The expression of the <I>glycogen phosphorylase- 2</I> gene (<I>gp2</I>) is initiated during early development and regulated by the extracellular morphogens cAMP and Differentiation Inducing Factor (DIF-1) [1-3]. Glycogen phosphorylase- 2 catalyzes the breakdown of glycogen reserves in developing cells to generate glucose precursors required for the synthesis of the end products of differentiation [4-6]. Thus, the expression of <I>gp2</I> is a significant event for cellular differentiation. The sequence of the <I>gp2</I> promoter, like other <I>Dictyostelium</I> promoters, has an AT-rich bias (88%) [7]. Previous deletional analyses of the promoter provided a map of the regions that contained transcriptional regulatory elements. The regions thus identified contained either "TAAAAATGGA" or C-rich repeat sequences [2]. These regions were dissected further by site-directed mutagenesis (SDM) to better define the physical boundaries of the regulatory elements. It was shown that the mutation of either one of the C-rich repeats resulted in a dramatic drop of about 95% in reporter gene levels. These data strongly suggested that both the C-rich repeats of <I>gp2</I> functioned as transcriptional regulatory elements. I have identified and purified a factor called TF2 that demonstrates a high specificity for a C-rich transcriptional regulatory element, the 5' C box. TF2 was first detected with electrophoretic mobility shift assays of DEAE chromatographic fractions of cell-free extracts. The specificity of TF2 for the 5' C box was tested by competition analysis using six other oligonucleotides. Purification of TF2 was achieved by ion-exchange chromatography, DNA affinity chromatography, gel filtration chromatography, and preparative SDS-PAGE. SDS-PAGE analysis indicated an apparent subunit molecular weight of 28 kDa. The apparent molecular weight of the native protein as estimated by gel filtration was about 53 kDa. This suggested that TF2 binds gp2 as a homodimer. A cDNA clone of the tf2 gene was provided by the Japanese <I>Dictyostelium</I> cDNA project. This allowed me to synthesize probes for Southern and Northern blot analyses. Southern blot analysis indicated that there is only one form of the <I>tf2</I> gene. Northern analysis showed little or no expression of <I>tf2</I> in undifferentiated cells. During development <I>tf2</I> expression increases up to a maximum at 8 h, then decreases in later stages. Attempts to disrupt the gene suggest that <I>tf2</I> mutation may be lethal. / Ph. D.

Pink heelsplitter (Potamilus alatus) mussels collected from Kentucky Lake, TN were held at two bottom locations (0.6 m, 2.5 m) and suspended in pocket nets (at depth about 1.0 - 1.5 m) in a pond at the Freshwater Mollusk Conservation Center (FMCC), Virginia Tech, for 1 yr. Survival of mussels after 1 yr was significantly different, with poorest survival (30 %) in the bottom of the deep end; and no difference between the shallow end (83.3 %) and the suspended pocket nets (63.3 %). Survival of mussels was inversely related to water temperature (r = - 0.72); lowest monthly survival occurred in summer, resulting in a significant difference among the three locations with a similar trend after 1 yr. The glycogen reserves of mussels in captivity for 1 yr differed by pond location, higher in mussels at the shallow end than those in suspended pocket nets and at the deep end. Therefore, the shallow end of pond was more suitable for holding mussels long-term, while the suspended pocket nets are an alternative site for holding captive mussels. Additionally, dissolved oxygen was very low at the deep end (1.9 mg/L) in summer, while it was adequate (range from 5.7 - 6.4 mg/L) at the location of suspended pocket nets, and 5.0 mg/L at the shallow end (24.7 °C). Data for 40 specimens indicated that sexual dimorphism in valve shape occurred in P. alatus. Female mussels had a significantly (p < 0.0001) greater ratio of height (H) to length (L) (52.3 %) and width (W) to length (31.8 %) than males (H/L: 48.4 %; W/L: 28.8 %), respectively. The posterior ends were somewhat round to oval in males and bluntly squared or truncated in females. Female mussels were more inflated than males. These morphological differences can be used to distinguish females from males during field collections. The red drum (Sciaenops ocellatus) was identified as a new fish host for P. alatus, as 48 active juveniles were transformed by this species, which is not a natural host. Four glochidia were observed on the fins versus 2,307 on the gills of five red drum. Freshwater drum also was verified as a suitable host fish, but black crappie (Pomoxis nigromaculatus), banded sculpin (Cottus carolinae), yellow perch (Perca flavescens) and nile tilapia (Oreochromis nilotica) did not support transformation of glochidia to juveniles. Survival and growth of propagated juveniles of P. alatus were assessed regarding the effects of algal diets (Nannochloropsis oculata and Neochloris oleoabundans) and substrate type (fine sediment and sand). Overall, survival of juveniles after 17 d ranged from 23.8 to 66.8 %, with mean of 48.5 %; however, survival dramatically declined during the next 2 wk period to only 5.8 % (range of 1.8 to 7.8 %). Survival rate of juveniles was significantly different (p = 0.027) between substrates, but not in diets (p = 0.520), with the lowest survival rate of 23.8 % in sand substrate and fed N. oculata. Juveniles grew faster in fine sediment (23.0 % increase in shell length) than in sand substrates (10.5 % increase) (p = 0.002). Moreover, mean growth rate of juveniles was 4.9 &#956;m/d during the first 2 wk, but decreased to 0.2 &#956;m/d in the remaining 2 wk. Therefore, fine sediments seemed more appropriate for juvenile culture compared to sands. Both species of algae, N. oculata and N. oleoabundans, can be used to feed juveniles in the laboratory. Adult pink heelsplitters were used to study feasibility of pearl production by using two surgical implants (non-nucleated implant = NNI, and image pearl implant = IPI) in two ponds of different nutrient levels (FMCC pond and Duck pond). NNI and IPI pearls with purple or purplish luster were successfully produced in P. alatus. Pearl weight was not significantly different (p = 0.562) between two ponds. No differences in monthly survival rates of mussels were observed in either pond (p = 0.051), or among mussels with surgical implants and the no-surgery control mussels (p = 0.881). Consequently, P. alatus can be considered a potential species for producing purple pearls in pearl culture. Additionally, mussels in the Duck pond had higher (p < 0.0001) glycogen levels, similar to those in wild collected mussels, than those in the FMCC pond, indicating that this pond environment may be more suited for holding implanted mussels in captivity. / Master of Science

The gene encoding the Dictyostelium replication protein A large subunit (DdRPA1) has been cloned by screening of an EcoR I partial genomic library and a Hind III genomic sub-library. The complete nucleotide sequence, including the promoter region of the gene has been obtained by sequencing. Though the DdRPA1 protein has a size shift during development, 62 kDa in undifferentiated cells and 81 kDa in differentiated cells; they are the products of the same gene. Northern blot analysis revealed that the expression level of the DdRPA1 was constant throughout differentiation and the size of mRNA is the same at all stages, corresponding to a 81 kDa protein. Thus, it seems that the size change between the 62 kDa and 81 kDa is probably due to posttranslational modification, most likely, proteolytic cleavage. The transcription start site for both sizes of DdRPA1 has been identified at 306 bp upstream of the coding sequence by primer extension reaction. A PCR fragment representing 27% of the gene encoding the DdRPA middle size subunit (DdRPA2) has been generated by using the degenerate primers. This PCR fragment has been cloned and sequenced. The mRNA for this subunit corresponds to a protein of about 35 kDa. A decrease of the DdRPA2 mRNA expression level during differentiation was found by comparison between undifferentiated and differentiated cells. In Dictyostelium, replication protein A is a heterotrimeric protein that can bind with specific DNA sequences in a stage-dependent pattern. These DNA sequences were identified as the cis-acting regulatory sites in differentiation-related genes, including the glycogen phosphorylase 2 gene (gp2). Therefore, it is possible that DdRPA is not only a single-stranded DNA binding protein that is used in multiple essential DNA metabolic processes, such as DNA replication, repair and recombination in undifferentiated cells, but also involved in the transcriptional regulation process during differentiation. / Master of Science

The cellular slime mold, Dictyostelium discoideum, provides an ideal model system to study eukaryotic development, cell differentiation, and aging. A crucial developmental event in Dictyostelium is glycogen degradation. The degradation of glycogen provides glucose monomers that are used to synthesize structural components necessary for cellular differentiation. Glycogen degradation is catalyzed by glycogen phosphorylase, and two developmentally regulated glycogen phosphorylase activities have been discovered in Dictyostelium. Glycogen phosphorylase 1 (gp-1) activity is predominant early in development, and is dependent upon 5’ AMP as a positive allosteric modifier; glycogen phosphorylase 2 (gp-2) activity peaks late in development and is independent of 5° AMP. I showed that these two glycogen phosphorylase activities are associated with unique proteins that are the products of two distinct, but related, genes. Both genes were observed to be typical Dictyostelium genes in a number of respects. The gp-1 and gp-2 enzymes were also found to be similar to glycogen phosphorylases from other organisms. I also examined the developmental expression of these genes and found that both mRNAs are developmentally regulated; gp-1 mRNA levels fluctuate during development, while gp-2 mRNA levels increase late in development. The expression of the gp-1 and gp-2 genes is regulated by exogenous cAMP. Exogenous cAMP enhances the level of gp-1 mRNA, apparently through a mechanism that requires intracellular cAMP signaling. Specific DNA sequence elements appear to be required for maximal vegetative and late developmental expression of gp-1. Exogenous cAMP induces the appearance of gp-2 mRNA via a mechanism that appears to be independent of intracellular cAMP signaling. Repeated TA-rich sequences located between nucleotides 193 and 305 upstream of the transcriptional start site are necessary for maximal cAMP induction of gp-2. I also examined the cell type specific expression of gp-1 and gp-2. gp-1 is expressed predominantly in pre-stalk cells. gp-2 1s expressed in both cell types in a temporally regulated fashion; this type of expression has not been reported for other Dictyostelium genes, but, given the importance of glycogen degradation in both stalk and spore cells, it is not inconceivable that such regulation 1s necessary. / Ph. D.

The purpose of this investigation was to determine the effects of post-exercise carbohydrate-protein feedings on muscle glycogen restoration following exhaustive cycle ergometer exercise. Seven male collegiate cyclist (age=25.6±3.3y, ht.=180.9±8.5cm, wt.=75.4±10.7kg, VO2max=4.20±0.4 1•miri 1) performed three trials, each separated by -lwk, 1) 100% (x-D glucose (CHO), 2) 70% carbohydrate-20% protein-10% fat (CHOPRO), and 3) 86% carbohdyrate-14% amino acid (CHO-AA). All feedings were eucaloric, based upon 1.0 g•kgb.W.'1•hr"1 of carbohydrate, and administered every half hour during a four hour muscle glycogen restoration period in an 18% wt./vol. solution. Muscle biopsies were obtained immediately and four hours post exercise. Following the exhaustive exercise and every half hour for four hours a blood sample was drawn. Muscle glycogen concentrations increased 53%, 47%, and 57% for the CHO, CHO-PRO, and CHO-AA feedings, respectively, however no differences among the feedings were apparent in muscle glycogen restoration. The plasma glucose and insulin concentrations demonstrated no differences throughout the restoration period among the three feedings. These results suggest that muscle glycogen restoration does not appear to be enhanced with the addition of either protein or amino acids to an eucaloric carbohydrate feeding following an exhaustive cycle exercise. However, it appears that if adequate amounts of carbohydrates are consumed (greater than 0.70 g•kgb,W,."'•hf' carbohydrate) following exhaustive exercise, maximal muscle glycogen restoration occurs. / School of Physical Education

Branched chain sugars display a varied and valuable range of biological activities. This thesis concerns the synthesis of 3,5-di-C-methyl-D-glucose, a potential inhibitor of glycogen phosphorylase (GP), and therefore a proposed therapeutic agent for type 2 diabetes. Chapter 1 looks at the occurrence of branched sugars in the natural world and current therapies for type 2 diabetes. Inhibition of GP is explored, and the molecular modelling studies which led to the design of the project target. Chapter 1 also looks into the development of new foodstuffs, the chemistry and biochemistry of imino sugars and branched hydroxy proline analogues. In Chapter 2, a range of different approaches to 3,5-di-C-methyl-D-glucose are investigated. Most of the initial investigations were carried out on the L-enantiomer, a readily available test system deriving from 2-C-methyl-D-ribono lactone. 2-C-Methyl-D-ribono lactone is synthesised rapidly from D-glucose in a one-pot reaction; as the key starting material for this work, the scalability of this process was investigated. One of the attempted syntheses of di-C-methyl glucose lead to the development of a route towards 3,5-di-C-methyl fructose, a novel dibranched ketose sugar. It was envisaged that through an enzymatic transformation, it might be possible to produce 3,5-di-C-methyl glucose stereoselectively. Synthesis of both enantiomers of 3,5-di-C-methyl glucose and mannose are reported, alongside results of GPb inhibition studies. Analysis of the preferred ring size of a range of di-C-methyl branched sugars and sugar lactones generated in this work is also presented. Chapter 3 explores the chemistry of 2,4-di-C-methyl-L-arabinono lactone, a key intermediate in the synthesis of 3,5-di-C-methyl-L-glucose. From this lactone a novel deoxy sugar, 2-deoxy-2,4-di-C-methyl-L-arabinono lactone, was generated. Routes towards a selection of imino sugars were explored, resulting in the synthesis of a methyl branched isofagomine analogue. A substituted aziridine was synthesised, from which a route to a di-C-methyl branched piperidine was proposed, and a pyrrolidine. Also presented is a synthesis of a dihydroxy di-C-methyl branched proline analogue. Detailed NMR analysis of several of the sugars generated in this work was carried out by Dr M. Wormald, of the University of Oxford Biochemistry department. The results of these investigations are presented in the Appendix. Throughout this work, the presence of quaternary centres has posed a problem with the assignment of relative configuration. As a result, this work has been greatly supported by X-ray crystallography, and the structures shown herein were wholly generated by me. Several other crystals were run during the course of this work, not all pertaining to these projects, and are provided in the CD appendix.

A depressão é o mais comum dos distúrbios afetivos. Afeta ao menos 10% da população idosa do Brasil. Nos idosos, alguns fatores ligados ao metabolismo parecem estar bastante relacionados a esse transtorno, como uma menor concentração de noradrenalina e serotonina (5-HT) e uma maior atividade da monoaminooxidase em relação a adultos jovens. Os inibidores seletivos da recaptação da serotonina (ISRS), principalmente a sertralina, são a primeira opção no tratamento da fase aguda e manutenção dos episódios depressivos em idosos. As plaquetas vêm sendo amplamente utilizadas como modelo para estudar na periferia alterações que ocorrem no sistema nervoso central. A 5-HT apesar de ser primordialmente expressa no cérebro, também pode ser encontrada em plaquetas. Este neurotransmissor está envolvido em inúmeros aspectos do funcionamento normal do cérebro desde a regulação do humor até a regulação hormonal. A deficiência nos níveis de 5-HT pode estar intimamente ligada a alguma anormalidade na atividade da glicogênio sintase quinase 3B(GSK3B). Esta enzima exerce funções no metabolismo celular que vão desde sobrevivência celular, metabolismo e processamento de proteínas, até processos cognitivos. A atividade da GSK3B é estreitamente regulada pela fosforilação. Fosforilação no sítio ser9 inativa a enzima, enquanto que a desfosforilação neste mesmo sítio ativa a enzima. Diversos estudos têm mostrado que a forma inativa da enzima exerce um efeito neuroprotetor. O objetivo do presente estudo foi verificar a influência do tratamento com sertralina, em pacientes idosos com diagnóstico de depressão maior, sobre a 5- HT e GSK3B após 3 e 12 meses de tratamento. A quantificação da 5-HT foi realizada por HPLC e da GSK3B plaquetária, pelos métodos de ELISA e blotting, que se revelaram equivalentes. Após um ano de tratamento encontramos uma diminuição da 5-HT plaquetária nos pacientes com depressão maior com relação aos níveis basais, bem como um aumento da forma total da enzima GSK3B (GSKT), uma diminuição da forma fosforilada (pGSK) e da razão entre pGSK e GSKT (rGSK). Quando comparados os níveis de GSK3B de pacientes tratados por um ano e controles, observamos uma maior expressão de GSKT em pacientes; enquanto a pGSK e rGSK se mostraram equivalentes. Pudemos observar, portanto, uma modulação da 5-HT e da GSK3B pelo uso de sertralina. Essa modulação pode indicar que a ação antidepressiva deste fármaco pode estar associada a essas vias de sinalização / Depression is the most common affective disorders. It affects at least 10% of the elderly population of Brazil. In the elderly, some factors related to metabolism appear to be closely related to this disorder, such as lower concentration of noradrenaline and serotonin (5-HT) and increased monoamine oxidase activity in relation to young adults. The selective serotonin reuptake inhibitors (SSRI), especially sertraline are the first choice in treating acute and maintenance of depressive episodes in the elderly. Platelets have been widely used as a model to study in peripheral changes that occur in Central Nervous System. Although 5-HT is primarily expressed in the brain, it can also be found in platelets. This neurotransmitter is involved in numerous aspects of normal brain function since the regulation of mood to the hormonal regulation. A deficiency in 5-HT levels may be closely related to an abnormality in glycogen synthase kinase 3B (GSK3B) activity. This enzyme plays several functions in cell metabolism, ranging from cell survival, metabolism and protein processing, to cognitive processes. The GSK3B activity is tightly regulated by phosphorylation. Phosphorylation on Ser9 site inactives the enzyme, whereas dephosphorylation in the same site actives the enzyme. Several studies have shown that the inactive form of the enzyme plays a neuroprotective effect. The objective of this study was to investigate the influence of sertraline in elderly patients diagnosed with major depression, on platelet 5-HT and GSK3B after 3 and 12 months of treatment. Quantification of 5-HT was performed by HPLC and GSK3B by ELISA and western blotting. The methods for platelet GSK3B determination showed to be equivalent. After one year of treatment we found a decrease of platelet 5-HT in patients with major depression relative to their baseline levels, as well as an increase in the total form of GSK3B enzyme (GSKT), a decrease in phosphorylated form (pGSK) and the ratio between pGSK and GSKT (rGSK). Comparing the levels of GSK3B of patients with one year of treatment and controls, we found a higher GSKT expression in patients; while pGSK and rGSK showed to be equivalent. Therefore we observed a modulation of 5-HT and GSK3B by sertraline. This modulation may indicate that the antidepressant action of this drug may be associated with these signaling pathways

O processo de secreção salivar é dependente de energia, consome glicose e pode mobilizar glicogênio na glândula submandibular. Nos ratos diabéticos a produção de saliva estimulada é reduzida e ocorre acúmulo de glicogênio nas glândulas parótida e submandibular. O objetivo deste trabalho foi avaliar in vivo o metabolismo de glicogênio das glândulas salivares, submandibular e parótida, de ratos diabéticos após o estimulo com agonistas colinérgico e adrenérgico e também analisar se os animais alimentados ou com restrição alimentar overnight apresentam diferenças no metabolismo de glicogênio das glândulas salivares nas condições estudadas. Os ratos foram divididos em grupos controles (C) e diabéticos (D). Após 30 dias da indução do diabete com estreptozotocina i.p. 60mg/kg p.c., os animais foram subdivididos em alimentados ou em jejum, anestesiados com pentobarbital 50mg/kg p.c. e hidrato de cloral 400mg/kg p.c, administrado i.p. 7,5 mg/kg p.c de pilocarpina ou 5 mg/kg p.c. de isoproterenol, os ratos foram eutanasiados 0(T0), 30(T30), 60(T60) and 120(T120) minutos após a injeção do agonista. As glândulas SM e P foram removidas e analisadas quanto ao conteúdo de proteína total, glicogênio, atividade da glicogênio sintase (GS) e da glicogênio fosforilase (GP), ativa (a) e total (t). Os dados foram analisados estatisticamente pelo ANOVA e o teste de Tukey (p>0,05). A concentração de proteína total não foi afetada pela doença diabetes, nem pela administração dos agonistas, mas apresentou-se maior nos grupos em jejum quando comparados aos grupos alimentados. A concentração de glicogênio inicial foi maior nos ratos diabéticos quando comparados ao controles nas glândulas SM e P. O estímulo com a pilocarpina e com o isoproterenol na SM dos ratos alimentados e em jejum promoveu a degradação do glicogênio observada em T30 e posterior recuperação do conteúdo até o T120 nos grupos controles e diabéticos. Na P os agonistas não mobilizaram o glicogênio no grupo controle e sim no grupo diabético. As enzimas GP e GS tiveram a atividade alterada pelos agonistas e pela doença diabetes, porém não apresentaram um padrão nas condições estudadas. Os animais em jejum apresentaram menor conteúdo de glicogênio que os diabéticos nas glândulas SM e parótida e as enzimas GS apresentou um aumento na relação da forma ativa e total nos grupos em jejum e a GP apresentou menores valores que foi mais evidente na glândula SM. A injeção dos sialogogos apresentou efeitos diferentes no metabolismo de glicogênio das glândulas P e SM, assim como nos animais diabéticos / The salivary secretion process is energydependent, consumes glucose and might mobilize glycogen in the submandibular glands. In diabetics rats the stimulated saliva flow rate is reduced and accumulate glycogen in submandibular (SM) and parotid (P). The aim of this work was evaluated in vivo glycogen metabolism in the SM and P of diabetic rats stimulated with adrenergic or cholinergic agonists, and to analyze if there are any differences in the glycogen metabolism in fed or unfed (alimentary fasting overnight) animals.The rats were divided in control (C) and diabetic (D) groups. Thirty days after diabetes induction with streptozotocin (60mg/kg b.w. i.p.), the animals were subdivided in fed or unfed, anaesthetized with pentobarbital (50mg/kg b.w. i.p.). and chloral hydrate (400mg/kg b.w. i.p.), injected pilocarpine (7.5mg/kg b.w.) or isoproterenol (5mg/kg b.w.) intraperitoneally, and euthanized 0(T0), 30(T30), 60(T60) and 120(T120) minutes post-injection of the agonists. SM and P were excised and assessed for glycogen and protein content and glycogen synthase (GS) and phosphorylase (GP) active (a) and total (t) activities. Data was statistically analyzed by ANOVA and Tukeys test (p<0.05). Protein concentration didn´t alter by diabetes or agonist injections but was higher in unfed when compared to the fed rats. Increased initial glycogen content was found in both groups of glands in diabetic rats when compared to the control group. Pilocarpine and isoproterenol stimulus promoted glycogen degradation in SM of fed and unfed rats on T30 and the T120 SM control and diabetic groups recovered glycogen content as the initial T0 values. In P the agonist mobilized glycogen just in diabetic group. The GP and GS activities were different and didnt present a pattern in this study´s condition. The unfed animals present glycogen content diminished when compared to fed animal in both glands and the relation of active and total glycogen synthase was higher in fast animals and lesser specific activities of active and total glycogen phosphorilase that were more evident in submandibular glands. The secretagogues injection presents different effects on glycogen metabolism of P and SM even so in diabetic animals.

Objetivo: A glicogênio sintase quinase-3 beta (GSK-3B) é uma enzima presente em diversos sistemas biológicos e está envolvida na fisiopatologia de vários transtornos neuropsiquiátricos, incluindo o transtorno bipolar. No entanto, estudos in vivo da GSK-3B que envolvam pacientes bipolares são escassos. O objetivo do presente estudo foi avaliar a atividade da GSK-3B em plaquetas de pacientes idosos com transtorno bipolar em tratamento, em comparação com idosos saudáveis não medicados. Métodos: Foram obtidas amostras de plaquetas de 63 idosos (transtorno bipolar=31, grupo controle=32). A atividade enzimática foi estimada pela razão entre a expressão da forma fosforilada (inativa) da GSK-3B em relação à expressão de ambas as formas (ativa e inativa) da enzima (GSK-3B total), que fornece uma estimativa inversa da atividade enzimática (um aumento da razão indica menor atividade da GSK-3B). A intensidade dos sintomas foi avaliada pela Escala de Depressão de Hamilton de 21 itens e pela Escala de Mania de Young, e o desempenho cognitivo foi avaliado pelo Cambridge Cognitive Test e pelo Mini- Exame do Estado Mental. Resultados: A forma fosforilada da GSK-3B (fosfo-GSK-3B) e a razão da GSK-3B estavam elevadas em pacientes com transtorno bipolar, quando comparadas aos idosos do grupo controle (p=0,018 e p=0,016, respectivamente). Na avaliação por subgrupos, observaram-se níveis da fosfo-GSK-3B e da razão da GSK-3B mais elevados nos pacientes com transtorno bipolar em uso de lítio, quando comparados aos controles (p=0,030 e p=0,023, respectivamente), mas não quando comparados aos pacientes com transtorno bipolar que não usavam lítio. O uso das demais medicações avaliadas (anticonvulsivantes, antipsicóticos, antidepressivos e 16 benzodiazepínicos) não estava associado a diferenças na fosfo-GSK-3B ou na razão da GSK-3B, quando comparado aos controles. Conclusões: A atividade da GSK-3B está diminuída no presente grupo de idosos com transtorno bipolar em tratamento medicamentoso. A ausência de um grupo de pacientes com transtorno bipolar não medicado, e a não uniformidade das medicações utilizadas não nos permitem afirmar se essa redução se deve à características da doença bipolar em si ou seria influência dos medicamentos utilizados / Objective: Glycogen synthase kinase-3 beta (GSK-3B) is an important enzyme present in various biological systems and it is involved in the pathophysiology of many prevalent neuropsychiatric diseases, including bipolar disorder. However, human studies addressing GSK-3B activity in vivo are scarce. The aim of the present study was to evaluate GSK-3B activity in platelets of elderly patients with bipolar disorder undergoing clinical treatment as compared to healthy older adults unmedicated. Methods: Platelets samples where obtained from 63 older adults (bipolar disorder=31, comparison group=32). Enzymatic activity was estimated by means of the ratio between the expression of the phosphorylated (inactive) form of GSK-3B to the expression of both forms (active and inactive) of the enzyme (total GSK-3B), yielding an inverse estimate of enzymatic activity (higher ratio indicating lower GSK- 3B activity). The magnitude of mood symptoms was evaluated by the Hamilton Depression Scale and Young Mania Rating Scale, and the cognitive performance was assessed by the Cambridge Cognitive Test and the Mini-Mental State Examination. Results: The phosphorylated form of GSK-3B (phospho-GSK-3B) and the GSK-3B ratio were elevated in patients with bipolar disorder as compared to healthy controls (P=.018 and P=.016, respectively). When analyzed by subgroups, phospho-GSK-3B and the GSK-3B ratio were elevated in bipolar patients undergoing lithium treatment as compared to healthy controls (P=.030 and P=.023, respectively), but not when compared to bipolar patients without lithium treatment. The use of other drugs evaluated (anticonvulsants, antipsychotics, antidepressants and benzodiazepines) was not associated with distinct values of either phospho-GSK-3B or GSK-3B ratio, when compared to controls. 18 Conclusions: GSK-3B activity is decreased in this group of older adults with bipolar disorder undergoing pharmacological treatment. The absence of a group of unmedicated bipolar patients and the non-uniform pattern of treatment do not allow us to say whether this reduction is due to characteristics of bipolar illness itself or an influence of the therapeutic drugs in use

A depressão é o mais comum dos distúrbios afetivos. Afeta ao menos 10% da população idosa do Brasil. Nos idosos, alguns fatores ligados ao metabolismo parecem estar bastante relacionados a esse transtorno, como uma menor concentração de noradrenalina e serotonina (5-HT) e uma maior atividade da monoaminooxidase em relação a adultos jovens. Os inibidores seletivos da recaptação da serotonina (ISRS), principalmente a sertralina, são a primeira opção no tratamento da fase aguda e manutenção dos episódios depressivos em idosos. As plaquetas vêm sendo amplamente utilizadas como modelo para estudar na periferia alterações que ocorrem no sistema nervoso central. A 5-HT apesar de ser primordialmente expressa no cérebro, também pode ser encontrada em plaquetas. Este neurotransmissor está envolvido em inúmeros aspectos do funcionamento normal do cérebro desde a regulação do humor até a regulação hormonal. A deficiência nos níveis de 5-HT pode estar intimamente ligada a alguma anormalidade na atividade da glicogênio sintase quinase 3B(GSK3B). Esta enzima exerce funções no metabolismo celular que vão desde sobrevivência celular, metabolismo e processamento de proteínas, até processos cognitivos. A atividade da GSK3B é estreitamente regulada pela fosforilação. Fosforilação no sítio ser9 inativa a enzima, enquanto que a desfosforilação neste mesmo sítio ativa a enzima. Diversos estudos têm mostrado que a forma inativa da enzima exerce um efeito neuroprotetor. O objetivo do presente estudo foi verificar a influência do tratamento com sertralina, em pacientes idosos com diagnóstico de depressão maior, sobre a 5- HT e GSK3B após 3 e 12 meses de tratamento. A quantificação da 5-HT foi realizada por HPLC e da GSK3B plaquetária, pelos métodos de ELISA e blotting, que se revelaram equivalentes. Após um ano de tratamento encontramos uma diminuição da 5-HT plaquetária nos pacientes com depressão maior com relação aos níveis basais, bem como um aumento da forma total da enzima GSK3B (GSKT), uma diminuição da forma fosforilada (pGSK) e da razão entre pGSK e GSKT (rGSK). Quando comparados os níveis de GSK3B de pacientes tratados por um ano e controles, observamos uma maior expressão de GSKT em pacientes; enquanto a pGSK e rGSK se mostraram equivalentes. Pudemos observar, portanto, uma modulação da 5-HT e da GSK3B pelo uso de sertralina. Essa modulação pode indicar que a ação antidepressiva deste fármaco pode estar associada a essas vias de sinalização / Depression is the most common affective disorders. It affects at least 10% of the elderly population of Brazil. In the elderly, some factors related to metabolism appear to be closely related to this disorder, such as lower concentration of noradrenaline and serotonin (5-HT) and increased monoamine oxidase activity in relation to young adults. The selective serotonin reuptake inhibitors (SSRI), especially sertraline are the first choice in treating acute and maintenance of depressive episodes in the elderly. Platelets have been widely used as a model to study in peripheral changes that occur in Central Nervous System. Although 5-HT is primarily expressed in the brain, it can also be found in platelets. This neurotransmitter is involved in numerous aspects of normal brain function since the regulation of mood to the hormonal regulation. A deficiency in 5-HT levels may be closely related to an abnormality in glycogen synthase kinase 3B (GSK3B) activity. This enzyme plays several functions in cell metabolism, ranging from cell survival, metabolism and protein processing, to cognitive processes. The GSK3B activity is tightly regulated by phosphorylation. Phosphorylation on Ser9 site inactives the enzyme, whereas dephosphorylation in the same site actives the enzyme. Several studies have shown that the inactive form of the enzyme plays a neuroprotective effect. The objective of this study was to investigate the influence of sertraline in elderly patients diagnosed with major depression, on platelet 5-HT and GSK3B after 3 and 12 months of treatment. Quantification of 5-HT was performed by HPLC and GSK3B by ELISA and western blotting. The methods for platelet GSK3B determination showed to be equivalent. After one year of treatment we found a decrease of platelet 5-HT in patients with major depression relative to their baseline levels, as well as an increase in the total form of GSK3B enzyme (GSKT), a decrease in phosphorylated form (pGSK) and the ratio between pGSK and GSKT (rGSK). Comparing the levels of GSK3B of patients with one year of treatment and controls, we found a higher GSKT expression in patients; while pGSK and rGSK showed to be equivalent. Therefore we observed a modulation of 5-HT and GSK3B by sertraline. This modulation may indicate that the antidepressant action of this drug may be associated with these signaling pathways