Cushing's syndrome and glucocorticoid therapy lead to central obesity, insulin resistance, and symptoms of altered energy regulation similar to those observed in the metabolic syndrome. We hypothesized that excess glucocorticoids alter energy sensing/signaling in skeletal muscle through mediation of the LKB1/AMPK signaling pathway. To test this hypothesis, three 100 mg pellets of corticosterone were implanted subcutaneously in each of nine rats for two weeks. Responses were compared with sham operated controls fed ad libitum or food restricted to produce the body weights similar to the treatment group rats. After the treatment period, animals were anesthetized and the right gastrocnemius-plantaris and soleus were removed for analysis. After tibial nerve stimulation for 5 min, the left gastrocnemius-plantaris and soleus were also removed. We assessed AMPK activity and subunit expression, as well as several metabolic indicators including ATP, creatine phosphate, creatine, glycogen, and malonyl-CoA levels in rested and stimulated gastrocnemius-plantaris and soleus muscles. We found that high levels of glucocorticoids decreased AMPKγ3 subunit expression in the gastrocnemius-plantaris. We also observed reduced AMPKα2 activity in the stimulated gastrocnemius-plantaris, but not the soleus; and that this decreased activity corresponded to a significant reduction in phosphorylated TBC1D1, a protein involved in signaling GLUT-4 translocation. Finally, in the gastrocnemius-plantaris, we also noted an increase in glycogen stores in the hypercorticosteronemic rats. Our data suggest that altered energy sensing/signaling associated with high levels of glucocorticoids may be due in part to inhibition of AMPKα2 activity and the high energy state produced by increased glycogen stores. We also conclude that high levels of glucocorticoids decrease the levels of AMPKγ3 and diminish insulin/contraction signaling through phosphorylated TBC1D1.

Although enhanced biological phosphorus removal (EBPR) and complete biological nutrient removal (BNR) systems can be operated successfully by experienced operators, the accuracy of design and strength of the scientific background need to be reinforced to enable accurate modeling and economically optimal design. One way to accomplish this would be through a better understanding of the biochemical mechanisms and microbial population dynamics that determine the reliability and efficiency of EBPR, and the utilization of this information to improve the design and operation of BNR plants. Such knowledge will also contribute to better structure of modeling tools that are used for design and educational purposes. The current body of knowledge is limited to observational studies that lack detailed biochemical explanations backed with a series of well planned experiments, and this has introduced uncertainties and inaccuracies into the biochemical and design models. Therefore, this study mainly covers a biochemical survey of the underlying metabolisms of active populations in BNR sludges. BNR biomass with biological phosphorus removal (BPR) capability was cultivated in continuous flow reactor (CFR) systems, configured as either University of Cape Town (UCT) and anoxic/oxic (A/O) systems. Following an acclimation period at 20°C, low temperature stress (5°C) was imposed on one UCT system for investigation of the response of the microbial consortium responsible from EBPR activity under cold temperature. Once a stable population with EBPR capabilities is established in each system, activities of ten enzymes that are hypothesized to be taking part in the EBPR metabolism were measured. These enzymes were selected among those that take part in major known pathways of bacterial energy and growth metabolism. Also, 13C-NMR was used as a tool to monitor the flux of labeled carbon in and out of pools of cellular storage; i.e. glycogen and polyhydroxyalkanoates (PHA). Combining the gathered information, accurate mass balances of carbons and reducing equivalents were calculated, eventually leading to determination of the biochemical pathways utilized by the EBPR consortium. Additionally, anaerobic stabilization of COD, a long debated but empirically established phenomenon, was addressed during the study. Considering the pathways proposed to be operative under different conditions imposed on the EBPR systems, a biochemical explanation for the occurrence of COD stabilization in wastewater treatment systems that incorporate anaerobic zones was proposed. Accordingly, depending on the pathways actively used by a microbial consortium, electrons stored in NADH and FADH2 can either be transferred to the terminal electron acceptor, oxygen, or they can be incorporated into storage polymers such as glycogen for future use. Such differences in metabolism reflect in the quantity of the oxygen consumed in the aerobic reactors. Thus, the correct incorporation of anaerobic stabilization of COD into process design would reduce design aeration requirements and result in economic savings during both construction and operation. / Ph. D.

Development of multicellular organisms is one of the most fundamental but least understood biological processes. Due to its simple life cycle, the lower eukaryote Dictyostelium has been used as a model system to study several basic biological problems, such as cell differentiation, cell motility, cell adhesion, signal transduction, and especially gene regulation. Glycogen phosphorylase is the enzyme that initiates one of the key biochemical pathways, glycogen degradation, during Dictyostelium discoideum development. Two forms of glycogen phosphorylase, gpl and gp2, exist in D. discoideum with gp1 being active in vegetative cells and gp2 in differentiating cells. Study of glycogen phosphorylase gene regulation clearly will provide insight into the molecular mechanism of D. discoideum development and facilitate understanding of development in general. Two distinct genes that encode the two forms of glycogen phosphorylase were cloned. The nucleotide sequence analysis of the gp2 gene revealed an open reading frame of 2976 bp, that consists of three exons separated by two introns. An interesting feature in the gene is a 45 bp sequence in the second exon that contains 11 CAA trinucleotide repeats. The entire 5' and 3' non-coding regions of the gp2 gene and the whole 5' noncoding region of the gp1 gene have also been cloned. The regulation of the gp2 gene by Dictyostelium developmental signals was studied. Both cyclic AMP (cAMP) and Differentiation Inducing Factor (DIF) were discovered to induce gp2 gene expression during differentiation. DIF was also found to inhibit the cAMP responsiveness of the gene. Both cAMP and DIF induction of the gene were repressed by NH₃. Another developmental signalling molecule, adenosine, was involved in gp2 gene regulation through the inhibition of the DIF-mediated expression. The cell-type-specificity of the gp2 gene were also investigated. The gene was found to be expressed in both prestalk/stalk and prespore/spore cells. This is in agreement with the cAMP and DIF inducibility of the gene since the former molecule is a spore-cell morphogen, while the latter is a stalk-cell morphogen. A model of gp2 gene regulation during development is proposed, based on these findings. The two gp? introns and the 45 bp CAA repeat were studied by deletion of these elements. However, there were no alterations of gp2 gene expression observed after these deletions. Also investigated was genomic structural alteration in gp1- mutants that were obtained through homologous recombination and antisense RNA. Southern analysis revealed that the normal gp1 gene was disrupted in all homologous recombination transformants and in half of the antisense RNA transformants. Finally, for the first time, an extrachromosomal luciferase reporter vector has been established for the study of cis-acting regulatory elements in D. discoideum. / Ph. D.

Inledning: Uthållighetsträning definieras som den typ av träning där flertalet stora muskelgrupper används och är i behov av hjärt-kärlsystemets kapacitet för att transportera syre till musklerna. Att fasta innebär i de flesta studier att ingen typ av föda intas före träningspassen medan att inte fasta vanligtvis innebär att 90 minuter före ett träningspass intas en kolhydratrik måltid. Insulin är viktig i fett-, protein- och kolhydratmetabolismen samt i uppsamlingen av socker i blodet. Glykogen fungerar som en energireserv i skelettmusklerna. Fria fettsyror är viktiga för att ge energi till den cellulära metabolismen som ska kunna fortgå. Syfte: Syftet med denna studie var att ta reda på om kroppen påverkas på olika sätt av uthållighetsträning på fastande respektive icke-fastande mage vad gäller insulin, muskelglykogen samt fria fettsyror hos människor. Metod: Litteratursökning i OneSearch med sökorden training* AND fasted state där sex artiklar valdes ut. Cohen’s D användes för att ge studiernas resultat en effektstorlek. Konfidensintervall användes för att bestämma om studierna var statistiskt signifikanta. Resultat: Tre studier undersökte insulin där resultaten visade att samtliga grupper som inte fastade före träningspassen hade lägst koncentration av insulin i viloperioden efter eftertestet. Samtliga sex studier undersökte muskelglykogen där resultaten visade att koncentrationen av muskelglykogen var högst efter eftertestet i gruppen som fastade före träningspassen i fem av sex studier. Tre studier undersökte fria fettsyror där resultaten visade att koncentrationen av fria fettsyror var lägst i eftertestet i gruppen som fastade före träningspassen i två av tre studier. Diskussion: Träning i icke-fastat tillstånd leder till att glykogennivåerna sparas vilket i sin tur leder till att oxidationen av insulin ökar vilket resulterar i lägre koncentrationer av insulin efter eftertestet. Träning i fastat tillstånd resulterar i ökad koncentration av muskelglykogen till energiunderhåll samt högre nivåer av β-hydroxyacyl coenzym A dehydrogenas (β-HAD), citratsyntasaktiviteten (CS-aktiviteten) samt succinatdehydrogenasaktiviteten vilket leder till ökad muskulär oxidativ kapacitet. Intag av kolhydrater före ett träningspass leder till ökat glykogenbesparande vilket resulterar i lägre koncentrationer av muskelglykogen jämfört med icke-fastat tillstånd. Högre koncentration av muskelglykogen före förtestet leder till högre koncentrationer av muskelglykogen i eftertestet. Träning i fastat tillstånd resulterar i lägre koncentrationer av fria fettsyror på grund av ökad FATmax (den maximala hastigheten av fettförbränning), ökad maximal oxidativ enzymaktivitet, uppreglering av hormonkänsligt lipas (HSL), ökat proteinuttryck av fettsyra-translokas / CD36 (FAT / CD36) samt ökat membranbundet proteinbindande protein som leder till ökad fettförbränning. Koncentrationen av fria fettsyror höjdes även i en studie på grund av att insulinutsöndringen inte skulle sjunka för lågt i gruppen som fastade före träningspassen. Konklusion: Resultaten visade att i tre av tre studier var insulinkoncentrationen lägst efter eftertestet i gruppen som intog en kolhydratrik måltid före träningspassen. I fem av sex studier var koncentrationen av muskelglykogen högst efter eftertestet i gruppen som fastade före träningspassen. I två av tre studier var koncentrationen av fria fettsyror lägst efter eftertestet i gruppen som fastade före träningspassen. Dessa resultat ökar förståelsen av hur träning på fastande respektive icke-fastande mage påverkar kroppen med avseende på insulin, muskelglykogen samt fria fettsyror. Vidare forskning skulle kunna undersöka dessa tre fysiologiska markörer under sex månader för att se om resultaten blir desamma i dessa två grupper. / Introduction: Endurance training is defined as the type of exercise where most major muscle groups are used in need of the cardiovascular system’s ability to transport oxygen to the muscles. Fasting means in most studies that no type of food is taken before the workout while not fasting usually means that a carbohydrate meal is taken 90 minutes before a workout. Insulin is important in fat, protein and carbohydrate metabolism as well as in the collection of sugar in the blood. Glycogen acts as an energy reserve in the skeletal muscles. Free fatty acids are important to provide energy for the cellular metabolism that can continue. Aim of the study: The aim of the study was to find out if the body is affected in different ways by endurance training exerted in fasted respectively non-fasting state regarding insulin, muscle glycogen and free fatty acids in humans. Methods: Literature search in OneSearch with the words training* AND fasted state where six articles were selected. Cohen’s D was used to give the results of the studies an effect size. Confidence interval was used to determine if the studies were statistically significant. Results: Three studies investigated insulin, where the results showed that all groups that did not fast before the workouts had the lowest concentration of insulin during the rest period after the test. All six studies investigated muscle glycogen, where the results showed that the concentration of muscle glycogen was highest after post-test in the group that fasted before the workouts in five out of six studies. Three studies investigated free fatty acids where the results showed that the concentration of free fatty acids were lowest in the post-test in the group that fasted before the workouts in two of three studies. Discussion: Training in the non-fasting state causes the glycogen levels to be saved, which in turn leads to increased oxidation of insulin resulting in lower concentrations of insulin after the post-test. Training in the fasted state results in increased concentration of muscle glycogen for energy maintenance and higher levels of β-hydroxyacyl coenzyme A dehydrogenase (β-HAD), citrate synthase activity (CS activity) and succinate dehydrogenase activity, leading to increased muscular oxidative capacity. Intake of carbohydrates prior to a workout leads to increased glycogen saving, resulting in lower concentrations of muscle glycogen compared to non-fasted state. Higher concentration of muscle glycogen before the pretest leads to higher concentrations of muscle glycogen in the post-test. Training in fasted state results in lower concentrations of free fatty acids due to increased FATmax (maximum rate of fat burning), increased maximal oxidative enzyme activity, hormone sensitive lipase (HSL) upset, increased fatty acid translocase / CD36 protein expression (FAT / CD36) as well as increased membrane bound protein binding protein which leads to increased fat burning. The concentration of free fatty acids was also increased in a study due to the fact that insulin secretion should not fall too low in the group that fasted before the workouts. Conclusion: The results showed that in three out of three studies, the insulin concentration was lowest after the post-test in the group who took a carbohydrate meal before the workouts. In five out of six studies, the concentration of muscle glycogen was highest after the post-test in the group that fasted before the workouts. In two out of three studies, the concentration of free fatty acids was lowest after the post-test in the group that fasted before the workouts. These results increase the understanding of how exercise in the fasted state and non-fasted state affects the body with regard to insulin, muscle glycogen and free fatty acids. Further research could investigate these three physiological markers for six months to see if the results are the same in these two groups.

A Doença de Alzheimer (DA) é uma desordem neurodegenerativa progressiva que causa comprometimento cognitivo e demência. O diagnóstico é baseado em parâmetros clínicos, mas sua confirmação é post-mortem, após avaliação patológica durante a autópsia. Os tratamentos disponíveis para a DA são os inibidores da colinesterase (IChEs) e os antagonistas de receptores de N-metil-D-aspartato (NMDA), sendo que os IChEs compõe o principal grupo. Diversos estudos tem mostrado um efeito neuroprotetor dos IChEs, levando a alterações na patogênese da DA. Avaliar e mensurar essas alterações são papeis atribuídos aos biomarcadores. Neste sentido podemos destacar a fosfolipase A2 (PLA2), a principal responsável pelo metabolismo de fosfolípides de membrana, e que tem sido achada diminuída na DA, assim como a glicogênio sintase-quinase (GSK), responsável pela fosforilação da proteína Tau, que é um dos processos alterados na DA. O objetivo deste trabalho foi avaliar o efeito do tratamento com IChE sobre a atividade da PLA2 e expressão da GSK3B em plaquetas de 30 pacientes com DA após 3 e 6 meses de tratamento. Como grupo controle foram investigados 42 individuos idosos sem doença neurodegenerativa. Encontramos nos pacientes com DA antes do tratamento uma diminuição da atividade da iPLA2 quando comparada ao grupo controle. Após três e seis meses de tratamento a PLA2 aumentou, voltando ao nível dos controles. Os pacientes que apresentaram um aumento maior da iPLA2 apos 3 meses de tratamento apresentaram melhora cognitiva mais marcante após seis meses de tratamento, avaliado pelo CAMCOG. Apos 6 meses de tratamento encontramos um inativação da GSK3B, medida por um aumento em sua forma fosforilada. Nossos resultados sugerem que o donepezil apresenta propriedades modificadoras na doença de Alzheimer, e ainda que a medida da atividade da iPLA2 poderia ser usada como marcador de resposta terapêutica ao donepezil e, possivelmente, a outros IChEs, na doença de Alzheimer / Alzheimer\'s disease (AD) is a progressive neurodegenerative disorder that causes dementia and cognitive impairment. The Diagnosis is based on clinical parameters, but confirmation is post-mortem after pathologic evaluation during autopsy. The treatments available for AD are cholinesterase inhibitors (IChEs) and N-methyl-D-aspartate (NMDA) antagonists. The main group comprises the IChEs. Several studies have shown a neuroprotective effect of IChEs, leading to alterations in the pathogenesis of AD. Evaluate and measure these changes are assigned to biomarkers. In this regard we can highlight the phospholipase A2 (PLA2) the main enzyme in membrane phospholipids metabolism and that has been found decreased in AD as well as Glycogen Synthase kinase (GSK), a major responsible for tau phosphorylation which is one processes altered in AD. The objective of this study was to evaluate the effect of treatment with IChE on PLA2 activity and GSK3B expression in platelet of 30 AD patients after 3 and 6 months of treatment. The control group comprised 42 elderly individuals without neurodegenerative disease The results obtained were a decreased iPLA2 activity in patients with AD before treatment as compared to controls. After 3 and 6 months of treatment, we observed a significant increase in iPLA2 activity, restoring enzymatic activity similar to that observed among control. The patients who showed higher iPLA2 activity in the first three months were those showing cognitive improvement after six months of treatment, measured by CAMCOG. After 6 months of treatment a GSK3B inactivation were found, measured by an increase in its phosphorylated form. Our results suggest that donepezil present modifying properties in Alzheimer disease and that iPLA2 activity measurement could be used as a marker of therapeutic response to donepezil and possibly other IChEs in Alzheimer\'s disease

A Doença de Alzheimer (DA) é um transtorno progressivo que acomete o Sistema Nervoso Central, causando demência em idosos. A doença leva a uma diminuição da memória, dificuldade no raciocínio e pensamento e alterações comportamentais. A fisiopatologia da doença corresponde ao aumento na concentração do peptídeo -amilóide com consequente deposição e formação da placa amiloide; e também ao aparecimento dos emaranhados neurofibrilares, que são agregados de proteína tau hiperfosforilada. A enzima glicogênio sintase cinase 3 beta (GSK-3) está diretamente envolvida nos dois processos e, por isso, a busca por novos inibidores dessa enzima é uma importante estratégia terapêutica para o tratamento da doença. Neste trabalho utilizou-se a triagem virtual em 7 bancos de dados de moléculas aplicando cinco diferentes estratégias in silico, através de planejamento de fármacos baseado em ligantes e baseado em estrutura, combinada com estudos in silico de farmacocinética, toxicidade e atividade biológica, seguido de posteriores ensaios de inibição enzimática in vitro. Obteve-se três compostos pela metodologia de farmacóforo, (Estratégia 3) dos quais dois deles demonstraram atividade inibitória interessante para GSK-3, na faixa de micromolar. A partir das outras quatro estratégias foram selecionados 16 compostos que futuramente serão também testados utilizando o mesmo protocolo de ensaio in vitro aqui utilizado. / Alzheimer\'s disease (AD) is a progressive disorder that affects the Central Nervous System, causing dementia in the elderly. The disease leads to decreased memory, difficulty in reasoning and thinking, and behavioral changes. The pathophysiology of the disease corresponds to the increase in -amyloid peptide concentration with consequent deposition and formation of the amyloid plaque and to the appearance of neurofibrillary tangles, which are aggregates of hyperphosphorylated tau protein. The enzyme glycogen synthase kinase-3 beta (GSK-3) is directly involved in both processes and, therefore, the search for new inhibitors of this enzyme is an important therapeutic strategy for the treatment of the disease. In this work, we used virtual screening in 7 molecule databases applying five different in silico strategies, using the ligand-based and structure-based drug design methodologies, combined with in silico studies of pharmacokinetics, toxicity and biological activity, followed by subsequent assays enzymatic inhibition in vitro. We obtained three compounds by the pharmacophore methodology (Strategy 3) of which two of them demonstrated interesting inhibitory activity for GSK-3 in the micromolar range. From the other four strategies, 16 compounds were selected which in future will also be tested using the same in vitro assay protocol used here.

A doença de Alzheimer (DA) é uma doença neurodegenerativa caracterizada pelo declínio progressivo da memória e de outras funções cognitivas, acometendo, sobretudo, indivíduos idosos. A anormalidade do metabolismo da proteína precursora do amilóide (APP) e a hiperfosforilação da proteína TAU são processos celulares característicos desta doença. A enzima glicogênio sintase quinase 3B (GSK3B) é altamente expressa no sistema nervoso central e apresenta grande importância na regulação da plasticidade neuronal e também nos mecanismos de sobrevivência celular. Estudos têm associado a GSK3B aos mecanismos que levam à formação das placas senis e dos emaranhados neurofibrilares na DA. Diante da dificuldade para se estabelecer com precisão o diagnóstico clínico da DA, sobretudo nas fases iniciais da doença, a identificação de biomarcadores se torna particularmente importante, principalmente em tecidos periféricos. Este trabalho avaliou, por meio de dois preparos distintos, dois possíveis candidatos a marcadores bioquímicos para a DA. Em plaquetas de pacientes com DA, comprometimento cognitivo leve (CCL) e idosos saudáveis, determinamos: (1) a razão de APP (APPr), que consiste na proporção entre fragmentos de 130kDa e 110kDa da APP; e (2) a expressão das formas fosforilada (fosfo-GSK3B) e total (total-GSK3B) da enzima GSK3B, possibilitando o cálculo da razão de GSK3B (fosfo-GSK3B/total-GSK3B). Ambas as razões foram avaliadas por Western Blot utilizando anticorpos específicos. Não observamos diferença estatisticamente significante nos valores de APPr entre os três grupos estudados (p=0,847). Para o cálculo da razão de GSK3B, foram necessárias adaptações do protocolo de preparo e análise de plaquetas, prevenindo a ativação plaquetária durante o procedimento, bem como a degradação do substrato pela ação de proteases e fosfatases presentes nesta matriz biológica; deste modo foram encontradas diferenças estatisticamente significantes entre os grupos nas médias de GSK3B total e da razão de GSK3B (p=0,05 e p=0,06 respectivamente). Não foi encontrada correlação entre a razão de APP e a razão de GSK3B. Contudo, a razão de GSK3B mostrou correlação com o desempenho em testes de memória, segundo o escore na bateria cognitiva CAMCOG. Estes resultados sugerem que a razão de GSK3B em plaquetas sinaliza algumas alterações biológicas que ocorrem na progressão do CCL para a demência na DA / Alzheimer\'s disease (AD) is a neurodegenerative disease characterized by progressive decline of memory and other cognitive functions, affecting mainly elderly. The abnormal metabolism of amyloid precursor protein (APP) and protein TAU hyperphosphorylation are cellular hallmarks of this disease. Glycogen synthase kinase 3B (GSK3B) is an enzyme highly expressed in the central nervous system and of great importance in the regulation of neuronal plasticity and also the mechanisms of cell survival. Studies have associated GSK3B with the mechanisms that lead to the formation of senile plaques and neurofibrillary tangles in AD. Given the difficulty to establish the precise clinical diagnosis of AD, especially in the early stages of the disease, identification of biomarkers is particularly important, especially in peripheral tissues. This work evaluated two candidate biochemical markers for AD, using two different preparations. We investigated the ratio between 130kDa and 110kDa APP fragments (APPr) and between phosphorylated and total GSK3B (phospho-GSK3B / total-GSK3B) in platelets of patients with AD and mild cognitive impairment (MCI), comparing their results with those from healthy older adults (controls). The expression of APP fragments and GSK3B was assessed by Western blot using specific antibodies. No statistically significant differences in APPr were found between the study groups (p=0.847). We found statistically significant differences in mean total GSK3B and GSK3B ratio across disgnostic groups (p=0.05 and p=0.06, respectively). APPr and GSK3B ratio were not correlated, but the latter parameter did correlate with the performance on memory tests, as shown by the CAMCOG sub-score. The present data indicate that platelet GSK3B ratio may indicate biological changes that occur in the MCI-AD continuum

For the development of a G6PD-deficient mouse model, we introduced the mutant Gpdxa-m1Neu allele (a severe ENU-induced mutation that results in 13-15% G6PD activities of wild type littermates) into the C57L/J background (a strain that constitutively exhibits low G6PD activity) through a breeding program. Of significance is that 78% of the F2 generation had G6PD activities <2 U/g Hb, levels similar to those of severe G6PD deficiency in human. The efficacy of this model was preliminary verified by the known haemolytic agent, naphthalene, as demonstrated by the decrease of GSH/GSSG ratio by 24.6% (P=0.032) and increase of methaemoglobin by 4.5 fold (P=0.8) when compared with the respective control without treatment. / Genetic analysis of 14 mutation hotpots was performed on 98 hemi-/homozygous and 17 heterozygous G6PD-deficient human subjects. We developed a novel Multiplex Primer Extension Reaction (MPER) assay and detected seven specific mutations in 97 subjects: c.1376G>T (33.7%), c.1388G>A (29.6%), c.871G>A + c.1311C>T (12.3%), c.95A>G (9.2%), c.392G>T (7.1%), c.1024C>T (6.2%) and c.1360C>T (1.0%). For the genotyping of 15 heterozygous female, all mutations were identified as follows: c.1376G>T/Normal (33.3%), c.1388G>A/Normal (26.7%), c.871G>A/Normal + c.1311C>T/Normal (20.0%), c.95A>G/Normal (13.3%) and c.392G>T./Normal (6.7%). The c.871G>A and 'silent' mutation c.1311 C>T was newly found to coexist in a high proportion of genotype in our population. / Glucose-6-phosphate dehydrogenase (G6PD)-deficient subjects are vulnerable to chemical-induced haemolysis if exposed to oxidative agents. Little is known, however, of the haemolytic effects of Chinese herbal medicine on G6PD-deficient subjects. Only one case study has reported that a G6PD-deficient newborn developed severe haemolysis after ingestion of Rhizoma Coptidis. Besides, recent studies reported that green tea and its constituents exerted pro-oxidative effects on cellular systems in culture. / Glucose-6-phosphate dehydrogenase deficiency is a genetic disorder inherited in the X-linked manner. The condition is prevalent in the Mediterranean region, Africa and Southeast Asia. In Hong Kong, the frequency of G6PD deficiency is around 4.5% in males and 0.3% in females. Over 140 specific mutations of the X-linked gene for G6PD have been characterized in various geographic regions. However, the local mutation pattern has not been clearly determined. / In conclusion, some Chinese herbal medicine, tea and tea polyphenols significantly altered the oxidative status of G6PD-deficient erythrocytes in vitro. Their in vivo effects on G6PD-deficient individuals would be further investigated by the novel G6PD-dificient mouse model. / In this study, we aim (1) to investigate effects of (a) a panel of Chinese Herbal Medicine (CHM), (b) tea and its constituents, on the oxidative status of human G6PD-deficient erythrocytes in vitro ; (2) to characterize the genotype of G6PD-deficiency in the Chinese population and their specific response to oxidative stress; (3) to develop a novel strain of mice as a model for study of chemicals agents on G6PD-deficient red cell in vivo. / Our results showed that six of eighteen CHM significantly reduced GSH levels in the G6PD-deficient erythrocytes (p<0.05, n=10). After exposure to 1 mg/mL of Rhizoma Coptidis, GSH levels in G6PD-deficient erythrocytes was decreased by 48.9 +/- 5.4% (P<0.001, n=10). At 5 mg/mL of Cortex Moutan, Radix Rehmanniae, Radix Bupleuri, Rhizoma Polygoni Cuspidati and Flos Chimonanthi, GSH levels were decreased significantly (P=0.001 to 0.004) by 51.8 +/- 7.6%, 25.9 +/- 6.7%, 21.0 +/- 6.9%, 17.5 $ 6.7% and 8.7 +/- 6.8% respectively. There were noticeable increases in levels of methaemoglobin by 2.8 fold (5 mg/mL, P=0.012) and 3.4 fold (10 mg/mL, P=0.016) in the presence of Rhizoma Coptidis and Cortex Moutan, respectively, in G6PD-deficient erythrocytes. / We also investigated the pro-oxidative effect of tea and its polyphenolic components on G6PD erythrocytes from G6PD-deficient (n=8) and normal adult (n=8) subjects. The tea extracts significantly reduced GSH and increased GSSG levels in G6PD-deficient erythrocytes in a dose-dependent manner (0.5-10 mg/mL), but not in normal erythrocytes. Similar dose-dependent responses to (-)-Epigallocatechin (EGC) and (-)-Epigallocatechin-3gallate (EGCG), but not to the other polyphenols, were observed. In G6PD-deficient cells, GSH was reduced by 43.3% (EGC at 0.05 mg/mL) and 33.3% (EGCG at 0.5 mg/mL), compared with pre-challenged levels. The concentration of methaemoglobin was increased significantly when these cells were challenged with tea extracts, and EGC. Plasma haemoglobin levels were higher in G6PD-deficient samples after exposure to tea extracts, EGCG, EGC and gallic acid, compared with those in normal blood. / Ko Chun Kay. / "August 2006." / Advisers: Tai Fai Fok; Kwai Har Karen Li. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1577. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. xxii-xliii). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

A Doença de Alzheimer (DA) é uma desordem neurodegenerativa progressiva que causa comprometimento cognitivo e demência. O diagnóstico é baseado em parâmetros clínicos, mas sua confirmação é post-mortem, após avaliação patológica durante a autópsia. Os tratamentos disponíveis para a DA são os inibidores da colinesterase (IChEs) e os antagonistas de receptores de N-metil-D-aspartato (NMDA), sendo que os IChEs compõe o principal grupo. Diversos estudos tem mostrado um efeito neuroprotetor dos IChEs, levando a alterações na patogênese da DA. Avaliar e mensurar essas alterações são papeis atribuídos aos biomarcadores. Neste sentido podemos destacar a fosfolipase A2 (PLA2), a principal responsável pelo metabolismo de fosfolípides de membrana, e que tem sido achada diminuída na DA, assim como a glicogênio sintase-quinase (GSK), responsável pela fosforilação da proteína Tau, que é um dos processos alterados na DA. O objetivo deste trabalho foi avaliar o efeito do tratamento com IChE sobre a atividade da PLA2 e expressão da GSK3B em plaquetas de 30 pacientes com DA após 3 e 6 meses de tratamento. Como grupo controle foram investigados 42 individuos idosos sem doença neurodegenerativa. Encontramos nos pacientes com DA antes do tratamento uma diminuição da atividade da iPLA2 quando comparada ao grupo controle. Após três e seis meses de tratamento a PLA2 aumentou, voltando ao nível dos controles. Os pacientes que apresentaram um aumento maior da iPLA2 apos 3 meses de tratamento apresentaram melhora cognitiva mais marcante após seis meses de tratamento, avaliado pelo CAMCOG. Apos 6 meses de tratamento encontramos um inativação da GSK3B, medida por um aumento em sua forma fosforilada. Nossos resultados sugerem que o donepezil apresenta propriedades modificadoras na doença de Alzheimer, e ainda que a medida da atividade da iPLA2 poderia ser usada como marcador de resposta terapêutica ao donepezil e, possivelmente, a outros IChEs, na doença de Alzheimer / Alzheimer\'s disease (AD) is a progressive neurodegenerative disorder that causes dementia and cognitive impairment. The Diagnosis is based on clinical parameters, but confirmation is post-mortem after pathologic evaluation during autopsy. The treatments available for AD are cholinesterase inhibitors (IChEs) and N-methyl-D-aspartate (NMDA) antagonists. The main group comprises the IChEs. Several studies have shown a neuroprotective effect of IChEs, leading to alterations in the pathogenesis of AD. Evaluate and measure these changes are assigned to biomarkers. In this regard we can highlight the phospholipase A2 (PLA2) the main enzyme in membrane phospholipids metabolism and that has been found decreased in AD as well as Glycogen Synthase kinase (GSK), a major responsible for tau phosphorylation which is one processes altered in AD. The objective of this study was to evaluate the effect of treatment with IChE on PLA2 activity and GSK3B expression in platelet of 30 AD patients after 3 and 6 months of treatment. The control group comprised 42 elderly individuals without neurodegenerative disease The results obtained were a decreased iPLA2 activity in patients with AD before treatment as compared to controls. After 3 and 6 months of treatment, we observed a significant increase in iPLA2 activity, restoring enzymatic activity similar to that observed among control. The patients who showed higher iPLA2 activity in the first three months were those showing cognitive improvement after six months of treatment, measured by CAMCOG. After 6 months of treatment a GSK3B inactivation were found, measured by an increase in its phosphorylated form. Our results suggest that donepezil present modifying properties in Alzheimer disease and that iPLA2 activity measurement could be used as a marker of therapeutic response to donepezil and possibly other IChEs in Alzheimer\'s disease

Le diabète est une maladie chronique avec une progression alarmante. L’insuline-résistance et la diminution de la masse fonctionnelle des cellules beta, associée à l'inflammation des îlots, sont les principaux défauts impliqués dans la pathogenèse du diabète de type 2 (DT2). La compréhension des mécanismes impliqués dans l'inflammation des îlots pancréatiques, et l'identification de cibles moléculaires à visée anti-inflammatoire, sont des approches intéressantes pour le traitement du diabète. La glycogène synthase kinase 3 (GSK3), est une sérine-thréonine kinase qui régule des fonctions cellulaires essentielles. Cette enzyme a été récemment décrite comme un régulateur important de l'inflammation dans différentes conditions pathologiques. Cependant, l'implication potentielle de GSK3beta dans l'inflammation des îlots au cours du diabète reste inexplorée. Le but de ce travail était d'étudier l'implication de GSK3beta dans l'inflammation des îlots pancréatiques et d'évaluer l'impact de l'inhibition de GSK3beta dans l’amélioration de l’hyperglycémie du rat diabétique Goto-Kakizaki. Le rat Goto-Kakizaki (GK) est un modèle spontané de DT2, avec une hyperglycémie chronique apparaissant au sevrage, une masse beta cellulaire réduite et une altération profonde de la sécrétion d'insuline en réponse au glucose. Peu après le sevrage, l'inflammation se développe dans les îlots du rat GK et participe au dysfonctionnement des cellules beta. Nous avons traité les rat GK mâles avec du chlorure de lithium (LiCl), un inhibiteur de GSK3. Le traitement chronique de jeunes rats GK a permis d’éviter l’installation de l’hyperglycémie chronique qui se développe normalement dans ce modèle chez les adultes. A la fin du traitement, la glycémie basale des rats GK traités par le LiCl était fortement réduite, en comparaison avec celle des rats GK non traités. Ces améliorations étaient associées à une réduction de l'expression des cytokines et des chimiokines pro-inflammatoires dans les îlots. L’inhibition de GSK3 a également diminué la fibrose des îlots et rétabli partiellement la sensibilité à l’insuline et la sécrétion d'insuline induite par le glucose chez les rats GK. De plus, des études ex vivo sur des îlots humains et des îlots de rats Wistar, exposés à un environnement inflammatoire en culture, ont révélé l'implication directe de GSK3 dans la réponse inflammatoire autonome des îlots. Ceci était entre autres associée à l’activation du facteur de transcription STAT3. En conclusion, nous montrons pour la première fois que GSK3beta est impliquée dans l’inflammation des îlots pancréatiques humains et de rongeurs. L’inhibition de GSK3beta atténue fortement l’inflammation insulaire, et prévient l’installation de l’hyperglycémie chronique chez le rat GK. L’ensemble des résultats de ce travail nous permet de proposer GSK3beta comme une cible potentielle pour le développement de traitements anti-inflammatoires dans le contexte du diabète de type 2 / Diabetes Mellitus (DM) is a chronic disabling disease with epidemic dimension. It is now established that islet inflammation is associated with defective functional beta cell mass in type 2 diabetes. The understanding of the mechanisms that govern diabetes-associated inflammation in pancreatic islets, and the identification of molecular targets to dampen inflammation are important steps to address this pathological condition. GK rat is a spontaneous model of type 2 diabetes with impaired beta cell function and mass, closely associated with islet inflammation. Glycogen Synthase Kinase 3 (GSK3) is a multi-tasking serine-threonine kinase which regulates crucial cellular functions. In recent years, GSK3beta has been found to be an important regulator of inflammation in different diseased conditions. However, the potential role of GSK3beta in the context of islet inflammation remains unexplored. In this study, we tested the potential of lithium, an inhibitor of GSK3, in improving islet inflammation and glucose metabolism in the GK rat. In vivo, treatment of young GK rats prevented the development of overt diabetes which normally occurs in adult individuals. Lithium improved the glycemic status of the GK rats after few weeks of treatment. At the end of the protocol, GK rats treated with lithium had a blood glucose levels that were significantly lower than that of age-matched untreated GK rats, which were overtly diabetic at this stage. Lithium treatment resulted in reduced expression of pro-inflammatory cytokines and chemokines, decreased fibrosis and reduced macrophage infiltration in the islets. Lithium partially restored the pancreatic insulin content, the insulin sensitivity and the glucose induced insulin secretion in the GK rats. Moreover, ex vivo studies in non-diabetic human and rat islets exposed to inflammatory environment in culture, revealed the direct implication of GSK3 in the islet autonomous inflammatory response. Moreover, we showed that GSK3 controls the islet inflammatory response at least in part by regulating the activity of the pro-inflammatory transcription factor STAT3. Taken together, our results identified GSK3 as a viable target to treat diabetes-associated inflammation, and could have potential clinical application in the treatment of diabetes and metabolic syndrome