Introdução: A glicogenose hepática tipo 1 (GSD I) é um erro inato do metabolismo (EIM), ocasionada pela presença de mutações patogênicas em genes que codificam enzimas envolvidas no catabolismo do glicogênio, levando ao acúmulo desse substrato e de gordura no fígado, rins e mucosa intestinal, ocasionando alterações metabólicas importantes que comprometem significativamente a qualidade de vida do indivíduo. O tratamento da GSD I é essencialmente dietético, e objetiva proporcionar uma fonte contínua de glicose para evitar a hipoglicemia e prevenir distúrbios metabólicos secundários, através da administração frequente de amido de milho cru (AMC) e/ou dieta contínua noturna administrada por sonda nasogástrica ou gastrostomia, além da restrição de frutose, sacarose e lactose, bem como suplementação de vitaminas e minerais. Em pacientes com complicações graves, o transplante hepático pode ser indicado. A não adesão aos tratamentos é um problema de saúde pública de magnitude mundial e sabe-se que a baixa adesão às intervenções prescritas é um problema complexo e presente especialmente em pacientes com doenças crônicas. Não foram encontrados estudos sobre adesão ao tratamento em glicogenoses hepáticas. Estudos realizados sobre adesão relacionada à fenilcetonúria (EIM com tratamento com restrição alimentar semelhante, sob alguns aspectos, às glicogenoses) indicaram, para essa doença, que o nível de adesão depende de múltiplos fatores e também foi evidenciado um alto nível de não adesão. Objetivo: Caracterizar a adesão ao tratamento de pacientes com GSD I e identificar os fatores que influenciam a adesão desses pacientes. Métodos: Estudo transversal cuja amostra foi selecionada por conveniência, incluindo indivíduos com diagnóstico de GSD Ia e GSD Ib acompanhados no ambulatório de EIM do Serviço de Genética Médica do Hospital de Clínicas de Porto Alegre. Os dados foram coletados através de revisão de prontuário e entrevista com pacientes ou familiares. Dois questionários foram aplicados a eles contendo questões sobre hábitos de vida, suporte social, percepção da dieta e conhecimento sobre a doença. A adesão foi avaliada por meio dos marcadores biológicos de tratamento e as variáveis foram analisadas estatisticamente para verificar possíveis associações. Resultados: Dezoito pacientes foram incluídos. A mediana de idade foi de 6,5 anos (IQ = 9 a 34 anos). Onze pacientes foram classificados como aderentes ao tratamento. A totalidade dos pacientes de GSD subtipo Ib foi aderente. O convívio com o pai e a mãe foi um dos fatores associados à adesão (p=0,049). Três pacientes tiveram um ótimo nível de 6 adesão. A maioria apresentou conhecimento satisfatório sobre a doença. Restrição dietética, acordar durante a madrugada, palatabilidade do AMC, custo da dieta e a distância da residência do paciente ao ambulatório foram as dificuldades mais relatadas. Conclusão: O estudo teve um índice maior de adesão do que o referenciado pela literatura para doenças crônicas. A adesão ao tratamento em GSD I é um tema complexo e que necessita de mais estudos. Compreender os fatores associados à adesão é necessário para a efetividade do tratamento. Nesse estudo, obtiveram-se informações importantes que possibilitam melhor compreensão sobre possíveis fatores que podem contribuir para não adesão ao tratamento das GSD I. Estratégias devem ser elaboradas pelo sistema de saúde e pelos profissionais com o intuito de que as dificuldades associadas ao tratamento sejam trabalhadas e minimizadas em conjunto com os pacientes ou familiares. / Introduction: Glycogen Storage Disease type I (GSDI) is an inborn error of metabolism (IEM) due to the presence of pathological mutations on genes that code enzymes involved in glycogen’s metabolism that leads to the accumulation of this substrate and fat in the liver, kidneys and intestinal mucosa and causes important metabolic alterations that significantly compromise the individual's quality of life. The treatment for GSDI is essentially dietetic and aims to provide a continuous source of glucose to avoid hypoglycemia and also to prevent secondary metabolic disorders, through the frequent administration of uncooked cornstarch (UCCS) and/or continuous nocturnal nasogastric feeding or gastrostomy, restricting fructose, saccharose and lactose besides supplementation of vitamins and minerals. Liver transplant may be indicated for more severely acute patients. Non-adherence to treatments is a world-wide public health matter and it is known that low adherence to prescribed interventions is a complex problem that is present especially in chronic disease patients. To date, no studies were found on adherence to treatment on hepatic glycogen storage disease. Studies on adherence to treatment related to phenylketonuria (EIM with dietetic restriction) indicate that, for this disease, the adherence level depends on multiple factors and a high level of non-adherence has been evidenced. Objective: To characterize adherence to treatment of GSDI patients and identify the factors that influence those patients' adherence. Methods: Convenience sampled in cross-sectional study, including individuals diagnosed with GSDIa and GSDIb followed by the IEM Ambulatory of the Medical Genetics Service of the Hospital de Clínicas de Porto Alegre. The data was collected through medical records review and interviews with patients and/or family members. Two questionnaires were administered with questions about life habits, social support, diet perception and knowledge about the disease. Adherence was evaluated by biochemical biomarkers and the variables were statistically analysed to verify possible associations. Results: 18 patients were included. The median age was 6,5 years. Eleven patients were classified as adherents to treatment. All the GSD patients with subtype Ib were adherent. Living with both parents was one of the factors associated to adherence. Three patients had an optimal level of adherence. Most patients showed satisfying knowledge about the disease. The most reported difficulties were dietetic restriction, waking up in the middle of the night, UCCS 8 palatability, diet cost and distance from the patient’s residence to the ambulatory clinic. Conclusions: The study had a higher index of adherence than referenced by the literature for chronic diseases. Adherence to treatment in GSDI is a complex theme that needs more research. It is necessary to understand the factors associated with adherence to result in effect treatment. In this study, important data was obtained that provides better comprehension of the possible factors that can contribute to non-adherence for the treatment of the GSDI types. Strategies must be considered and incorporated into standards of care by the Health Care System and health professionals to insure that patients and their families understand requirements needed to improve and minimize the difficulties associated with the treatment.

Lam Ching-wan. / "May 2000." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (p. 91-101). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: INTRODUCTION: Glycogen synthase kinase-3 (GSK-3) is a serine-threonine protein kinase that was first discovered as a regulator of glycogen synthase thus playing a role in glycogen synthesis (Embi et al. 1980). GSK-3 has also been shown to down regulate the expression of SERCA-2a (a calcium ATPase pump) thus playing a role in myocardial contractility (Michael et al. 2004). However, SERCA-2a activity is regulated by phospholamban (PLM) and sarcolipin (SLN) (Asahi et al. 2003). GSK-3 is constitutively active in cells and can be acutely inactivated by insulin through phosphorylation by PKB/Akt. However, GSK-3 is known to phosphorylate and inhibit IRS-1 protein, thus disrupting insulin signaling (Eldar-Finkelman et al. 1996). In addition, abnormally high activities of GSK-3 protein has been implicated in several pathological disorders which include type 2 diabetes, neuron degenerative and affective disorders (Eldar-Finkelman et al 2009). This led to the development of new generations of inhibitors with specific clinical implications to treat these diseases (Martinez 2008). GSK-3 inhibition has been shown to improve insulin and blood glucose levels and to be cardioprotective during ischemia/reperfusion (Nikoulina et al. 2002; Kumar et al. 2007). AIMS: To determine whether myocardial GSK-3 protein and its substrate proteins are dysregulated in obesity and insulin resistance, and whether a specific GSK-3 inhibitor can prevent or reverse the cardiovascular pathology found in obese and insulin resistant animals. OBJECTIVES: To correlate the alterations in expression and activation of GSK-3 protein in a well characterised rat model of obesity coupled to insulin resistance with: i) myocardial contractile dysfunction and an inability of hearts to withstand ischemia/reperfusion, ii) the activation and expression of phospholamban and SERCA-2a in the sarcoplasmic reticulum, iii) the activation of intermediates (IRS-1, IRS-2 and PKB/Akt) that lie upstream in the activation pathway of GSK-3 and iv) to determine the effects of inhibition of GSK-3 on the abovementioned parameters. METHODS: Age and weight matched male Wistar rats (controls and diet induced obese (DIO) animals) were used in the present study. Controls were fed normal rat chow, while DIOs were fed a rat chow diet supplemented with sucrose and condensed milk, for 8 or 16 weeks. Half of each group of animals were treated with the GSK-3 inhibitor for 4 weeks (from 12 to 16 weeks). After the feeding and treatment period, animals were weighed, sacrificed, hearts removed and freeze clamped immediately or perfused with Krebs-Henseleit buffer and subjected to low flow ischemia (25 min) followed by 30 min reperfusion. Biometric (body weight, intraperitoneal fat, ventricular weight and tibia length) and biochemical (fasting blood glucose and insulin levels) parameters were determined. Expression of GSK-3, PKB/Akt, IRS-1, IRS-2, SERCA-2a and Phospholamban were determined by Western blotting. Ca2+ ATPase activity was determined spectrophotometrically. RESULTS: At both 8 and 16 weeks DIO animals were significantly bigger than control animals and this was associated with increased intraperitoneal fat in DIOs. In DIO animals: IRS-1 was downregulated at 8 weeks and both IRS-1 and IRS-2 as well as PKB/Akt at 16 weeks. There was an increased tendency of GSK-3 expression at both 8 and 16 weeks in DIO animals while SERCA-2a was severely downregulated from 8 weeks onwards and associated with lower Ca2+-ATPase activity. PLM expression was upregulated but its phosphorylation was attenuated. At 16 weeks, baseline heart rate (225 vs 275 in control, P<0.0001, n=6) and rate pressure product (21000 vs 30000 in control, P=0.019, n=6) were significantly lower in hearts from DIO animals. Functional recovery was unchanged but the time to ischemic contracture development was increased (11.6±0.4 control vs 16.2±0.5 min DIO, P<0.01, n=6). Treatment had no effect on total GSK-3 expression. However, GSK-3 phosphorylation was significantly increased in treated controls, while there was no significant difference in DIO animals. However, there was a tendency for an increased GSK-3 phosphorylation in treated DIO animals. GSK-3 inhibitor, improved hypertrophy in DIO animals, while it led to its development in control animals. GSK-3 inhibitor improved IRS-2 expression in both control and DIO animals while it had no effect on IRS-1 and SERCA-2a expression and activity. However, GSK-3 inhibition increased PKB/Akt and phospholamban phosphorylation in DIO animals. CONCLUSION: These findings show that high calorie diet as well as imbalance between energy intake and expenditure lead to the development of obesity and insulin resistance in male Wistar rats. We showed that GSK-3 and its substrate proteins are dysregulated in obesity and insulin resistance. The reduced SERCA-2a expression at baseline may have a negative impact on cardiac function. By treating the animals with GSK-3 inhibitor, we showed that GSK-3 protein may not be responsible for changes seen at baseline. The decreased IRS-1 and SERCA-2a expression may have been caused by a different mechanism other than the actions of GSK-3. However, according to this study, GSK-3 may play a role in regulation of IRS-2 expression but not in IRS-1. Increased PKB/Akt phosphorylation may contribute to the GSK-3 inhibition. In addition, GSK-3 inhibition may reverse cardiac hypertrophy in DIO animals, thus acting as a negative regulator of hypertrophy. / AFRIKAANSE OPSOMMING: Inleiding: Glikogeen sintase kinase-3 (GSK-3), 'n serien/threonien proteïen kinase, is oorspronklik ontdek as 'n rolspeler in glikogeen sintese, aangesien dit 'n reguleerder van glikogeen sintase is (Embi et al.1980). Intussen is dit ook bevind dat GSK-3 die uitdrukking van SERCA-2a ('n kalsium ATPase pomp) kan afreguleer en dus sodoende 'n rol speel in miokardiale kontraktiliteit (Michael et al. 2004). Die aktiwiteit van SERCA-2a kan egter ook gereguleer word deur fosfolamban (PLM) en sarkolipin (Asahi et al. 2003). GSK-3 is deurgaans aktief, maar kan tydelik geïnaktiveer word onder kondisies van insulien stimulasie deur PKB/Akt gemedieerde fosforilering. Aan die ander kant is dit bekend dat GSK-3 die IRS-1 proteïen kan fosforileer om dus sodoende insulien sein-transduksie af te reguleer (Eldar-Finkelman et al. 1996). Daarmee saam is abnormaal hoë vlakke van GSK-3 aktiwiteit geassosieer met verskeie patologiese versteurings, insluitend tipe 2 diabetes, neuron degeneratiewe en affektiewe versteurings (Eldar-Finkelman et al. 2009). Daar is dus nuwe generasies GSK-3 inhibitore ontwikkel met die kliniese potensiaal om hierdie patologieë te behandel (Martinez 2008). Dit is al bevind dat GSK-3 inhibisie geassosieer kan word met beide die normalisering van plasma insulien- en glukose vlakke, asook kardiobeskerming in die konteks van iskemie/herperfusie (Nikoulina et al. 2002; Kumar et al. 2007). Doelwitte: Om te bepaal of GSK-3 proteïen en sy substraat proteïene gedisreguleer is onder kondisies van obesiteit en insulien weerstandigheid, asook om vas te stel of 'n spesifieke GSK-3 inhibitor die kardiovaskulêre patologie wat gevind word in obese en insulien weerstandige diere kan verhoed of omkeer. Mikpunte: Om veranderinge in uitdrukking en aktiwiteit van GSK-3 proteïen in 'n goed gekarakteriseerde rotmodel van obesiteit, gekoppel aan insulien weerstandigheid, te korreleer met die volgende: i) miokardiale kontraktiele disfunksie en onvermoë om kardiale iskemie/herperfusie besering te weerstaan, ii) aktivering en uitdrukking van PLM en SERCA-2a in die sarkoplasmiese retikulum, iii) die aktivering van intermediêres wat proksimaal geleë is in die insulienseintransduksiepad van GSK-3 (IRS-1, IRS-2 en PKB/Akt) en iv) om die effek van behandeling met 'n spesifieke inhibitor van GSK-3 op die bogenoemde punte te bepaal. Metodes: Ouderdoms- en gewigsgepaarde manlike Wistar rotte (kontrole en dieet geïnduseerde obees (DIO) diere) is in die studie gebruik. Kontrole diere was normale rotkos gevoer, terwyl die DIO diere op 'n dieet van rotkos aangevul met sukrose en kondensmelk geplaas is vir 'n periode van 8 of 16 weke. Helfte van die diere van elke groep is behandel met die GSK-3 inhibitor vir 4 weke (vanaf week 12 tot 16). Na afloop van die voer- en behandelingsperiode is die diere geweeg, doodgemaak en die harte verwyder om dan of onmiddelik gevriesklamp te word, of retrograad geperfuseer te word met Krebs-Hensleit buffer. Ex vivo geperfuseerde harte is dan blootgestel aan 25 minute lae vloei iskemie gevolg deur 30 minute herperfusie. Biometriese (liggaamsgewig, intraperitoneale vet, ventrikulêre gewig en tibia lengte) en biochemiese (vastende bloedglukose en -insulien vlakke) parameters is telkens bepaal. Western klad tegnieke is gebruik om die uitdrukking en fosforilering van GSK-3, PKB/Akt, IRS-1, IRS-2, SERCA-2a en PLM te bepaal. Ca2+-ATPase aktiwiteit is spektrofotometries bepaal. Resultate: Na beide 8 en 16 weke was die DIO diere beduidend swaarder as die kontrole diere. Hierdie gewigstoename was geassosieer met meer intraperitoneale vet in die DIO diere. Verder, in die DIO diere was IRS-1 afgereguleer na 8 weke, terwyl beide IRS-1 en IRS-2 asook PKB/Akt afgereguleer was na 16 weke. GSK-3 uitdrukking het 'n neiging getoon om toe te neem na beide 8 en 16 weke in die DIO diere, terwyl SERCA-2a beduidend afgereguleer was reeds vanaf 8 weke, geassosieer met laer Ca2+-ATPase aktiwiteit. PLM uitdrukking het toegeneem en die fosforilering daarvan was verlaag. Op 16 weke was die basale harttempo (225 vs 275 in die kontrole groep, P<0.0001, n=6) en tempo druk produk (21000 vs 30000 in die kontrole groep, P=0.019, n=6) betekenisvol laer in die DIO diere. Funksionele herstel het onveranderd gebly, alhoewel die tyd tot iskemiese kontraktuur toegeneem het in die DIO groep (kontrole: 11.6±0.4 min vs DIO: 16.2±0.5 min, P<0.01, n=6). Toediening van die inhibitor het geen effek op totale GSK-3 uitdrukking gehad nie. Fosforilering van GSK-3 was egter wel beduidend verhoog in die behandelde kontrole diere, terwyl daar geen verskille in die DIO groep was nie. Die fosforilering van GSK-3 het wel geneig na 'n toename in die behandelde DIO diere. Die GSK-3 inhibitor het kontrasterende effekte op hipertrofie gehad: dit het dit omgekeer in die DIO groep, maar veroorsaak in die kontrole diere. Daarmee saam het die inhibitor die uitdrukking van IRS-2 in beide DIO en kontrole diere gestimuleer, maar geen effek op IRS-1 en SERCA-2a uitdrukking en aktiwiteit gehad nie. GSK-3 inhibisie het wel PKB/Akt en PLM fosforilering in die DIO diere verhoog. Gevolgtrekking: Hierdie bevindinge toon dat 'n hoë kalorie dieet, tesame met 'n wanbalans tussen energie inname en verbruiking, lei tot die ontwikkeling van obesiteit en insulien weerstand in manlike Wistar rotte. Die studie het ook getoon dat GSK-3 en sy substraat proteïene wel gedisreguleer is in obesiteit en insulien weerstandigheid. Die verlaagde basale uitdrukking van SERCA-2a mag dalk 'n negatiewe impak hê op kardiale funksie. Behandeling van die diere met 'n GSK-3 inhibitor het getoon dat GSK-3 moontlik nie verantwoordelik is vir die basislyn veranderinge nie. Die afname in IRS-1 en SERCA-2a uitdrukking kan moontlik toegeskryf word aan ander meganismes buiten die effekte van GSK-3. Hierdie studie toon wel dat GSK-3 moontlik 'n rol speel in die regulering van die uitdrukking van IRS-2, maar nie IRS-1 nie. Verhoogde PKB/Akt fosforilering mag dalk bydra tot die inhibisie van GSK-3. Daarmee saam blyk dit dat GSK-3 inhibisie hipertrofie kan omkeer in DIO diere, om dan sodoende op te tree as 'n negatiewe reguleerder van hipertrofie, maar in normale kontrole diere, hipertrofie in die hand werk. / South African Medical Research Council / University of Stellenbosch, Dept. of medical Physiology

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.

Thirty-eight female Wistar rats were studied to determine the response of citrate synthase (CS) and glycogen (GLY) to two similar programs of endurance training. Animals were randomly assigned to one of three groups: run-trained (RUN), swim-trained (SWIM) or sedentary control (CON). The treadmill trained animals ran at a speed of 27 m/min. up an eight degree incline. The swim-trained animals swam with 2% of body weight attached to their tails. The duration of the exercise protocols was 2 hours/day, the frequency 5 days/week and the length of the training regimen was 10 weeks. Liver GLY content (mmoles/g) for the exercise trained groups was significantly higher (p < 0.01) than CON. There were no significant differences between RUN and SWIM animals in the GLY levels of the hindlimb muscles. The GLY levels of the forelimb muscles were significantly greater (p0.01) in the SWIM animals compared to the RIJN animals, apart from the pectoralis (EEC). The CS activity in the soleus (SOL) and red -vastus (RV) of the RUN animals was significantly larger (p <; 0.01) than SWIM. The plantaris (PLANT) of the SWIM animals had significantly greater CS activity than the RUN animals. In the forelimb muscles, only -the deltoid (DEL) of the SWIM group was higher in CS activity than the RUN groups. The results of this study indicate that the mechanisms responsible for increased GLY storage in skeletal muscle are under independent control to those factors governing the changes in the oxidative enzyme CS. Differences in muscle GLY levels and CS activity between RUN and SWIM rats can be explained by contrasting mechanics in these two (nodes of exercise and the resulting fiber recruitment patterns.

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.

Call number: LD2668 .T4 1986 N48 / Master of Science / Animal Science and Industry

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

As glândulas salivares são glândulas exócrinas que vertem seus produtos para cavidade oral. As principais glândulas são as parótidas, sublinguais e submandibulares sendo elas as responsáveis pela contribuição do maior volume de saliva durante o processo de secreção que, assim como todas as atividades que nosso corpo exerce, também dependem de energia. A secreção salivar consome glicose e mobiliza o glicogênio para adquirir energia, e este processo pode sofrer influencia de alguns fatores dentre eles o estado diabético e o ritmo circadiano. O diabetes altera todo o metabolismo de carboidratos e diminui o fluxo salivar. Já o ritmo circadiano promove uma alteração fisiológica no fluxo e composição da saliva de acordo com o horário do dia. Desta forma o objetivo deste trabalho foi em um primeiro momento observar o comportamento da concentração de glicogênio em glândulas parótidas e submandibulares de ratos com diferentes idades e condições alimentares em um determinado período do dia. Em um segundo momento observar as alterações que ocorrem na concentração de glicogênio em ratos diabéticos durante o dia. Na primeira fase do estudo foram utilizados ratos saudáveis com 21, 30 e 60 dias de vida, divididos em grupos alimentado e alimentados com restrição. No grupo com restrição de alimento os animais ficaram restritos a alimentação noturna (19 7 horas) desde 2 dias antes do sacrifício. Na segunda fase do estudo, com ratos diabéticos, foram utilizados animais com 60 dias de vida e a indução do diabetes foi realizada através de uma injeção intraperitoneal de estreptozotocina (65 mg/Kg p.c.). 30 dias após a indução os animais foram sacrificados. Em todos os grupos o sacrifício foi realizado nos seguintes horários - 7, 9, 11, 13, 15, 17 e 19 horas. As glândulas submandibulares e parótidas foram removidas imediatamente para posterior análise da concentração de glicogênio. Os dados foram analisados estatisticamente pelos testes ANOVA e o teste de Tukey (p<0.05). Os resultados obtidos com os animais normais mostra uma variação da concentração de glicogênio durante o período analisado nas duas glândulas, sendo mais evidente quando os ratos não foram submetidos a restrição de alimento. Nesta condição a variação da concentração de glicogênio em glândulas parótidas pouco alterou independente da idade. Nos animais diabéticos observamos um acúmulo de glicogênio nas glândulas parótidas e uma diminuição da concentração de glicogênio em submandibulares. Durante o período analisado também houve pouca variação da concentração de glicogênio, assim como nos animais não diabéticos, sendo evidente a menor interferência do ritmo circadiano no estado diabético. Esse estudo nos mostrou que o ritmo circadiano interfere na concentração de glicogênio das glândulas salivares parótidas e submandibulares de ratos durante o período analisado e que a restrição de alimento durante o dia alterou a concentração de glicogênio principalmente nas glândulas parótidas. Observamos também que no estado diabético ocorre um acúmulo do glicogênio em glândulas parótidas e uma diminuição nas glândulas submandibulares. / The secretory process is very important for oral health. The majors salivary glands (parotid, submandibular and sublingual) are the most important for secretion of saliva, for this activity the glands needs energy and a common way obtain is using glucose from the mobilization of glycogen during the fast period. Because of the importance of glycogen in this process, this study aimed to analyze the behavior of glycogen concentration during the day (7 to 19 hours) in parotid, and submandibular glands of rats. In the first moment the study was made with health male rats in different ages (21, 30 and 60 days old), divided in a group with free access to food and other group fed only during the night (19 to 7 hours). The second part of the study was made using diabetic male rats to search for alterations caused by this disease in glycogen concentration. All animals were killed during the day in different hours (7, 9, 11, 13, 15, 17,19 hours), their glands were removed and clamped between aluminium plates pre-cooled in dry ice. The frozen glands were then stored at -80oC until analysis of glycogen concentration. The statistical analyses was made using ANOVA test and Tukey (p<0,5). In the group of health rats we observed a variation of the glycogen concentration during the period analyzed in both glands, but it became more evident when the animals had free access to food. When they were fed during the night the variation of the glycogen concentration in parotid glands were not so evident. The results with diabetics rats showed a higher accumulation of glycogen in parotid glands and a lower concentration of glycogen in submandibular glands. In this period we could not see the variation of glycogen that happen in health rats. This study showed that circadian rhythm modify the concentration of glycogen in parotid and submandibular glands in health rats and the restriction of food made alterations in glycogen concentration of parotid glands. In diabetic rats was possible to see a higher concentration of glycogen in parotid and lower concentration in submandibular gland compared with health rats.

O Sagui-de tufo-branco (Callithrix jacchus) vem sendo utilizado como modelo experimental em inúmeras pesquisas. Entretanto, características anatômicas e fisiológicas dessa espécie são pouco conhecidas. O propósito do estudo foi avaliar microscopicamente o fígado de Callithrix jacchus criados em cativeiro. Neste estudo utilizou 31 saguis-do-tufo-branco do Centro de Primatas da Alemanha. Após a eutanásia, os fígados foram resfriados, dissecados, mensurados e rocessados por meio de técnicas histológicas. As colorações Hematoxilina e Eosina, Picrossírius, Fucsina Ácida, Gordon & Sweet´s e o Ácido Periódico de Schiff (PAS) foram usadas para observar respectivamente o arranjo do parênquima hepático, colágeno, reticulina e glicogênio. As mensurações foram organizadas em grupos conforme o sexo, a idade e o exame. A idade dos animais foi de 4,3&plusmn;2,4 anos. A massa corpórea foi de 427&plusmn;66g, com diferença significativa nos grupos exames. A massa do fígado foi 25,6&plusmn;13,1g. O fígado de Callithrix jacchus apresenta lóbulos hepáticos pouco definidos. Os hepatócitos apresentaram-se poliédricos, mono ou binucleados. O colágeno estava nos capilares sinusóides, no espaço porta-hepático e na veia centrolobular, com valor de 6,59&plusmn;3,66% (Picrossirius) e 5,89&plusmn;3,37% (Fucsina Ácida). A reticulina mostrouse presente principalmente nos capilares sinusóides, com valor de 18,86&plusmn;6,47%. O glicogênio estava distribuído ao longo do lóbulo hepático e com concentrações próximas à veia centrolobular, com valor de 23,92&plusmn;7,72%. A alimentação e hábitos em cativeiro podem interferir no aumento de massa e no desenvolvimento de doenças. O parênquima hepático de Callithrix jacchus não apresentou grandes alterações nos diferentes grupos formados, além de se assemelhar a de outras espécies, o que reforça o uso dessa espécie como modelo experimental. / The marmoset-white stuff (Callithrix jacchus) has been used as an experimental model in many studies. However, anatomical and physiological characteristics of this species are poorly known. The purpose of this study was to evaluate microscopically the liver of captive Callithrix jacchus. Thirty one Callithrix jacchus from Primate Center in Germany were studied. After euthanasia, livers were cooled, dissected, measured and processed by histological techniques. The hematoxylin and eosin staining, Picrosirius, Acid Fuchsin, Gordon & Sweet\'s and periodic acid-Schiff (PAS) were used respectively to observe the arrangement of the hepatic parenchyma, collagen, reticulin and glycogen. The data collected were organized into groups according to sex, age and examination. The average age of the animals was 4.3&plusmn;2.4 years. The average body weight was 427&plusmn;66g, with a significant difference in the groups exams. The average mass of the liver was 25.6&plusmn;13.1g. Despite Callithrix jacchus presented liver lobe, it was not well defined. Morphologically the hepatocytes were polyhedral, mono or binucleate. The collagen was in the sinusoidal capillaries in the portal space and the hepatic central vein, with a value of 6.59&plusmn;3.66% (Picrosirius) and 5.89&plusmn;3.37% (Acid Fuchsin). The reticulin was present mainly in the capillary sinusoids, with a concentration of 18.86&plusmn;6.47%. The glycogen was distributed throughout the liver lobule and near the central vein, with a value of 23.92&plusmn;7.72%. The feeding habits can interfere with the increase in mass and in the occurrence of disease on captive animals. The hepatic parenchyma of Callithrix jacchus showed no major changes in the different groups of this study. Additionally the hepatic parenchyma of Callithrix jacchus resembles other species, which reinforces the use of this species as an experimental model.