Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb. 7, 2008). Includes bibliographical references.

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

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.

No description available.

Atherosclerosis is a major underlying cause of cardiovascular disease; however, the molecular mechanisms by which cardiovascular risk factors promote the development of atherosclerosis are poorly understood. Recent evidence from our laboratory suggests that endoplasmic reticulum (ER) stress signaling through glycogen synthase kinase (GSK)-3α/β is involved in the activation of pro-atherosclerotic processes. Previous studies from our lab show that myeloid-specific deletion of GSK3α attenuates the progression of atherosclerosis. However, the precise role(s) of GSK3α/β in atherosclerotic regression is not known. The primary goal of this thesis is to investigate the role(s) of GSK3α/β in lesional macrophages and atherosclerotic regression. Initially, we have targeted the ER stress- GSK3α/β pathway by supplementing the drinking water of low-density lipoprotein receptor (Ldlr)-/- mice with the small molecules 4-phenylbutyric acid or valproate. The results suggest that ER stress or GSK3α/β inhibition can attenuate the growth of existing atherosclerotic lesions and appear to increase lesion stability. From this study it remains unclear whether these interventions can promote atherosclerotic regression. Next, to investigate the role(s) of GSK3α/β in pro-atherogenic processes, bone marrow derived macrophages were isolated from myeloid-specific GSK3α- and/or GSK3β-deficient mice. The effects of GSK3α/β-deficiency on signaling pathways regulating atherogenic functions in macrophage were analyzed. This study revealed that GSK3α and GSK3β play distinct, and often opposing roles in macrophage polarization, inflammatory response, lipid accumulation and migration. Furthermore, both GSK3α and GSK3β appear to play redundant roles macrophage viability, proliferation, and metabolism. Lastly, we investigated the effect of macrophage-specific deletion of GSK3α and/or GSK3β on atherosclerotic regression in Ldlr−/− mice. A novel inducible knock out mouse model has been created in which GSK3α and/or GSK3β expression can be ablated by treating the mice with tamoxifen. These mice were fed a high fat diet to promote the development of atherosclerosis, and then mice were treated with tamoxifen to induce GSK3α/β deletion and switched to a chow diet for 12 weeks. All mice were sacrificed at 33 weeks of age and atherosclerotic plaques were analysed. Female mice with induced macrophage-specific GSK3α deficiency, but not GSK3β deficiency, showed regression of existing atherosclerotic lesions. Together, these studies begin to delineate the specific roles of GSK3α and GSK3β in atherosclerotic regression. Furthermore, these data suggest that GSK3α inhibition could be an effective strategy for the treatment of atherosclerotic cardiovascular disease. / Thesis / Doctor of Philosophy (PhD) / Atherosclerosis is a disease involving the build-up of fatty plaques in the arteries, making them hard and narrow, which leads to damage in the heart, coronary or peripheral blood vessels. This can cause acute cardiovascular complications (heart attacks or stroke) and potentially death. We suspect that protein named glycogen synthase kinase (GSK)-3α/β is involved in the development of atherosclerosis. The purpose of this research is to see if we can treat atherosclerosis by blocking GSK3α/β’s functions. The findings of this study demonstrate that blocking GSK3α reduces inflammation, which is a primary cause of atherosclerosis. Furthermore, blocking GSK3α promotes the regression of atherosclerotic plaques and may lower the risk of cardiovascular disease. This knowledge could aid in the development of medications to treat atherosclerosis and reduce the number of individuals who die from heart attacks or strokes.

Obesity has been related to a greater incidence of more aggressive, advanced stage prostate cancer. It is expected that serum adipokines related to obesity will promote a more aggressive phenotype in PC cells in vitro. Patient serum (n = 80) was obtained for analysis and divided into four patient groups based on obesity and prostate cancer status. Characteristics of serum-treated PC cells in vitro were measured. In a separate set of analyses, LNCaP and PC3 cells were treated with adiponectin and resistin in vitro, and cell characteristics were analyzed. Serum from obese PC patients induces greater amounts of cell migration and lower amounts of cell proliferation and invasion in vitro. Exogenous treatment of adiponectin on PC cells in vitro does not affect cell migration or invasion. However, adiponectin modulates cytosolic protein levels of soluble β-catenin and GSK-3β, indicating that its mechanism of action may be through the Wnt signalling pathway.

A via canônica Wnt/beta-catenina regula várias funções em vertebrados, incluindo diferenciação de células T, bem como a proliferação, sobrevivência, morfogênese e migração de vários tipos celulares. As células T CD4+ é fundamental para a competência imunológica. Foi observado pelo nosso grupo que células T CD4+ humanas apresentam ativação da via Wnt/beta-catenina após tratamento com sais de lítio ou outros agonistas da via. A ativação desta via induziu a proliferação de células T CD4+ naive e de memória central. Em conjunto, estes dados sugerem um importante papel da via Wnt/beta-catenina na homeostase de células T CD4+ humanas. Seria importante avaliar o papel da via Wnt/beta-catenina nas células do sistema imune no modelo murino, já que pouco se sabe sobre seu efeito na homeostase de células T CD4+ murinas. A ativação da via Wnt/beta-catenina pode ser induzida com inibidores da proteína Glicogênio sintase quinase 3beta (GSK3beta), por exemplo, os sais de lítio (LiCl e Li2CO3) e inibidores específicos (SB, CHIR) em vários tipos celulares. Neste trabalho, avaliamos o efeito de inibidores de GSK3? na ativação da via Wnt/beta-catenina canônica em esplenócitos e células T CD4+, através da realização de experimentos in vivo e in vitro, avaliando a expressão de seus genes alvo HIG2, Bcl-xL, Ciclina D1 e c-myc. Verificou-se que o tratamento in vivo agudo (2-12 h após a administração) ou crônico (administração diária por 30 dias) de camundongos não é capaz de ativar a via Wnt/beta-catenina in vivo em células esplênicas e células T CD4+, embora o mesmo tratamento induza a expressão dos genes alvo da via no tecido cerebral (córtex e hipocampo). Além disso, também não foi possível verificar ativação da via em esplenócitos e células T CD4+ após tratamento in vitro das mesmas com LiCl ou os inibidores específicos de GSK3beta testados(CHIR99021, SB-216763), embora essa ativação tenha sido observada na linhagem celular HEK293. Nossos resultados sugerem que a via Wnt/beta-catenina (canônica) não é induzível em células T CD4+ murinas maduras, com os agonistas testados. Isso pode ter implicações fisiológicas, por exemplo sobre a homeostase de células T CD4+, já que a proliferação homeostática de células T, influenciada em humanos pela via Wnt/beta-catenina, é menos importante em camundongos / The Wnt/beta-catenin pathway regulates many functions in vertebrates, including T cell differentiation, as well as proliferation, morphogenesis and migration in different cell types. CD4+ T cells play is fundamental for immunological competence. Our group has observed that human CD4+ T cells present activation of the Wnt/beta-catenin pathway after treatment with lithium salts or other pathway agonists. The activation of this pathway induced proliferation in naive and central memory CD4+ T cells. Together, these results suggest an important role for the Wnt/beta-catenin pathway in the homeostasis of human CD4+ T cells. It would be very important to evaluate the role of the Wnt/beta-catenin pathway in T cells in the mouse model, since little is known about its effect in mice CD4+ T cell homeostasis. The activation of the Wnt/beta-catenin pathway may be induced with Glycogen Synthase Kinase 3B (GSK3beta) inhibitors, i.e., lithium salts as mentioned above, and specific GSK3beta inhibitors (SB, CHIR) in different cell types. In this work, we evaluated the effect of GSK3beta inhibitors in the activation of the canonical Wnt/beta-catenin in splenocytes and CD4+ T cells, by conducting experiments in vivo and in vitro, evaluating the expression of its target genes HIG2, Bcl-xL, Cyclin D1 and c-myc. We verified that acute (2-12 hours after administration) or chronic (daily administration for 30 days) treatment of mice with lithium salts is not able to activate the Wnt/beta-catenin pathway in splenocytes and CD4+ T cells, although we could observe activation in brain tissues (cortex and hypothalamus). Besides, no activation of the Wnt/beta-catenin pathway was observed in these cell types after in vitro treatment with LiCl or the specific inhibitors of GSK3beta (CHIR99021, SB-216763), while the pathway was activated by the same treatments in HEK293 cells. Our results suggest that the Wnt/beta-catenin pathway is not inducible in murine mature CD4+ T cells with the tested agonists. This may have physiological implications, for instance on the homeostasis of CD4+ T cells, where homeostatic proliferation - influenced the Wnt/beta-catenin pathway in human T cells - is less important in the maintenance of the murine peripheral T cell pool

In a 4-cell C. elegans embryo the ventral blastomere EMS requires polarity signaling from its posterior sister cell, P2. This signaling event enables EMS to orient its division spindle along the anterior-posterior (A/P) axis and to specify the endoderm fate of its posterior daughter cell, E. Wnt pathway components have been implicated in mediating P2/EMS signaling. However, no single mutants or various mutant combinations of the Wnt pathway components disrupt EMS polarity completely. Here we describe the identification of a pathway that is defined by two tyrosine kinase related proteins, SRC-1 and MES-1, which function in parallel with Wnt signaling to specify endoderm and to orient the division axis of EMS. We show that SRC-1, a C. elegans homolog of c-Src, functions downstream of MES-1 to specifically enhance phosphotyrosine accumulation at the P2/EMS junction in order to control cell fate and mitotic spindle orientation in both the P2 and EMS cells. In the canonical Wnt pathway, GSK-3 is conserved across species and acts as a negative regulator. However, in C. elegans we find that GSK-3 functions in a positive manner and in parallel with other components in the Wnt pathway to specify endoderm during embryogenesis. In addition, we also show that GSK-3 regulates C. elegans germline development, a function of GSK-3 that is not associated with Wnt signaling. It is required for the differentiation of somatic gonadal cells as well as the regulation of meiotic cell cycle in germ cells. Our results indicate that GSK-3 modulates multiple signaling pathways to regulate both embryogenesis and germline development in C. elegans.

A via canônica Wnt/beta-catenina regula várias funções em vertebrados, incluindo diferenciação de células T, bem como a proliferação, sobrevivência, morfogênese e migração de vários tipos celulares. As células T CD4+ é fundamental para a competência imunológica. Foi observado pelo nosso grupo que células T CD4+ humanas apresentam ativação da via Wnt/beta-catenina após tratamento com sais de lítio ou outros agonistas da via. A ativação desta via induziu a proliferação de células T CD4+ naive e de memória central. Em conjunto, estes dados sugerem um importante papel da via Wnt/beta-catenina na homeostase de células T CD4+ humanas. Seria importante avaliar o papel da via Wnt/beta-catenina nas células do sistema imune no modelo murino, já que pouco se sabe sobre seu efeito na homeostase de células T CD4+ murinas. A ativação da via Wnt/beta-catenina pode ser induzida com inibidores da proteína Glicogênio sintase quinase 3beta (GSK3beta), por exemplo, os sais de lítio (LiCl e Li2CO3) e inibidores específicos (SB, CHIR) em vários tipos celulares. Neste trabalho, avaliamos o efeito de inibidores de GSK3? na ativação da via Wnt/beta-catenina canônica em esplenócitos e células T CD4+, através da realização de experimentos in vivo e in vitro, avaliando a expressão de seus genes alvo HIG2, Bcl-xL, Ciclina D1 e c-myc. Verificou-se que o tratamento in vivo agudo (2-12 h após a administração) ou crônico (administração diária por 30 dias) de camundongos não é capaz de ativar a via Wnt/beta-catenina in vivo em células esplênicas e células T CD4+, embora o mesmo tratamento induza a expressão dos genes alvo da via no tecido cerebral (córtex e hipocampo). Além disso, também não foi possível verificar ativação da via em esplenócitos e células T CD4+ após tratamento in vitro das mesmas com LiCl ou os inibidores específicos de GSK3beta testados(CHIR99021, SB-216763), embora essa ativação tenha sido observada na linhagem celular HEK293. Nossos resultados sugerem que a via Wnt/beta-catenina (canônica) não é induzível em células T CD4+ murinas maduras, com os agonistas testados. Isso pode ter implicações fisiológicas, por exemplo sobre a homeostase de células T CD4+, já que a proliferação homeostática de células T, influenciada em humanos pela via Wnt/beta-catenina, é menos importante em camundongos / The Wnt/beta-catenin pathway regulates many functions in vertebrates, including T cell differentiation, as well as proliferation, morphogenesis and migration in different cell types. CD4+ T cells play is fundamental for immunological competence. Our group has observed that human CD4+ T cells present activation of the Wnt/beta-catenin pathway after treatment with lithium salts or other pathway agonists. The activation of this pathway induced proliferation in naive and central memory CD4+ T cells. Together, these results suggest an important role for the Wnt/beta-catenin pathway in the homeostasis of human CD4+ T cells. It would be very important to evaluate the role of the Wnt/beta-catenin pathway in T cells in the mouse model, since little is known about its effect in mice CD4+ T cell homeostasis. The activation of the Wnt/beta-catenin pathway may be induced with Glycogen Synthase Kinase 3B (GSK3beta) inhibitors, i.e., lithium salts as mentioned above, and specific GSK3beta inhibitors (SB, CHIR) in different cell types. In this work, we evaluated the effect of GSK3beta inhibitors in the activation of the canonical Wnt/beta-catenin in splenocytes and CD4+ T cells, by conducting experiments in vivo and in vitro, evaluating the expression of its target genes HIG2, Bcl-xL, Cyclin D1 and c-myc. We verified that acute (2-12 hours after administration) or chronic (daily administration for 30 days) treatment of mice with lithium salts is not able to activate the Wnt/beta-catenin pathway in splenocytes and CD4+ T cells, although we could observe activation in brain tissues (cortex and hypothalamus). Besides, no activation of the Wnt/beta-catenin pathway was observed in these cell types after in vitro treatment with LiCl or the specific inhibitors of GSK3beta (CHIR99021, SB-216763), while the pathway was activated by the same treatments in HEK293 cells. Our results suggest that the Wnt/beta-catenin pathway is not inducible in murine mature CD4+ T cells with the tested agonists. This may have physiological implications, for instance on the homeostasis of CD4+ T cells, where homeostatic proliferation - influenced the Wnt/beta-catenin pathway in human T cells - is less important in the maintenance of the murine peripheral T cell pool

Le facteur de transcription T-bet joue un rôle central dans la régulation de la différenciation des lymphocytes T. La protéine tyrosine kinase, la glycogène synthase kinase 3 (GSK-3), inhibe l'activation des lymphocytes T et contrôle l'expression de leurs récepteurs inhibiteurs PD-1 et LAG- 3. Bien que l'inhibition de GSK-3 puisse augmenter l'expression de T-bet, l'interrelation entre T-bet et GSK-3 dans l'immunité tumorale est inconnue. Dans cette étude, nous montrons que les souris knock-out T-bet (Tbet - / -) sont compromises dans leur capacité à contrôler la croissance des cellules tumorales du mélanome B16. Cependant, l'injection d'une petite molécule inhibitrice (SMI) de GSK-3 inverse cette condition compromise entraînant le contrôle de la croissance tumorale similaire à celle observée chez les souris de type sauvage. Un examen de Tbet - / - a montré une perte de cellules dendritiques (DC) et de cellules leucocytes polymorphonucléaires (PMN) potentiellement suppressives et de lymphocytes tumoraux T (TILs) CD4 + accompagnée d'une augmentation de cellules T CD8 +. L'analyse viSNE (avancé tSNE) a en outre montré une réduction de la population effectrice expérimentée à l'antigène dans les TILs CD8 + chez Tbet -/-. Cette population est marquée par la réduction de CD44. L'inhibition de GSK-3 n'a montré aucun effet sur la perte de DC, TILs CD4 +, PMN et les TILs CD8 + ainsi que l’expression de Granzyme B (GZMB) sur les cellules T CD8 +. La seule exception était une augmentation mineure néanmoins statistiquement significative du facteur de transcription Eomesdermin (Eomes) dans les TILs CD8 +. L'étude démontre un effet compensatoire inattendu de l'inhibition de GSK-3 sur la perte de T-bet. Il reste à élucider la nature complète du parcours de cette compensation. / The transcription factor T-bet plays a central role in regulating T-cell differentiation, while the protein tyrosine kinase, glycogen synthase kinase 3 (GSK-3) inhibits T-cell activation and controls the expression of inhibitory receptors PD-1 and LAG-3 on T-cells. Although GSK-3 inhibition can increase T-bet expression, the inter-relationship between T-bet and GSK-3 in tumor immunity is unknown. In this study, we show that T-bet knock-out (Tbet-/-) mice are compromised in their ability to control the growth of the B16 melanoma tumor cells. However, the injection of a small molecule inhibitor (SMI) of GSK-3 reverses this compromised condition resulting in the control of tumor growth similar to that seen in wild type mice. An examination of Tbet-/- showed a loss of dendritic cells (DC) and potentially suppressive polymorphonuclear leucocytes (PMN) and CD4+ cell tumor infiltrating lymphocytes (TILs) accompanied by an increase in CD8+ cells. viSNE analysis (advanced tSNE- t-Distributed Stochastic Neighbor Embedding) further showed a reduction of antigen experienced effector marker CD44 in CD8+ TILs in Tbet-/-. GSK-3 inhibition showed no effect on the loss of DCs, CD4+ TILs or the presence of PMNs or CD8+ T-cells or the loss of Granzyme B (GZMB) on CD8+ cells. The one exception was a minor but statistically significant increase in the transcription factor Eomesodermin (Eomes) in CD8+ TILs. The study demonstrates an unexpected compensatory effect of GSK-3 inhibition on the loss of T-bet. The full nature of the pathway that accounts for this compensation remains to be elucidated.