Thesis (M.Sc.)--York University, 2009. Graduate Programme in Higher Education. / Typescript. Includes bibliographical references (leaves 76-87). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR51589

Indiana University-Purdue University Indianapolis (IUPUI) / Glycogen, a branched polymer of glucose, serves as an osmotically neutral means of storing glucose. Covalent phosphate is a trace component of mammalian glycogen and has been a point of interest with respect to Lafora disease, a fatal form of juvenile myoclonus epilepsy. Mutations in either the EPM2A or EPM2B genes, which encode laforin and malin respectively, account for ~90% of disease cases. A characteristic of Lafora disease is the formation of Lafora bodies, which are mainly composed of an excess amount of abnormal glycogen that is poorly branched and insoluble. Laforin-/- and malin-/- knockout mice share several characteristics of the human disease, formation of Lafora bodies in various tissues, increased glycogen phosphorylation and development of neurological symptoms. The source of phosphate in glycogen has been an area of interest and here we provide evidence that glycogen synthase is capable of incorporating phosphate into glycogen. Mice lacking the glycogen targeting subunit PTG of the PP1 protein phosphatase have decreased glycogen stores in a number of tissues. When crossed with mice lacking either laforin or malin, the double knockout mice no longer over-accumulate glycogen, Lafora body formation is almost absent and the neurological disorders are normalized. Another question has been whether the abnormal glycogen in the Lafora disease mouse models can be metabolized. Using exercise to provoke glycogen degradation, we show that in laforin-/- and malin-/- mice the insoluble, abnormal glycogen appears to be metabolically inactive. These studies suggest that a therapeutic approach to Lafora disease may be to reduce the overall glycogen levels in cells so that insoluble, metabolically inert pools of the polysaccharide do not accumulate.

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Indiana University-Purdue University Indianapolis (IUPUI) / Glycogen is a major energy reserve in most eukaryotes and its rate of synthesis is controlled by glycogen synthase. The activity of eukaryotic glycogen synthase is regulated by the allosteric activator glucose-6-phosphate, which can overcome the inhibitory effects of phosphorylation. The effects of phosphorylation and glucose-6-phosphate on glycogen synthase are mediated by a cluster of six arginines located within a stretch of 12 amino acids near the C-terminus of the enzyme’s polypeptide chain. We studied isoform-2 of yeast glycogen synthase as a model to study the structural and molecular mechanisms that underlie the regulation of the eukaryotic enzymes and our primary tools of investigation were macromolecular X-ray crystallography, site-directed mutagenesis, intein-mediated peptide ligation and enzyme assays. We have solved the tetrameric structure of the yeast enzyme in two different activity states; the resting enzyme and the activated state when complexed with glucose-6-phosphate. Binding of glucose-6-phosphate to glycogen synthase induces large conformational changes that free the active site of the subunits to undergo conformational changes necessary to catalyze the reaction. Further, using site directed mutagenesis and intein-mediated peptide ligation to create specific phosphorylation states of the enzyme we were able to define specific roles for the arginine residues that mediate the regulatory effects of phosphorylation and glucose-6-phosphate activation. Based on these studies, we propose a three state structural model for the regulation of the enzyme, which relate the observed conformational states to specific activity levels. In addition to these regulatory studies, we have also solved the structure of the enzyme complexed with UDP and with substrate analogs, which provide detailed insight into the catalytic mechanism of the enzyme and the ability of glycogen synthase to remain tightly bound to its substrate glycogen.

Thesis (MScMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Introduction: The worldwide escalation in the incidence of obesity and its strong association with insulin resistance, type 2 diabetes and the cardiovascular complications that accompany these disease states have elicited interest in the underlying mechanisms of these pathologies. Preliminary data generated in our laboratory showed that obesity is associated with abnormalities in the insulin signalling pathway. Specifically, we found a down-regulation of protein kinase B (PKB/Akt), which is known to mediate the metabolic effects of insulin. One of the downstream targets of PKB/Akt is glycogen synthase kinase-3 (GSK-3), which is inhibited by this phosphorylation. Detrimental effects of unopposed activity of GSK-3 have recently been described. This may play a pivotal role in some of the adverse consequences of insulin resistance in the heart. Hypothesis: Chronic inhibition of GSK-3 will induce myocardial hypertrophy or exacerbate the development of existing hypertrophy in a pre-diabetic model of diet induced obesity and insulin resistance. Objectives: (1) Assess the extent of the development of myocardial hypertrophy in a rat model of diet induced obesity (DIO) and insulin resistance. (2) Assess the effect of inhibition of GSK-3 protein on the development of myocardial hypertrophy. Methods: Two groups of age-matched male Wistar rats were used. Control animals received standard rat chow, while obese animals received a high caloric diet for 20 weeks. After 12 weeks, half of the animals in both groups received GSK-3 inhibitor treatment (CHIR118637, 30mg/kg/day, Novartis). At the end of 20 weeks, three series of experiments were conducted. (i) The animals were subjected to echocardiography to determine in vivo myocardial function, and biometric, metabolic and biochemical parameters were evaluated. (ii) The ability of the cardiomyocytes to accumulate deoxy-glucose after stimulation with insulin was determined, and (iii) the localization of key proteins was monitored using fluorescence microscopy and cell size was determined using light microscopy and flow activated cell sorter analysis. Results and discussion: The high caloric diet increased body weight (p<0.005) and intraperitoneal fat mass (p<0.01) when compared to controls. Complications associated with obesity, such as impaired glucose tolerance (p<0.05), hyperinsulinemia (p<0.0005) and an increased HOMA-IR index (p<0.01) were observed. Additionally, cardiomyocytes from the DIO animals had a significantly impaired response to insulin, specifically when 10nM (p<0.05) and 100nM (p<0.05) of insulin were used as stimulus. We also found a dysregulation in PKB/Akt, indicated by a down-regulation of phosphorylated PKB/Akt (p<0.01). The diet promoted the development of myocardial hypertrophy, since the ventricular weight (p<0.05) and ventricular weight to tibia length ratio were increased (p<0.01). Echocardiography experiments showed an increase in end diastolic diameter in the DIO animals (p<0.05). Additionally, there was an increase in the cardiomyocyte cell width in the DIO rats (p<0.0001) and a tendency for peri-nuclear localization of NFATc3. GSK-3 inhibition promoted the development of insulin resistance in control animals, as indicated by an increase in the body weight (p<0.05), serum insulin levels (p<0.01) and HOMA-IR index (p<0.01). In the DIO animals, the GSK-3 inhibitor treatment improved insulin resistance, as a decrease in serum insulin concentration (p<0.05) was observed. The cardiomyocytes from the treated DIO animals also showed an increase in glucose uptake (p<0.05) when stimulated with 100nM of insulin. The GSK-3 inhibitor promoted the development of myocardial hypertrophy in the control animals, indicated by an increase in ventricular weight (p<0.05) and cardiomyocyte cell width (p<0.0001), but did not exacerbate hypertrophy in the DIO animals. Conclusion: Both the high caloric diet and the GSK-3 inhibitor promoted the development of insulin resistance and myocardial hypertrophy in the rats. In the DIO animals the GSK-3 inhibitor treatment ameliorated insulin resistance and did not promote the further development of myocardial hypertrophy. / AFRIKAANSE OPSOMMING: Inleiding: Die huidige styging in vetsugtigheid en die sterk assosiasie daarvan met insulien weerstandigheid, tipe 2 diabetes en kardiovaskulêre komplikasies soos hipertrofie, het ‘n belangstelling in die onderliggende meganismes van hierdie siektetoestande ontlok. Voorlopige data uit ons laboratorium het getoon dat vetsug geassosieerd is met abnormaliteite in die insulien seintransduksie-pad soos byvoorbeeld ‘n afregulering van miokardiale proteïen kinase B (PKB/Akt), wat bekend is om die metaboliese effekte van insulien te medieer. Een van die proteïene wat deur PKB/Akt gefosforileer en daardeur geïnhibeer word, is glikogeen sintase kinase-3 (GSK-3). Negatiewe effekte van onge-opponeerde aktiwiteit van GSK-3 is beskryf en dit mag ‘n sleutelrol speel in sommige van die nadelige gevolge van insulien weerstandigheid in die hart. Hipotese: Chroniese onderdrukking van GSK-3 sal miokardiale hipertrofie ontlok of die bestaande hipertrofie in ‘n pre-diabetiese model van dieet-geïnduseerde vetsug en insulien weerstandigheid vererger. Doelstellings: (1) Om die omvang van die ontwikkeling van miokardiale hipertrofie in ‘n rotmodel van dieet-geïnduseerde vetsug te ondersoek en (2) om die effek van inhibisie van GSK-3 op die ontwikkeling van hipertrofie te ondersoek. Metodes: Ouderdomsgepaarde manlike Wistarrotte is in hierdie studie gebruik. Die diere is vir ‘n periode van 20 weke aan verskillende diëte onderwerp, naamlik standaard kommersiële rotkos vir die kontrole diere en ‘n hoë kalorie dieet vir die eksperimenteel vet diere (DIO). Helfte van elke groep diere is vir 8 weke met ‘n GSK-3 inhibitor behandel (CHIR118637, 30mg/kg/day, Novartis). Na die 20 weke is 3 eksperimentele reekse uitgevoer: (i) Die diere is eggokardiografies ondersoek om in vivo miokardiale funksie te bepaal en biometriese, metaboliese en biochemiese parameters is evalueer. (ii) Die vermoë van kardiomiosiete om de-oksiglukose na insulien stimulasie te akkumuleer, is bepaal, en (iii) die lokalisering van sleutelproteïene is met behulp van fluoressensie mikroskopie en die selgrootte met behulp van ligmikroskopie bepaal. Resultate en bespreking: Die hoë kalorie dieet het gepaard gegaan met ‘n beduidende toename in liggaamsgewig (p<0.005) en intraperitoneale vetmassa (p<0.01) in vergelyking met diere op die kontrole dieet. Newe-effekte geassosieerd met vetsug nl. onderdrukte glucose toleransie (p<0.05), hiperinsulinemie (p<0.0005) en ‘n verhoogde HOMA-IR index (p<0.01) is ook waargeneem. Daar was ook ‘n beduidend ingekorte respons van glukose opname deur kardiomiosiete van die vet diere na stimulasie met 10nM (p<0.05) en 100nM (p<0.05) insulien. Disregulering van PKB/Akt is gevind in die vorm van ‘n afregulering van die fosforilering van die proteïen (p<0.01). Die dieet het ook gelei tot die ontwikkeling van miokardiale hipertrofie aangesien die ventrikulêre gewig (p<0.05) asook die verhouding van die ventrikulêre gewig teenoor tibia lengte beduidend toegeneem het (p<0.01). Eggokardiografie het ‘n toename in ventrikulêre end-diastoliese dimensie in die DIO diere aangetoon (p<0.05). Tesame hiermee het die breedte van kardiomiosiete van die DIO diere toegeneem (p<0.0001) en daar was ook ‘n peri-nukluêre lokalisering van NFATc3. Behandeling van kontrole diere met ‘n GSK-3 inhibitor het insulienweerstandigheid ontlok soos afgelei uit ‘n verhoging in liggaamsgewig (p<0.05), serum insulien-vlakke (p<0.01) en die HOMA-IR waarde (p<0.01). In teenstelling het behandeling van die DIO diere met die GSK-3 inhibitor tot ‘n verbetering van insulienweerstandigheid gelei aangesien ‘n verlaging in serum insulien konsentrasies gevind is (p<0.05). Kardiomiosiete vanaf die behandelde DIO diere het ook ‘n verhoogde insulien-gestimuleerde glukose opname met 100nM insulien getoon (p<0.05). Behandeling met die GSK-3 inhibitor het die ontwikkeling van miokardiale hipertrofie in die kontrole diere teweeggebring, soos aangetoon deur ‘n toename in die ventrikulêre gewig (p<0.05) en ‘n groter selwydte in kardiomiosiete terwyl dit geen invloed op die bestaande hipertrofie van die vet diere gehad het nie. Gevolgtrekking: Die huidige studie het getoon dat die betrokke dieet asook behandeling met ‘n GSK-3 inhibitor insulienweerstandigheid sowel as die ontwikkelling van miokardiale hipertrofie in rotte ontlok. In die DIO diere het die behandeling met die GSK-3 inhibitor bloedglukose en insulien-vlakke verlaag en het nie hipertrofie vererger nie.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: It is widely accepted that insufficient insulin-stimulated activation of muscle glycogen synthesis is one of the major components of non-insulin-dependent (type 2) diabetes mellitus. Glycogen synthase, a key enzyme in glycogen synthesis, is extensively regulated, both allosterically (by glucose-6-phosphate, ATP, and other ligands) and covalently (by phosphorylation). Although glycogen synthase has been a topic of intense study for more than 50 years, its kinetic characterization has been confounded by its large number of phosphorylation states. Questions remain regarding the function of glycogen synthase regulation and the relative importance of allosteric and covalent modification in fulfilling this function. The regulation of glycogen synthase and glycogen phosphorylase, the enzyme that catalyses the degradation of glycogen chains, are reciprocal in many respects. In the present research, using mathematical modelling, we aim to establish the function of the allosteric and covalent regulation of glycogen synthase and glycogen phosphorylase in muscle and, in the case of glycogen synthase, the relative importance of these two mechanisms in performing this function. In order to realize these aims it is essential that a detailed kinetic model of glycogen metabolism is constructed. We begin with a thorough review of the kinetics and regulation of glycogen synthase inwhich we propose that both allosteric and covalent modification of glycogen synthase can be described by a Monod-Wyman-Changeux model in terms of apparent changes to L0, the equilibrium constant between the T and R conformers. We then proceed to develop a rate equation according to the proposed Monod-Wyman-Changeux model and determine values for its kinetic parameters from published experimental data using non-linear least-squares regression. We show that the application of the Monod-Wyman-Changeux model to glycogen synthase kinetics also has important implications for the rate equations of enzymes that catalyse the phosphorylation and dephosphorylation of glycogen synthase. We formalize these implications for a generic protein that follows Monod-Wyman-Changeux-type conformational change and then also show how the findings apply to glycogen synthase. Taking into account the kinetic model of glycogen synthase and how it also influences the covalent regulation of the enzyme, we proceed to construct a detailed mathematical model of glycogen synthesis that includes the glycogen synthase phosphorylation cascade. A variation of this model in which glycogen synthase phosphorylation is described with a single parameter is also provided. We reuse an existing model of muscle glycogenolysis and also combine these models in an overall model of glycogen metabolism. Finally, we employ the theoretical frameworks of metabolic control analysis, supply-demand analysis, and co-response analysis to investigate the function of glycogen synthase and glycogen phosphorylase regulation. We show that the function of glycogen synthase regulation is not flux control, as assumed in the textbook view, but rather the maintenance of glucose-6-phosphate within a narrow range far from equilibrium. Similarly, we show that regulation of glycogen phosphorylase functions to minimize variation in cellular energy charge in the face of highly variable energy demand. We conclude with an appeal for a renewed interest in the enzyme kinetics of muscle glycogen metabolism. / AFRIKAANSE OPSOMMING: Daar word wyd aanvaar dat onvoldoende insulien-gestimuleerde aktivering van spierglikogeensintese een van die hoofkomponente van insulien-onafhanklike (tipe 2) diabetes mellitus is. Glikogeensintase, ’n sleutelensiem in glikogeensintese is onderworpe aan breedvoerige regulering, beide allosteries (deur glukose-6-fosfaat, ATP, en ander ligande) en kovalent (deur fosforilering). Alhoewel glikogeensintase reeds vir meer as 50 jaar deeglik bestudeer word, word die kinetiese karakterisering daarvan bemoeilik deur die groot aantal fosforilasiestate waarin die ensiem voorkom. Daar is steeds vrae betreffende die funksie van die regulering van glikogeensintase en die relatiewe bydrae van allosteriese en kovalente regulering in die vervulling van hierdie funksie. Die regulering van glikogeensintase en glikogeenfosforilase, die ensiem wat die afbraak van glikogeenkettings kataliseer, is in baie opsigte resiprook. In hierdie studie beoog ons om met die hulp van wiskundige modellering vas te stel watter funksie die regulering van glikogeensintase en glikogeenfosforilase vervul en, in die geval van glikogeensintase, wat die relatiewe belang is van allosteriese en kovalente regulering in die vervulling van hierdie funksie. Om hierdie oogmerke te verwesentlik is dit nodig dat ’n kinetiese model van glikogeenmetabolisme ontwikkel word. Ons begin met ’n omvattende oorsig van die kinetika en regulering van glikogeensintase waarin ons voorstel dat beide die allosteriese en kovalente regulering van glikogeensintase beskryf kan word met die Monod-Wyman-Changeux model in terme van oënskynlike veranderings aan L0, die ekwilibriumkonstante tussen die T en R konformasies. Ons gaan dan voort om ’n snelheidsvergelyking te ontwikkel volgens die voorgestelde Monod-Wyman-Changuex-model en bepaal ook die waardes van hierdie vergelyking se parameters vanaf gepubliseerde eksperimentele data deur middel van nie-lineêre kleinste-vierkantsregressie. Ons wys dat die toepassing van die Monod-Wyman-Changuex-model op glikogeensintase-kinetika belangrike gevolge het vir die snelheidsvergelykings van die ensieme wat die fosforilering en defosforilering van glikogeensintase kataliseer. Ons formaliseer hierdie gevolge vir ’n generiese Monod-Wyman-Changeux-tipe proteïen en wys dan ook hoe die bevindings op glikogeensintase van toepassing is. Met inagneming van die kinetiese model vir glikogeensintase en hoe dit die kovalente regulering van die ensiem be¨ınvloed, gaan ons voort om ’n gedetaileerde wiskundige model van glikogeensintese, wat ook die glikogeensintase-fosforileringskaskade insluit, te ontwikkel. ’n Variasie op hierdie model waarin die fosforilering van glikogeensintase deur ’n enkele parameter beskryf word, word ook voorsien. Ons herbruik ’n bestaande model van spierglikogenolise en kombineer ook hierdie modelle in ’n oorkoepelende model van glikogeenmetabolisme. Uiteindelik span ons die teoretiese raamwerke van metaboliese kontrole-analise, vraag-aanbod-analise, en ko-responsanalise in om die funksie van die regulering van glikogeensintase en glikogeenfosforilase te ondersoek. Ons wys dat die funksie van die regulering van glikogeensintase nie fluksiekontrole, soos algemeen in handboeke aangeneem word, is nie, maar liewer dat dit glukose-6-fosfaat handhaaf binne ’n noue band ver vanaf ekwilibrium. Insgelyks wys ons dat die regulering van glikogeenfosforilase funksioneer om variasie in sellulˆere energielading te beperk ten spyte van hoogs wisselende vlakke van energie-aanvraag. Ons sluit af met ’n pleidooi vir hernieude belangstelling in die ensiemkinetika van glikogeenmetabolisme in die spier. / National Research Foundation

Stress associated psychiatric disorders such as depression, anxiety, and post-traumatic stress disorder affect a large proportion of the population. Reductions in the complexity of neuronal morphology and reduced neurogenesis are commonly observed outcomes following stress exposure in rodent models and may represent a mechanism for the reduced brain volume in stress sensitive regions such as the hippocampus observed in individuals diagnosed with stress associated disorders. Multiple lines of evidence suggest that glycogen synthase kinase (GSK)-B may play a role in the neurodegenerative phenotype observed following stress exposure. GSK3B is atypical in that it is inhibited by phosphorylation. This inhibitory phosphorylation has typically been studied by examining the phosphorylation state of the serine 9 (S9) site. Inhibition of GSK3B is implicated in synaptic stabilization, increased expression of trophic factors that support dendritic complexity and neurogenesis, reduced apoptosis, and the antidepressive effects of currently implemented therapeutics. It is surprising then that little research has examined the regulation of GSK3B by stress. A novel GSK3B phosphorylation site, serine 389 (S389), has recently been described that is regulated by p38 mitogen activated protein kinase (MAPK) and is independent of S9 phosphorylation by AKT. p38 MAPK is implicated in the behavioral effects of stress exposure making an understanding of its interaction with GSK3B S389 phosphorylation during stress a compelling research target. The current studies examine GSK3B regulation following variate stress exposure in stress reactive brain regions, describe the anatomical specificity of GSK3B S389 phosphorylation in the brain, and detail the behavioral phenotype of a novel mutant mouse that cannot inhibit GSK3B by S389 phosphorylation (GSK3B KI). Region specific changes in GSK3B phosphorylation were observed following stress exposure, as well as voluntary exercise, a behavior that confers stress resistance. Elevated GSK3B S389 phosphorylation was associated with increased levels of phosphorylated p38 MAPK. This pathway is implicated in the response to DNA damage, and, surprisingly, we observed that histone H2A-variant-X (H2A.X), a marker of DNA damage, was elevated following stress and exercise. Accumulated DNA damage is a proposed driver of neurodegeneration suggesting that the pathway activated by stress may be engaged to protect against such decline. Consistent with a role in the response to DNA damage, we observed a primarily nuclear localization of GSK3B S389 phosphorylation in the brain while S9 phosphorylation was found in nuclear and cytosolic compartments. Further, we observed neurodegeneration in hippocampal and cortical regions of GSK3B KI mice supporting the idea that the inhibition of GSK3B by S389 phosphorylation observed following stress and exercise may be protective. Though largely similar to wild type mice in behavioral tests, increased auditory fear conditioning was evident in GSK3B KI mice. Contextual and cued freezing was prolonged in GSK3B KI mice, a phenotype that is commonly observed in stress models. Together these findings suggest that GSK3B S389 phosphorylation is playing a critical role in neuronal integrity that is independent of GSK3B S9 phosphorylation, and that the subset of neurons protected by GSK3B S389 phosphorylation may play an important role in preventing a portion of the maladaptive behavioral changes observed following stress exposure.

Apesar da elevada prevalência dos transtornos depressivos em idosos, os mecanismos fisiopatológicos subjacentes a estes quadros são pouco conhecidos. Atualmente, o principal foco dos estudos sobre a fisiopatologia da depressão geriátrica são as alterações cerebrovasculares associadas a estes quadros. Outros mecanismos fisiopatológicos têm sido estudados, como as alterações em cascatas neurotróficas e inflamatórias. A enzima glicogênio sintase quinase 3 beta (GSK-3B) tem sido implicada na patogênese de diversos transtornos mentais, em especial os transtornos afetivos (i.e. depressão maior e o transtorno afetivo bipolar) e doenças neurodegenerativas (i.e. doença de Alzheimer). Entretanto, não há estudos que avaliam o papel desta enzima nos pacientes idosos com depressão maior. Desta maneira, o objetivo principal deste trabalho é avaliar a atividade da GSK-3B em pacientes idosos com depressão maior. A hipótese deste estudo é que a atividade enzimática está aumentada nos pacientes idosos deprimidos em relação a idosos saudáveis. Para este estudo, recrutamos 40 idosos com depressão maior (de acordo com os critérios diagnósticos do DSM-IV) e que não estavam em uso de antidepressivos. O grupo comparativo foi constituído por 13 idosos saudáveis, sem evidências de transtornos cognitivos ou do humor. A gravidade da sintomatologia depressiva foi avaliada pela escala de depressão de Hamilton de 21 itens (HAM-D); o desempenho cognitivo dos pacientes e controles foi avaliado pelo teste cognitivo de Cambridge (CAMCOG) e pelo mini-exame do estado mental (MEEM). A expressão da GSK-3B foi determinada em plaquetas através de ensaio imunoenzimático (EIA), sendo estabelecido os níveis totais da GSK-3B (T-GSK-3B) e de sua forma fosforilada (P-GSK-3B), inativa. A atividade enzimática foi inferida indiretamente pela razão P-GSK- 3B / T-GSK-3B. Nos pacientes idosos com depressão maior, observou-se uma redução significante dos níveis P-GSK-3B (p=0,03) e da razão da GSK- 3B (p=0,03). Os pacientes com sintomatologia depressiva mais grave (HAMD > 21) e déficits cognitivos mais intensos (CAMCOG < 86) apresentaram maior atividade enzimática (p=0,03 e teste, p=0,01, respectivamente). Os resultados deste trabalho sugerem que a atividade da GSK-3B está significantemente aumentada em pacientes idosos com depressão maior e que está alteração é mais pronunciada nos pacientes com sintomatologia depressiva e déficits cognitivos mais graves. Neste contexto, a atividade da GSK-3B pode ser considerada um marcador de estado em pacientes idosos com episódios depressivos mais graves e ser um importante alvo para o desenvolvimento de estratégias terapêuticas para estes quadros / Despite the high prevalence of depressive disorders in the elderly, its main physiopathological mechanisms are largely unknown. In the recent years, most of the research efforts focused on the association between cerebrovascular changes and geriatric depression. Nonetheless, other mechanisms have been studied, such as changes in neurotrophic and inflammatory cascades. The enzyme glycogen synthase kinase 3 beta (GSK- 3B) has been implicated in many mental disorders, in particular affective disorders (i.e. major depression and bipolar disorder) and neurodegenerative disorders (i.e. Alzheimers disease). However, there is no study so far that addressed the role of this enzyme in elderly patients with major depression. Therefore, the main objective of this study was to evaluate if GSK-3B activity is changed in elderly patients with major. The working hypothesis is that enzyme activity is significantly increased in elderly patients with major depression as compared to elderly controls. We recruited 40 elderly patients with current major depressive episode (according to the DSM-IV criteria) that was not under antidepressant treatment. The comparison group included 13 healthy elderly subjects with no evidence of cognitive impairment or major psychiatric disorder. The severity of depressive symptoms was assessed by the Hamilton Depression Scale 21 items; cognitive performance was assessed by the Cambridge Cognitive test (CAMCOG) and the Mini-mental State Examination (MMSE). The levels of total and phosphorylated GSK-3B (T-GSK-3B and P-GSK-3B, respectively) levels were determined in platelets by immunoenzymatic assay (EIA). Enzyme activity was indirectly inferred by the ratio P-GSK-3B / T-GSK-3B. Elderly patients with major depression had a significant reduction in the P-GSK-3B levels (p = 0.03) and GSK-3B ratio (p= 0.03). The patients with severe depressive episode (HAM-D scores above 21 points) and cognitive impairment (CAMCOG scores below 86 points) presented the more significant reduction of GSK-3B ratio (p = 0.03 and p = 0.01, respectively). These data altogether suggest that GSK-3B activity is significantly increased in elderly patients with major depression, in particular in those with more severe depressive episode and worse cognitive performance. In this context, the increased enzyme activity may be regarded as a state marker of severe depressive episodes and may an important target to the development of therapeutic strategies to this disorder

While prompt activation of NF-κB is essential for optimal immune response, it is equally important to terminate the response to avoid tissue damage and perhaps even death resulting from organ failure. This thesis describes two inhibitors of NF-κB, A20 and IκBβ. A20 is an essential inhibitor of NF-κB mediated inflammation as mice lacking A20 die from multi-organ inflammation and cachexia. Multiple biochemical approaches have suggested that A20 functions as a deubiquitinase by disassembling K63-linked regulatory ubiquitin chains from upstream adapter molecules like RIP1. To determine the contribution of the deubiquitinase role of A20 in downregulating NF-κB, we generated and characterized a knock-in mouse lacking the deubiquitinase activity of A20. However, we find that these mice display normal NF-κB activation and show no signs of inflammation. Our results suggest that the deubiquitinase activity of A20 is dispensable for downregulating NF-κB. The second part of this thesis unravels a new biological pathway mediated by IκBβ. Unlike IκBα, which functions solely as an inhibitor of NF-κB, IκBβ can both inhibit and activate NF-κB depending on the physiological context. We hypothesized that this may be because IκBβ (unlike IκBα ) exists in two forms, a constitutively phosphorylated form and an unphosphorylated form. Prior work from our group has demonstrated that hypophosphorylated IκBβ complexes with p65:cRel and mediates the expression of certain inflammatory genes like TNFα . We report here that Glycogen Synthase Kinase 3β (GSK-3β ) interacts with and phosphorylates IκBβ at Serine-346. This phosphorylation masks the NLS of p65 in the phospho-IκBβ:p65:cRel complex, thereby sequestering the complex in the cytoplasm and mediating the anti-inflammatory role of IκBβ. We discovered a peptide that can inhibit this phosphorylation by abrogating the interaction between GSK-3β and IκBβ. Mice succumb to a sublethal dose of LPS when injected with this peptide because of increased production of TNFα (but not IL-6); thereby demonstrating the inflammatory role of unphosphorylated IκBβ in upregulating specific genes like TNFα. We propose a signaling model by which phosphorylation by GSK-3β can regulate the functions of IκBβ in response to LPS.

by Wong Kin Kwan Kelvin. / Thesis submitted in: September 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 91-101). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Abbreviations --- p.x / List of Figures --- p.xiii / List of Tables --- p.xiv / Chapter Chapter 1 --- Intorduction --- p.1 / Chapter 1.1 --- Alzheimer，s disease --- p.1 / Chapter 1.2 --- Histopathological features --- p.1 / Chapter 1.3 --- Tau protein pathology and AD --- p.4 / Chapter 1.4 --- Tau protein kinase I (TPKI)- GSK-3β --- p.6 / Chapter 1.5 --- Tau protein kinase II (TPKII)- Cyclin dependent kinase 5 (Cdk5) --- p.8 / Chapter 1.6 --- Available treatment --- p.9 / Chapter 1.7 --- Objectives of the present study --- p.12 / Chapter Chapter 2 --- Screening for GSK-3p inhibitors from Traditional Chinese Medicine (TCM) --- p.13 / Chapter 2.1 --- Introduction --- p.13 / Chapter 2.1.1 --- Phosphorylation of tau in AD --- p.13 / Chapter 2.1.2 --- Gsk-3p inhibitors --- p.14 / Chapter 2.1.3 --- Screening of GSK-3β inhibitor from TCM --- p.16 / Chapter 2.2 --- Material and Methods --- p.18 / Chapter 2.2.1 --- Preparation of extracts and fractions (AOF1-5) --- p.18 / Chapter 2.2.2 --- General cell culture techniques --- p.21 / Chapter 2.2.3 --- "3-(4,5-dimethyltiazoI-2-yl)-2, 5-diphenyl-tetrazolium (MTT) assay of AOF" --- p.23 / Chapter 2.2.4 --- Recombinant DNA techniques --- p.23 / Chapter 2.2.5 --- Transfection of GSK-3β and tau cDNA into COS7 cells --- p.28 / Chapter 2.2.6 --- Extraction of total proteins from culture cells --- p.28 / Chapter 2.2.7 --- Quantitation of protein by the Bradford method --- p.29 / Chapter 2.2.8 --- Protein separation by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.29 / Chapter 2.2.9 --- Western blot analysis --- p.31 / Chapter 2.2.10 --- GSK-3β kinase assay --- p.32 / Chapter 2.2.11 --- Determination of lithium content by atomic adsorption spectrophotometry --- p.34 / Chapter 2.3 --- Results --- p.35 / Chapter 2.3.1 --- Establishment of a co-transfected cell model for GSK-3β induced tau hyperphosphorylation --- p.35 / Chapter 2.3.2 --- Preliminary screening results of aqueous and ethanol extracts (AOF1 and AOF2) --- p.37 / Chapter 2.3.3 --- Ethanol extract of AOF inhibits GSK-3p induced tau phosphorylation in COS-7 cells --- p.40 / Chapter 2.3.5 --- Effect of the essential oils of AOF on GSK-3P induced tau phosphorylation --- p.46 / Chapter 2.3.6 --- The effect of AOF essential oil on GSK-3P activity in COS7 --- p.50 / Chapter 2.3.7 --- Lithium content of AOF extracts --- p.52 / Chapter 2.4 --- Discussion --- p.54 / Chapter Chapter 4 --- Evaluation of the in vivo efficacy of cryptotenshinone (CT) in Morris Water Maze Task (WMT) --- p.59 / Chapter 4.1 --- Introduction --- p.59 / Chapter 4.1.1 --- Involvement of Cholinergic system in cognitive dysfunction in AD --- p.59 / Chapter 4.1.2 --- Animal model for Alzheimer's disease --- p.60 / Chapter 4.1.3 --- Morris Watermaze Task (WMT) --- p.61 / Chapter 4.2 --- MATERIAL AND METHODS --- p.64 / Chapter 4.2.1 --- Morris Water maze setup --- p.64 / Chapter 4.2.2 --- Animal model --- p.66 / Chapter 4.2.3 --- Drug preparation --- p.67 / Chapter 4.2.4 --- Toxicity test of CT --- p.67 / Chapter 4.2.5 --- Water maze task (WMT) --- p.68 / Chapter 4.2.6 --- Visual acuity test --- p.73 / Chapter 4.3 --- RESULTS --- p.74 / Chapter 4.3.1 --- Chronic crytotanshinone treatment does not cause hepatic damages to the mice --- p.74 / Chapter 4.3.2 --- Training Session --- p.76 / Chapter 4.4 --- DISCUSSION --- p.85 / Chapter Chapter 5 --- General Discussion and Future Directions --- p.87 / Chapter 5.1 --- "AOF, the potential GSK-3 inhibitor" --- p.87 / Chapter 5.2 --- CT´ؤthe AChEI --- p.88 / References --- p.91 / Appendix --- p.102 / Chapter A1 --- Reagents for SDS-PAGE --- p.103 / Chapter A3 --- Solution components provided by QIAGEN Plasmid Maxipreps kit --- p.108 / Chapter A4 --- Reagents and medium for cell culture --- p.109 / Chapter A5 --- Reagents for kinase assay --- p.110 / Chapter A6 --- Raw data of figures --- p.112 / Chapter A7 --- Plasmid map of PCI-neo --- p.119