate the role of oxidative stress in these studies we used a line of transgenic mice with vascular smooth muscle cell-specific overexpression of the antioxidant catalase. The results of this study suggest that oxidative stress-mediated elastin degeneration within the aortic media is etiologic of altered aortic mechanics. Lastly, we sought to determine the independent mechanical contribution of the aortic adventitia and media tunica to overall aortic behavior. To accomplish this goal we compared the circumferential and axial mechanical behavior of aortas with and without collagenase treatment. The data demonstrated that the adventitia regulates the circumferential behavior of the aorta by preventing overstretch and the media regulates the axial behavior by maintaining tensile loading. This thesis demonstrates 1) that detecting early aneurysm progression in the form of mechanical or geometric changes may miss the window in which aneurysm pathology may be potentially reversed, 2) that mitigating oxidative stress within the aortic wall may provide protection against AAA, and 3) the adventitia is an important load bearing constituent of the arterial wall and plays a role in vascular adaptation to altered mechanical states. Overall our results impact understanding of early aneurysmal pathogenesis and may facilitate the development of preventative therapies for AAA progression and rupture.

Cyclic-AMP dependent protein kinase A (PKA) regulates traffic of multiple proteins at different stages along the constitutive secretory pathway. PKA effects are regulated by protein phosphatases, which reverse the actions of PKA by dephosphorylation of PKA-substrates. Localization of specific PKA effects is mediated by the binding of A-kinase anchoring proteins (AKAPs). Apolipoprotein E (apoE) is an important regulator of lipid metabolism and atherosclerosis, and represents a large proportion of total protein constitutively secreted from macrophages. The signalling and trafficking pathways regulating secretion of apoE are unknown. Catalase is a peroxisomal enzyme which contributes to defence against hydrogen peroxide (H2O2). The primary hypothesis of this thesis is PKA and related protein phosphatase pathways are involved in the regulation of apoE secretion. The secondary hypothesis is that these pathways also regulate cellular clearance of H2O2. In Chapter Three, I have investigated the role of PKA in apoE secretion from primary human macrophages. Structurally distinct inhibitors of PKA (H89, KT5720, inhibitory peptide PKI14-22) all decreased basal secretion of apoE by between 50-80% whereas apoE mRNA or cellular protein are unaffected. Disruption of PKA-AKAP anchoring also significantly inhibited apoE secretion from human macrophages. Secretion of apoE was not immediately stimulated by PKA activity, suggesting that although PKA activity may be permissive for apoE secretion, it is in itself insufficient to stimulate apoE secretion above basal levels. Data from confocal microscopy and live cell imaging revealed PKA inhibition paralysed apoE vesicular movement from and to the plasma membrane. In Chapter Four, I investigated the effects of protein phosphatase 2B (PP2B) inhibition on apoE secretion by cyclosporin A (CsA). This was found to dose- and time-dependently inhibit secretion of apoE from primary human macrophages and increased cellular accumulation of apoE without affecting apoE mRNA levels. The role of PP2B in regulating apoE secretion was confirmed by using additional peptide and chemical inhibitors of PP2B. This effect was independent of the known inhibition of ABCA1 by CsA. Live cell imaging and confocal microscopy all demonstrated that inhibition of PP2B did not affect the apparent cellular distribution of apoE. Biochemical and microscopy studies indicated distinct mechanisms for PKA and PP2B regulation of apoE secretion. Chapter Five identified PKA-anchoring AKAPs in human macrophages, and investigated AKAP220 expression and its role in PKA-dependent processes relevant to atherosclerosis. AKAP220 protein was absent in human monocytes but was detectable after their differentiation into macrophages, with stable expression during late stages of maturation. It was also present in Chinese Hamster Ovary cells (CHO) cells. AKAP220 silencing had no effects on lipoprotein cholesteryl ester accumulation, total cellular apoE levels, apoE secretion or cholesterol efflux from human macrophages. Confocal microscopy in CHO cells revealed peroxisomal localisation of AKAP220. Catalase activity was confirmed to be PKA-regulated process, and AKAP220 was found to be a negative regulator of catalase activity, such that cell lysate catalase activity increased during AKAP220 silencing. AKAP220 silencing also decreased basal secretion of H2O2, detected using a sensitive and specific Amplex?? Red assay kit from intact CHO monolayers. In conclusion, this thesis has provided evidence that apoE secretion occurs via PKA- and PP2B-dependent pathways in human macrophages, and has identified the A-kinase anchoring protein AKAP220 as a regulator of cellular H2O2 clearance. These results will provide a basis for future investigations into the roles of PKA-related pathways in apoE secretion and catalase activity.