The steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly stimulates the uptake of phosphate in isolated chick intestinal cells , while the steroid 24,25- dihydroxyvitamin D3 [24,25(OH)2D3] inhibits the rapid stimulation by l,25(OH)2D3. Earlier work in this laboratory has indicated that a cellular binding protein for the 24,25(OH)2D3 is the enzyme catalase. Since binding resulted in decreased catalase activity and increased H2O2 production, studies were undertaken to determine if pro-oxidant conditions mimicked the inhibitory actions of 24,25(OH)2D3, and anti-oxidant conditions prevented the inhibitory actions of 24,25(OH)2D3. An antibody against a putative 24,25(OH)2D3 binding protein was found to neutralize the inhibitory effect of the steroid on 1,25(OH)2D3-mediated 32P uptake (P2D3, each in Cells exposed to hormone alone again showed an increased accumulation of 32P from T=5-10 min, while cells treated with catalase inhibitor and hormone had uptake levels that were indistinguishable from controls. We tested whether inactivation of protein kinase C (PKC), the signaling pathway for 32P uptake, occurred. Incubation of cells with 100 nM phorbol-13-myristate (PMA) increased 32P uptake to 143% of controls, while cells pretreated with 50 μM H2O2 prior to PMA did not exhibit increased uptake. Likewise, PMA significantly increased PKC activity at T=1-3 min (P2O2 prior to PMA did not. It is concluded that catalase has a central role in mediating rapid responses to steroid hormones.

The application of Cerium oxide nanoparticles (CNPs) for therapeutic purposes requires a stable dispersion of nanoparticles in biological environment. The objective of this study is to tailor the properties of polyelectrolyte coated CNPs as a function of molecular weight to achieve a stable and catalytic active dispersion. This was achieved by coating CNPs with polyacrylic acid (PAA) which increased the dispersion stability of CNPs and enhanced the catalytic ability. The stability of PAA coating was analysed using the change in the Gibbs free energy computed by Langmuir adsorption model. The adsorption isotherms were determined using soft particle electrokinetics which overcomes the challenges presented by other techniques. The Gibbs free energy was highest for PAA coated CNPs by 250 kg/mole indicating the most stable coating. The free energy for PAA 100 kg/mole coated CNPs is 85% lower than the PAA250 coated CNPs. This significant difference is caused by the strong adsorption of PAA100 on CNPs. Catalytic activity of PAA-CNPs is accessed by the catalase enzymatic activity of nanoparticles. The catalase activity was higher for PAA coated CNPs as compared to bare CNPs which indicated preferential adsorption of hydrogen peroxide induced by coating. Apart from PAA coating the catalase activity is also affected by the structure of the coating layer.

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.

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.

FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O trabalho mostra o tratamento e os efeitos do l-deprenil (DEP), no aprendizado, na memÃria e na peroxidaÃÃo lipÃdica em cÃrebros de ratos submetidos à isquemia cerebral transitÃria (ICT). Os animais (ratos Wistar, fÃmeas, 200-250g) foram submetidos à isquemia cerebral transitÃria pela oclusÃo de ambas as artÃrias carÃtidas durante 20 minutos e tratados durante 5 dias com DEP (5 e 10 mg/kg). A temperatura retal foi monitorada e mantida em torno de 37ÂC atravÃs de uma luz incandescente. O mesmo procedimento foi feito no grupo controle + salina, Falso-operado + salina (FO) com exceÃÃo do clampeamento das artÃrias carÃtidas. No 6 dia apÃs a induÃÃo da isquemia, os animais foram submetidos aos testes de atividade locomotora e memÃria (esquiva passiva, labirinto em T elevado e labirinto aquÃtico de Morris), a seguir foram sacrificados e os cÃrebros dissecados sobre gelo (hipocampo e cÃrtex temporal) para as determinaÃÃes de MDA, nitrito/nitrato e atividade da catalase e atividade da protease caspase-3. No protocolo de avaliaÃÃo da Ãrea total do infarto encontramos apÃs 1 hora de ICT uma Ãrea de infarto 38,01  3,44% da Ãrea total do cÃrebro, e apÃs 24 horas de ICT uma Ãrea de infarto 22,00  2,90% da Ãrea total do cÃrebro. Os parÃmetros fisiolÃgicos estudados nÃo mostraram alteraÃÃes entre os grupos ICT e FO. Nenhuma alteraÃÃo na atividade locomotora foi detectada nos grupos FO, ICT, Dep 10 + ICT. PorÃm, um aumento na atividade locomotora foi observado no grupo Dep 5 + ICT (7,37  1, 77, p< 0,02) quando comparado com o grupo FO, tratado com salina, (4,66  1,54). No teste do Labirinto em T elevado (T Maze) a ICT afetou os processos de aquisiÃÃo e retenÃÃo de memÃria quando os animais foram testados no mesmo dia (esquiva 1 e 2) quando comparados com o grupo controle (FO). O teste de Kruskall-Wallis mostrou alteraÃÃo significativa na latÃncia da esquiva inibitÃria (esquiva 1 e 2 quando comparados com o treino) no falso-operado (FO - treino: 20,34  3,43 s; esquiva 1 - 231,6  34,81 s; esquiva 2 â 247,8  27,25 s; KW = 19,62, p< 0,001), e no grupo l-deprenil (5 e 10 mg/kg) + isquemia (Dep 5 â treino: 110,8  56,16 s; esquiva 1 - 299,8  0,16 s; esquiva 2 â 260  40,00 s; KW = 9,16, p< 0,01. Dep 10 â treino: 29,15  8,64 s; esquiva 1 â 299,80  0,25 s; esquiva 2 299,8  0,25 s; KW = 6,98, p< 0,05). Isto indica uma boa aquisiÃÃo de memÃria. Portanto, o resultado do grupo ICT + salina indicou um dÃficit da memÃria (ICT â treino: 37,75  11,52 s; esquiva 1 â 116,30  65,46 s; treino 2 â 195,00  64,10 s; KW = 3,90, p< 0,141). AlÃm disso, existiu uma diferenÃa significativa (Teste Mann-Whitney) entre os grupos na esquiva 3 (retenÃÃo) quando comparados com o grupo ICT (Dep 5, MW (3) = 18,483, p< 0,0003, Dep 10, MW (3) = 18,483, p< 0,003) significando que a retenÃÃo da memÃria foi aumentada pelo tratamento com a droga. No teste da esquiva passiva os animais do grupo controle (FO + salina) apresentaram uma boa retenÃÃo da memÃria, tanto na fase imediata (memÃria recente - MR), quanto na fase de consolidaÃÃo (memÃria tardia - MT), quando comparadas ao treino (ANOVA) (FO + salina (n-7)- treino - 15,94  4,40 s, MR - 138,84  34,60 s, MT - 196,32  34, 71, p< 0,006). Por outro lado, os animais que sofreram ICT nÃo apresentaram diferenÃa no tempo de latÃncia de entrada no lado escuro quando comparado com o treino, significando um dÃficit na aprendizagem e memÃria (ICT (n-7)- treino - 34,37  10,16 s, MR - 105,54  35,21 s, MT - 96,20  33, 44, p< 0,33), e, portanto dano na aquisiÃÃo e retenÃÃo da memÃria. Comparando os tratamentos observamos um aumento significativo, no tempo de latÃncia de entrada no lado escuro do aparelho, nos ratos tratados com l-deprenil 5 mg/kg quando avaliados na MR (FO + salina- 138,84  34,60s; ICT - 105,54  35,21; ICT + Dep 5- 198,88  38,42s; ICT + Dep 10- 188,06  34,60s; Kruskall-Wallis, KW-9,66, p<0,05, Mann-Whitney, Dep 5 vs ICT, p<0,05), enquanto na MT foi observada uma diminuiÃÃo significativa, no tempo de latÃncia de entrada no lado escuro do aparelho, nos ratos tratados com l-deprenil (5 e 10 mg/kg) (FO + salina- 196,32  34,71s; ICT - 96,20  33,44s; ICT + Dep 5- 299,83  0,16s; ICT + Dep 10- 264,70  35,28 s; Kruskall-Wallis, KW-14,57, p<0,05, Mann-Whitney, Dep 5 e Dep 10 vs ICT, p<0,05), significando melhora no aprendizado do animal fazendo-o lembrar o choque recebido durante o treino e indicando uma reversÃo da lesÃo sofrida com a ICT. No teste do Labirinto AquÃtico (Water Maze) a ICT promoveu um dano da retenÃÃo na memÃria dos animais em relaÃÃo ao grupo controle (FO), porÃm o l-deprenil conseguiu reverter o dano na aquisiÃÃo da memÃria induzida pela ICT em ambas as doses (5 e 10 mg/kg), observamos tambÃm que o grupo Dep 5 obteve um melhor desempenho na aquisiÃÃo da memÃria quando comparado com o grupo Dep 10. (FO (n-10): 5,4  0,84s; FO + DEP 10 (n-10): 9,7  2,28s; ICT (n-9): 32,44  2,95s; ICT + DEP 5 (n-8): 12,88  1,4s; ICT + DEP 10 (n-8): 4,5  0,70s; Kruskall-Wallis, KW-29,07, p<0,05, Mann-Whitney, FO + DEP 10, Dep 5 e Dep 10 vs ICT, p<0,05). Os ratos submetidos a ICT mostraram um aumento de 71% nos nÃveis de MDA no hipocampo quando comparados com o grupo controle (FO), e o tratamento com l-deprenil reverteu significativamente este efeito (p<0,05). Os valores dos nÃveis de MDA foram trazidos prÃximos aqueles valores do grupo controle (FO) em relaÃÃo aos grupos (ICT + DEP 5 e ICT + DEP 10, 34 e 38%, respectivamente) com ambas as doses de l-deprenil mais ICT (Hipocampo - FO (n-7): 45,4  4,45; ICT (n-7): 77,6  8,97; ICT + DEP 5 (n-7): 51,2  1,68; ICT + DEP 10 (n-7): 48,5  6,70 nmoles/g; p<0,05, ANOVA e Teste de Tukey). No cÃrtex temporal, a ICT nÃo aumentou os nÃveis de MDA quando comparados com o grupo controle. Portanto, os ratos submetidos a ICT e tratados com altas doses de l-deprenil (10 mg/kg) apresentaram nÃveis de MDA 30% menor que aqueles mostrados por ambos os grupos FO e ICT (CÃrtex temporal - FO (n-7): 46,8  4,36; ICT (n-7): 48,7  1,33; ICT + DEP 5 (n-7): 52,5  3,74; ICT + DEP 10 (n-7): 33,4  2,98 nmoles/g; p<0,05, ANOVA e Teste de Tukey). No hipocampo, os nÃveis de nitrito foram significativamente aumentados apÃs a ICT quando comparados com o grupo controle FO (82% aumento). O DEP 10 reverteu este efeito e os valores foram trazidos para aqueles do controle. Por outro lado, a isquemia nÃo afetou os nÃveis de nitrito no cÃrtex, entretanto o DEP 5 diminui significativamente os nÃveis de nitrito quando comparados com os grupos controle e ICT. A ICT mostrou um aumento em 50 % da atividade da protease caspase-3 no hipocampo; e o tratamento com l-deprenil (10 mg/kg) reverteu este efeito trazendo os valores prÃximos aos do grupo controle (FO), porÃm o tratamento com DEP 5 nÃo mostrou o mesmo (Valor da AbsorbÃncia: FO â 0,083  0,006; ICT - 0,124  0,017; ICT + DEP 10 â 0,080  0,007; ICT + DEP 5 â 0,125  0,007), porÃm nos animais controle que receberam tratamento com DEP 10 (FO + DEP 10) a atividade da caspase â 3 diminui em 99% em relaÃÃo ao grupo ICT. Em conclusÃo mostramos que a administraÃÃo do l-deprenil diariamente por 5 dias melhorou os danos da memÃria observados apÃs a isquemia cerebral transitÃria em ratos. A droga protegeu o cÃrebro contra a hiperperoxidaÃÃo e formaÃÃo de radicais livres observados apÃs o dano isquÃmico, como diminui a atividade da caspase â 3. Pelo menos parte desses efeitos à devido ao efeito antioxidante e conseqÃentemente inibiÃÃo da ativaÃÃo da produÃÃo de radicais livres pelo l-deprenil. / The present work shows the effects of l-deprenyl (DEP, 5 and 10 mg/kg, po) on memory, as well as on rat brain free radical formation after transient cerebral ischemia (TCI). Wistar rats were anesthetized and submitted to TCI by occlusion of both carotid arteries for 20 minutes. In another experiment, animals were submitted to surgery without ischemia (sham-operated). After surgery, ischaemic rats were treated with DEP (DEP, 5 and 10 mg/kg, po) once and daily for 5 days. One group of animals was left untreated (controls). The parameters studied were, memory acquisition and memory retention, locomotor activity and tiobarbituric acid reactive substances, as an index of lipid peroxidation. After treatment all, animals were submitted to passive avoidance test, water maze test and elevated T maze test, and 24 h later sacrificed and their hippocampi and temporal cortex dissected for evaluation of lipid peroxidation and used for catalase activity determinations. The protein concentration was measured according to the method described by Lowry (1951). In another set of experiments the animals were sacrificied forty eight hours after ischemia for caspase activity evaluation. Results show that DEP significantly reversed ischaemia-induced memory deficits. l-Deprenyl treatment significantly improved memory deficits as compared to ischemic group as measured by The elevated T maze and Water maze tests. A similar result was observed on the passive avoidance test where l-deprenyl improved late but not early memory as compared to the ischemic group. Except for an increased locomotor activity observed in the group treated with 5 mg/kg, no other alteration was detected in this behavioral test. Rats submitted to transient cerebral ischemia (and without l-deprenyl) showed an increase im MDA levels in the hippocampus and the treatment with l-deprenyl (5 and 10 mg/kg) significantly reversed this effect bringing values close to those of the sham-operated controls. A similar profile was observed with nitrite levels. Rats submitted to transient cerebral ischemia show an increase in caspase activity in the hippocampus and the treatment with l-deprenil (10 mg/kg) significantly reversed this effect bringing values close to those of the sham-operated controls. Moreover catalase activity in the hippocampi was not altered by ischemia. In conclusion, the work showed a signifant protective effect of l-deprenyl on memory deficits and lipid hyperperoxidation observed after cerebral ischemia. Possibly, the drug is acting at least in part through its antioxidant and antiapoptotic activities.

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.