Bakgrund: Diabetes Mellitus typ 1 debuterar under barn- och ungdomsåren. Behandlingen är egenvård som innebär blodglukoskontroller, läkemedelsbehandling och att följa rekommendationer om kost och motion. Sjuksköterskans yrkesområde innefattar barn- och ungdomar. Avvikande från egenvårdsaktiviteter och hemlighetsmakeri kring sjukdomen påverkar vårdkvaliteten för ungdomar. Syfte: Att beskriva ungdomars upplevelse av att leva med Diabetes Mellitus typ 1 samt att beskriva de granskade studiernas undersökningsgrupper. Metod: Deskriptiv litteraturstudie med tolv vetenskapliga artiklar med kvalitativ samt kvalitativ- och kvantitativ ansats. Samstämmigheter och olikheter i artiklarnas resultat har kategoriserats och teman och subteman har identifierats. Huvudresultat: En del ungdomar upplevde svårigheter att acceptera sin sjukdom och önskade ha kontroll över och ta eget ansvar för sin sjukdom och öka oberoendet. Ungdomar upplevde känslan av att vara annorlunda och undvek att utföra egenvård i sociala sammanhang. Svårigheter att följa kostrekommendationer varierade och att utföra blodglukoskontroller upplevdes ansträngande. Stöd från vänner och jämnåriga underlättade utförandet av egenvård. Det upplevdes positivt att sjuksköterskor visade intresse för ungdomarnas liv utöver deras DMT1. Sjuksköterskor upplevdes brista i att kunna sätta sig in i ungdomarnas situation och vara ett känslomässigt stöd. Slutsats: Det finns ett behov av ökat stöd och förståelse från sjuksköterskor. Genom ökad förståelse kan sjuksköterskan bidra med stöd för att underlätta överföringen av ansvar. Detta kan leda till en ökad trygghet och känsla av kontroll hos ungdomarna, vilket kan bidra till att de vågar dela med sig av sin sjukdom till omgivningen. / Background: Diabetes Mellitus Type 1 debuts in childhood and adolescence. The treatment is self-care that involves blood glucose control, drug treatment and following recommendations on diet and exercise. Nursing's professional area includes children and adolescents. Deviating from self-care activities and secrecy around the disease affects the quality of care for young people. Purpose: To describe the young people's experience of living with Diabetes Mellitus type 1 as well as describing the study groups of the studies examined. Method: Descriptive literature study with twelve scientific articles with qualitative as well as qualitative and quantitative approach. Consequences and differences in the results of the articles have been categorized and themes and subthemes have been identified. Results: Young people experienced difficulties in accepting their illness and wanted to have control over and take responsibility for their illness and increase independence. Young people experienced the feeling of being different and avoiding self-care in social contexts. Difficulties to follow dietary recommendations varied and blood glucose controls were exerted. Support from friends and peers facilitated the performance of self-care. It was positive that nurses showed interest in the lives of adolescents in addition to their DMT1. Nurses were found to fail to get into the situation of young people and to be an emotional support. Conclusion: There is a need for increased support and understanding from nurses. Through increased understanding, the nurse can help to facilitate the transfer of responsibility. This can lead to increased safety and sense of control among adolescents, which may help them dare to share their illness with the environment.

Bakgrund: Diabetes mellitus typ 1 är en kronisk sjukdom som i Sverige drabbar cirka 800 barn årligen och kräver daglig behandling med insulin. Sjukdomen innebär begränsningar i det dagliga livet. Ungdomar har ett ökat behov av att frigöra sig från föräldrar och att bli självständig är en naturlig del i ungdomars utveckling. Syfte: Beskriva ungdomars upplevelse av att leva med diabetes mellitus typ 1 samt att granska vilken undersökningsgrupp som återfinns i de inkluderade artiklarna. Metod: Beskrivande litteraturstudie. Databaserna Medline via Pubmed och Cinahl användes för att söka underlag till studien. Elva vetenskapliga artiklar återfinns i resultatet. Huvudresultat: Ungdomar med diabetes mellitus typ 1 upplevde en känsla av att vara annorlunda och att sjukdomen var en börda. Ungdomarna upplevde även att de hade fler konflikter med sina föräldrar än sina kamrater och att föräldrarna hade svårt att släppa kontrollen. För att acceptera sin sjukdom beskrev ungdomarna att de behövde integrera sjukdomen som en del i sin identitet och sitt dagliga liv. På så vis kunde ungdomarna nå självständighet och utveckla sin förmåga att ta eget ansvar gällande egenvård. Slutsats: Resultatet påvisade att majoriteten av ungdomar med diabetes mellitus typ 1 kände sig annorlunda i jämförelse med jämnåriga. Många ungdomar upplevde svårigheter i relationen till sina föräldrar relaterat till ansvar för egenvården. Ungdomarna beskrev processen att acceptera och integrera sin sjukdom som en del i deras identitet. Sjuksköterskor har en viktig uppgift att stötta och vägleda ungdomar med diabetes mellitus typ 1 mot självständighet och ge dem förutsättningar att klara av egenvård. / Background: Diabetes mellitus type1 is a chronic disease which yearly afflicts around 800 children in Sweden and requires daily insulin treatment. The disease entails limitations in everyday life. Adolescents have an increased need to liberate themselves from their parents and gaining independence is a natural part of youths’ development.  Aim: Describe adolescents´ experience of living with diabetes mellitus type 1 and to examine which research group is found in the included articles.  Method: Descriptive literature study. The databases Medline via Pubmed and Cinahl were used in searching for study material. Eleven scientific articles are included in the results. Main findings: Adolescents with diabetes mellitus type1 had an experience of being different and that the disease was a burden. The adolescents also had the experience of having more conflicts with their parents than with their friends, and that their parents had difficulties letting go of control. In order to accept the disease, the adolescents described the need to integrate the disease as a part of their identity and daily life. In this way the adolescents could gain independence and grow in their ability to take responsibility and manage their self care. Conclusion: The findings showed that the majority of adolescents with diabetes mellitus type1 had an experience of being different compared to their peers. Many adolescents experienced difficulties in the relationship with their parents regarding the responsibility for selfcare. The adolescents described to process of acceptans and the need to integrate their disease as part of their identity. Nurses have an important task of supporting and guiding adolescents with diabetes mellitus type 1 towards independence and giving them the conditions for coping with selfcare.

The Angiotensin II Type 1 (AT1) receptor is the main receptor responsible for the effects of the renin-angiotensin system, and its expression pattern is altered in several diseases. [11C]Methyl-Losartan has been developed based on the clinically used AT1 receptor antagonist Losartan. The aim of this work is to characterize the pharmacokinetics, repeatability and reliability of measurements, binding specificity and selectivity of [11C]Methyl-Losartan in rats using in vivo small animal positron emission tomography (PET) imaging, ex vivo biodistribution and in vitro autoradiography methods. Also, we aim to measure the presence of metabolites in the kidney and plasma using high-performance liquid chromatography. We have demonstrated in vivo that [11C]Methyl-Losartan is taken up in the AT1 receptor-rich kidneys and that it is displaceable by selective AT1 receptor antagonists. Using ex vivo biodistribution, we have confirmed these results and demonstrated that [11C]Methyl-Losartan binds selectively to the AT1 receptor over the AT2, Mas and β-adrenergic receptors. In vitro autoradiography results confirmed these renal binding selectivity studies. [11C]Methyl-Losartan was also shown to have one and two C-11 labeled metabolites in the plasma and kidneys, respectively. In conclusion, [11C]Methyl-Losartan is a promising agent for studying the AT1 receptor in rat models with normal and altered AT1 receptor expression using small animal PET imaging.

Aerobic exercise interventions involving individuals with type 1 diabetes have had little positive effect on blood glucose control as reflected by hemoglobin A1c. The few existing interventions involving resistance exercise, either alone or combined with aerobic exercise, while small in sample size, have had better outcomes. The purpose of this research program was to examine the changes in blood glucose levels during activity and for 24 hours post-exercise (as measured by continuous glucose monitoring) when resistance exercise is performed, either on its own or combined with aerobic exercise, as compared to aerobic exercise alone or no exercise. Twelve physically active individuals with type 1 diabetes performed 5 separate exercise sessions in random order separated by at least five days: 1) no exercise/control; 2) aerobic exercise (45 minutes of treadmill running at 60% VO2peak); 3) resistance exercise (45 minutes of weight lifting – 3 sets of 8 repetitions of 7 different exercises); 4) aerobic then resistance exercise (2 and 3 combined with the aerobic exercise first); 5) resistance then aerobic exercise (2 and 3 combined with the resistance exercise first). We found that resistance exercise was associated with a lower risk of hypoglycemia during exercise, less carbohydrate intake during exercise, less post-exercise hyperglycemia and more frequent (but less severe) nocturnal hypoglycemia than aerobic exercise. When aerobic and resistance exercise were combined, performing resistance exercise prior to aerobic exercise (rather than the reverse) resulted in attenuated declines in blood glucose during aerobic exercise, accompanied by a lower need for carbohydrate supplementation during exercise and a trend towards milder post-exercise nocturnal hypoglycemia.

Type 1 diabetes (T1D) is an autoimmune disease whereby the insulin-producing β-cells of the pancreas are destroyed by the immune system, possibly related to an inappropriate immune reaction to dietary antigens and/or microbes in the gut. We previously observed a deficit in gut-resident CD163+ M2 anti-inflammatory macrophages in BioBreeding diabetes-prone (BBdp) rats. Heme oxygenase-1 (HO-1) is the rate-limiting enzyme of the CD163 pathway and through the breakdown of toxic heme releases potent antioxidants. We hypothesized that the treatment of animals with cobalt protoporphyrin (CoPP), an inducer of HO-1 expression, would inhibit development of T1D through modulation of the CD163/HO-1 pathway and increase M2 macrophages. HO-1 expression was significantly increased in the pancreas and gut. T1D incidence was inhibited in CoPP-treated rats and these animals showed an unexpected increase in cells expressing CD68 (an M1 pro-inflammatory macrophage marker) in the pancreas and gut. CoPP induced the expression of cathelicidin anti-microbial peptide (CAMP) in the jejunum, which co-localized with CD163+ (M2) macrophages. KLF4, an M2 macrophage-specific transcription factor, was significantly upregulated in the pancreas and jejunum of CoPP-treated animals and co-localized with CD68 and HO-1 in the pancreas. We conclude that HO-1 induction prevented T1D through modulation of the gut immune system and potential recruitment of a unique population of anti-inflammatory M2 macrophages in the gut and pancreas

Le paludisme est la première endémie parasitaire mondiale causée par le protozoaire Plasmodium. Cette parasitose est responsable de 219 millions de cas et 660 000 décès par an. La prévalence et la mortalité élevées sont liées notamment à la résistance des parasites aux traitements existants, ce qui rend primordial le développement de nouvelles thérapeutiques. Pour ce faire, une meilleure connaissance de la biologie fondamentale du parasite est nécessaire. Dans ce contexte l’un des axes de recherche concerne la régulation du cycle cellulaire chez Plasmodium et notamment les mécanismes de phosphorylation/déphosphorylation qui sont essentiels.Parmi les nombreux acteurs des mécanismes de phosphorylation, la sérine/thréonine protéine phosphatase de type 2A (PP2A) est, avec PP1, l’une des phosphatases majeures. Cette phosphatase est impliquée dans de nombreux processus cellulaires notamment la mitose, la méiose ou encore l’apoptose. Elle est composée d’une sous-unité catalytique (PP2Ac), d’une sous-unité d’aide à l’agencement spatial (A) et d’une sous-unité régulatrice (B). Il existe quatre familles de sous-unités régulatrices contenant chacune plusieurs membres qui permettent de réguler la localisation, la spécificité et l’activité de PP2A. Il existe également des protéines régulatrices indépendantes, notamment les inhibiteurs 1 et 2, la protéine α4 et le PhosphoTyrosyl Phosphatase Activator (PTPA). Chez Plasmodium falciparum, la protéine phosphatase de type 2A ou PfPP2A a été identifiée et semble essentielle pour le développement asexué du parasite. Cependant, peu de choses sont connues sur sa régulation chez le parasite. En effet, seul l’inhibiteur 2 de PP2A a été décrit et caractérisé. Au cours de cette thèse, nous avons effectué par des études in silico un recensement des régulateurs putatifs de PfPP2A. Ces études nous ont permis d’identifier la sous-unité A et une unique sous-unité B. Parmi les régulateurs spécifiques, outre l’inhibiteur 2 déjà caractérisé, l’analyse du génome du parasite montre qu’il contient un orthologue de l’inhibiteur 1, d’α4 et de PTPA. Le projet de cette thèse s’articule autour de la caractérisation moléculaire et fonctionnelle de l’un de ces régulateurs : PfPTPA.La caractérisation moléculaire de PfPTPA a permis de montrer dans ce travail la conservation de cette protéine au cours de l’évolution. L’analyse de sa séquence a révélé que cinq des six motifs de fixation à la PP2A identifiés chez l’homme sont conservés. Par des études in vitro et in vivo dans un modèle hétérologue, nous avons pu confirmer le rôle d’activateur de PfPTPA vis-à-vis de la PP2A. Par une approche de mutation unique d’acides aminés, nous avons identifié trois résidus impliqués dans l’interaction et l’activité de PfPTPA notamment le résidu G292 qui est essentiel pour l’interaction PfPTPA/PfPP2A. Nous avons ensuite montré par des études de génétique inverse que PfPP2A et PfPTPA, qui sont présents dans le même compartiment cellulaire au cours du cycle érythrocytaire, sont essentielles pour la complétion du cycle intra-érythrocytaire du parasite. De plus, PfPTPA semble impliqué dans le cycle cellulaire chez le xénope.En parallèle, l’analyse de la séquence de PfPTPA, a révélé la présence, spécifique au parasite, d’un motif de fixation à la PP1 (motif RVxF). L’identification de ce motif, nous a incités à étudier la relation entre PfPTPA et PfPP1. Nous avons ainsi pu montrer que PfPTPA était capable de se lier à PfPP1 même si elle est incapable de réguler son activité.L’ensemble de ce travail de thèse a permis de caractériser chez Plasmodium falciparum un activateur de la protéine phosphatase de type 2A et de montrer sa spécificité par rapport à la protéine humaine. Nos résultats, et notamment l’implication de PfPTPA dans la régulation du cycle cellulaire, font de ce régulateur une cible thérapeutique potentielle. / Malaria is the most deadly parasitic disease in the world caused by the Apicomplexa protozoan Plasmodium falciparum. This parasite is responsible for 219 million cases and 660 000 deaths per year and the drug resistance increases the prevalence and the morbidity. The emergence of multi-drug resistance requires the development of new therapeutics. Hence, a better understanding of parasitic fundamental biology is necessary. In this context, one research axis is the cell cycle regulation of Plasmodium, notably phosphorylation/dephosphorylation mechanisms which are essential for the parasite.Among the actors of the reversible phosphorylation, the serine/threonine phosphatase type 2A (PP2A) in eukaryote is, with PP1, one of the major phosphatases. It is involved in several cell processes like mitosis, meiosis or apoptosis. PP2A is composed of a catalytic subunit (PP2Ac), a scaffold subunit (A) and a regulatory subunit (B). There are four regulatory subunit families which regulate location, specificity and activity of PP2A. Furthermore, several independent regulatory proteins including inhibitor 1 and 2, the α4 protein or the phosphotyrosyl phosphatase activator (PTPA) were identified.In Plasmodium falciparum, the protein phosphatase type 2A named PfPP2A has been characterized and seems to be essential for the parasite asexual development as shown by the inhibition of parasitic growth after treatment with natural toxins inhibiting phosphatases. However, its regulation is still poorly understood in Plasmodium. Indeed, only the PP2A inhibitor 2 is characterized in P. falciparum and in P. berghei (a rodent specific Plasmodium species). Using an in silico study, we have identified a putative scaffold subunit and only one B subunit. Among the regulatory proteins, we have identified orthologs of the inhibitor 1, α4 and PTPA. The purpose of this thesis is to study PfPTPA both of the molecular and functional levels.The molecular characterization of PfPTPA showed the evolutionary conservation of this protein. The PfPTPA sequence analysis revealed that five out of six amino acids involved in interaction with PP2A in human, are conserved in P. falciparum. In vitro binding and functional studies revealed that PfPTPA binds to and activates PfPP2A. Mutation studies showed that three residues (V283, G292 and M296) of PfPTPA are indispensable for the interaction and that G292 residue is essential for its activity. Localization studies indicated that PfPTPA and PfPP2A are localized in the same cellular compartment throughout the erythrocytic cycle of P. falciparum, suggesting a possible interaction of both proteins in vivo. In Plasmodium falciparum, genetic studies likely suggested the essentiality of PfPTPA for the completion of intraerythrocytic parasite lifecycle. Functionnal studies, using Xenopus oocyte, showed that PfPTPA blocked the G2/M transition. Further analysis of PfPTPA sequence revealed that PfPTPA, unlike its human counterpart, possess one of the most canonical binding motif to PP1 (RVxF motif). The identification of this RVxF motif led us to study the role PfPTPA on PfPP1. Thus, we have shown that PfPTPA interacts with PfPP1 but was unable to regulate PfPP1 activity in vitro. This work allowed characterizing the PfPTPA, an activator of protein phosphatase type 2A in Plasmodium falciparum and to show some specificities when compared to its human ortholog. Our data which suggest that this regulator could be involved in cell cycle regulation, together with its essentiality for the growth of P. falciparum strongly support the idea to explore it as potential drug target.

Background: In Sweden type 1-diabetes is the most common chronic disease among children. When a child suffers from type 1-diabetes the parents gets affected as well. The children’s need of support from the parents depends on the child’s age, maturity and individual needs. Aim: The purpose of this study was to describe the parents’ experience of living with a child that suffers from type 1-diabetes, as well as describe the articles research groups. Method: A descriptive literature study that’s based on 11 articles, nine of qualitative approach and two of qualitative- and quantitative approach. Result: Parents to children with type 1-diabetes experienced fear, grief, anxiety and frustration. Type 1-diabetes among children influences parents’ life in many ways. The disease required planning, attention and adjustment. Parents experienced that type 1-diabetes affected school, work, sleep, diet and physical activity. Conclusion: Parents of children with type 1 diabetes experiences that the disease required a great commitment and responsibility. The majority of the care and treatment is handled by the parents, due to the children's lack of self-care. The nurse therefore has an important role to provide information, support and encourage both to patients and their families regarding care and treatment. / Bakgrund: I Sverige är typ 1-diabetes den kroniska sjukdomen som är vanligast förekommande hos barn. När ett barn drabbas av typ 1-diabetes berörs även föräldrarna. Barns behov av stöd från föräldrar varierar efter barnets ålder, mognad och individuella behov. Syfte: Syftet med studien var att beskriva hur föräldrar upplever att det är att leva med ett barn med typ 1-diabetes samt att beskriva artiklarnas undersökningsgrupper. Metod: En beskrivande litteraturstudie som baserats på 11 artiklar, nio av kvalitativ ansats och två av kvalitativ- och kvantitativ ansats. Resultat: Föräldrar till barn med typ 1-diabetes upplevde rädsla, sorg, oro och frustration. Typ 1-diabetes hos ett barn påverkade föräldrarnas liv på många olika sätt. Sjukdomen krävde planering, uppmärksamhet och anpassning. Föräldrarna upplevde att typ 1-diabetes påverkade skola, jobb, sömn, kost och fysisk aktivitet. Slutsats: Föräldrar till barn med typ 1-diabetes upplevde att sjukdomen krävde ett stort engagemang och ansvar. Vård och behandling hanterades till större del av föräldrarna vilket berodde på barnens bristande egenvård. Sjuksköterskan har därmed en viktig roll att ge information, stötta och uppmuntra både patient samt dess anhöriga vad gäller vård och behandling.

Orientador: Carlos Eduardo Steiner / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T13:53:01Z (GMT). No. of bitstreams: 1 Assis_NilmaAlmeidade_M.pdf: 1405023 bytes, checksum: 12e3861c31dae0e759ab571d2d029476 (MD5) Previous issue date: 2006 / Resumo: A retinopatia diabética é uma complicação freqüente no diabetes melito tipo 1, acometendo quase a totalidade de pacientes, em graus variados, após 20 anos de doença. A interferência de fatores ambientais como a manutenção de um estado hiperglicêmico na sua fisiopatologia já foi comprovada, mas ainda não foi esclarecido porque alguns pacientes desenvolvem essa complicação de maneira grave e precoce. Nos últimos anos, diversos estudos têm sugerido a participação de fatores genéticos nesse processo. O fator de crescimento endotelial vascular (VEGF), potente indutor da angiogênese, foi associado à retinopatia diabética por alguns autores, pelo aumento da sua expressão, em virtude de mutações em sua região promotora. Neste trabalho foi realizada uma avaliação clínico-laboratorial, a análise do SNP rs833061 (- 460) e a pequisa da deleção de 18 pares de bases em -2549, ambas na região promotora do gene VEGF em 114 pacientes com diabetes melito tipo 1, de três centros de referência em diabetes no Brasil ¿ Hospital das Clínicas da Unicamp, Instituto Estadual de Diabetes e Endocrinologia Luiz Capriglione (RJ) e Santa Casa da Misericórdia de São Paulo (SP). Houve associação entre retinopatia diabética proliferativa e nefropatia, porém nenhum paciente apresentou a deleção em -2549, nem o alelo -460 C em homozigose. Tais resultados sugerem que esses polimorfismos na região promotora do gene VEGF não interferem na predisposição à retinopatia diabética na população estudada e que outros agentes ambientais e/ou genéticos devem ser significativos / Abstract: Diabetic retinopathy is a frequent complication of diabetes mellitus type 1 and almost all patients develop it after twenty years of disease. The causes of these complications are not clear, but several environmental factors such as chronic hyperglicaemia may act in this predisposition, however, it is not clearly understood why some individuals develop it in a severe and precocious way. The participation of genetic factors as the vascular endothelial growth factor (VEGF), a potent angiogenic mediator, was already confirmed for some authors. In this study we investigate whether polymorphisms in VEGF gene are associated with proliferative diabetic retinopathy. One hundred-fourteen patients with diabetes mellitus type 1 underwent a clinical and laboratorial study, as well as the analysis of two polymorphisms: rs833061 and the deletion of 18 bp at -2549 both on the promoter region of the VEGF gene. There was an association between nephropathy and retinopathy in our patients but none of the individuals presented the deletion at -2549 or the allele C in rs833061 in homozygous state. These results suggest that such polymorphisms in the promoter region of the VEGF gene do not interfere in the predisposition to diabetic retinopathy in our population and that other environmental and/or genetic factor may be more relevant / Mestrado / Genetica Medica / Mestre em Ciências Médicas

Orientador: Maria Luiza Silveira Mello / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T06:29:06Z (GMT). No. of bitstreams: 1 Ghiraldini_FlaviaGerelli_D.pdf: 75704253 bytes, checksum: 9d1afc3cebcffd05000b5efb25d3996d (MD5) Previous issue date: 2013 / Resumo: O diabetes mellitus do tipo I (DM1) caracteriza-se pela ocorrência de insulite com consequente hiperglicemia e poliuria. Alterações celulares estruturais e metabólicas decorrentes do aumento da glicemia podem provocar fenótipos de envelhecimento precoce. O envelhecimento celular e resultado de fatores intrínsecos e extrínsecos, que alteram a estrutura e a organização da cromatina e que, consequentemente, afetam a expressão gênica. As sirtuinas, deacetilases NAD+-dependentes, estão envolvidas na transcrição gênica, reparo de DNA, transcrição do rDNA, regulação metabólica e remodelação cromatinica. As sirtuinas nucleares, especialmente Sirt1 e Sirt6 estão envolvidas com o envelhecimento precoce, no metabolismo de glicose e na resposta a inflamação. O presente trabalho teve como objetivo geral comparar os processos de remodelação cromatinica em hepatocitos sob o efeito da hiperglicemia e do envelhecimento, usando-se modelo animal (camundongos). Um modelo de cultura celular (HepG2) foi também utilizado para estudo de efeitos da hiperglicemia, utilizando-se como metodologias analisem morfológicas e moleculares. Foi observado um aumento em conteúdo de DNA e em acessibilidade da cromatina a MNase mais acentuado em hepatocitos de animais DM1 do que de idosos. O aumento na abundancia de Sirt1 em animais hiperglicêmicos não refletiu em sua maior atividade, enquanto em idosos houve um decréscimo generalizado nesses parâmetros e aumento da aceptilação de sítios histónicos. Em animais DM1, Sirt6 apresentou abundancia semelhante à de Sirt1, possivelmente devido à alta fragmentação de DNA observada nesses animais, diferente do ocorrido em idosos. Ambos os animais DM1 e idosos apresentaram baixa relação área AgNOR+/área nuclear. Em animais diabéticos isto foi devido ao aumento na área nuclear, enquanto nos idosos, foi devido à diminuição na área AgNOR+ e aumento na área nuclear. O aumento na metilação de rDNA na porção 18S e a baixa abundancia de Sirt7 confirmam diminuição no metabolismo celular no envelhecimento. Em hepatocitos de camundongos DM1 e idosos foi observado genes diferencialmente expressos relacionados à inflamação. Admite-se que no primeiro caso este achado seja devido à natureza auto-imune da doença, enquanto no segundo possa ser um indício de inflamação naturalmente encontrada em processos de envelhecimento. Em animais DM1, a expressão diferenciada de genes envolvidos com metabolismo de lipídios poderia contribuir para com a peroxidação lipídica e produção de ROS levando a esteatose hepática. Nas células HepG2, alterações na expressão dos genes Apoe, Igfbp1 e Foxo1, ocorridas em meio de cultura hiperglicêmicas, tornaram-se revertidas quando as células foram retornadas a normoglicemia. Contudo, as abundancias das marcações epigenéticas nos promotores desses genes decresceram progressivamente, indicação de uma memória hiperglicêmica, dado não observado em modelo animal. A análise do fenótipo nuclear dessas células indicou possível indução da proliferação celular quando retornadas a normoglicemia. A inibição de sirtuinas aumentou o conteúdo Feulgen-DNA e o contraste entre cromatina condensada e não-condensada, indicativo de atuação na proliferação celular e na remodelação cromatinica. DM1 e envelhecimento, portanto, não podem ser considerados fenômenos idênticos, pois enquanto no primeiro ha um mecanismo compensatório que promove alterações genéticas, epigenéticas e remodelação cromatinica, no segundo ha um decréscimo generalizado no metabolismo celular levando a modificações diferentes nos mesmos parâmetros / Abstract: Diabetes mellitus type I (DM1) is characterized by insulitis and consequent hyperglycemia and polyuria. Structural and metabolic changes in the cell caused by hyperglycemia might induce an early-ageing phenotype. Both intrinsic and extrinsic agents might contribute to cellular ageing thus leading to chromatin structural changes and differential gene expression. Sirtuins, NAD+-dependent deacetilases, play a role in cell metabolism, transcription, DNA repair and chromatin remodeling. Sirt1 and Sirt6, especially, are nuclear proteins related to early-ageing, glucose metabolism and inflammatory response. The general purpose of the present work was to compare processes of chromatin remodeling in hepatocytes under the effects of hyperglycemia and aging, using mouse models. A model using cells in culture (HepG2) was also used to study the effects caused by hyperglycemia. The methodology used involved morphological and molecular analysis. An increase in DNA content and chromatin accessibility to MNAse was found more pronounced in hepatocytes from DM1 than from aged mice. Despite the high abundance of Sirt1 in DM1 animals, its activity was not proportionally high, whereas in old animals there was a reduction in these parameters, increasing the acetylation of Sirt1-histonic sites. In DM1 mice, Sirt6 presented similar abundance as Sirt1, possibly due to the high DNA fragmentation, different to what was found in aged animals. Both DM1 and normoglycemic old mice presented a decrease in AgNOR+ area/nuclear area ratio. While in DM1 animals it was a result from the increase in nuclear area, in old animals it was a combination of increased nuclear areas and decreased AgNOR+ areas. The DNA methylation increase in the 18S rDNA region and the decrease in Sirt7 abundance in the hepatocytes from old mice support the hypothesis of diminished cellular metabolism. Differential expression analysis for DM1 and old mouse hepatocytes presented a high number of genes involved in the inflammatory response. While in the former it could be an autoimmune characteristic of the disease, in the latter it might be an evidence of inflammatory state naturally associated with aging. Moreover, DM1 mice also presented differential gene expression related to lipid metabolism, which could contribute to increase lipid peroxidation and ROS production leading to hepatic steatosis. HepG2 cells showed changes in Apoe, Igfbp1 and Foxo1 expression in hyperglycemic medium and they were reverted when the cells returned to normoglycemic medium. The epigenetic marks, however, presented a progressive decrease in abundance, indicative of a hyperglycemic memory, which was not observed in DM1 animals. The nuclear phenotype in HepG2 cells under these same experimental conditions indicated a possible induction in cellular proliferation when the cells were returned to the normoglycemic medium. Inhibition of sirtuins increased the contrast between condensed and non-condensed chromatin and the Feulgen-DNA content, indicating a role in cell division and chromatin remodeling. Therefore, DM1 and ageing cannot be considered as identical processes, because while in DM1 there is a compensatory mechanism that induces changes in epigenetic marks, chromatin remodeling and differential gene expression, there is a general decrease in cell metabolism under aging that leads to different changes in the same parameters / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Perdas gestacionais, malformações, restrição de crescimento intrauterino (IUGR) são associadas a gestações diabéticas. Para ampliar o conhecimento nesse tema, nosso grupo desenvolveu um modelo de gestação complicada por diabetes tipo 1 em camundongos que, nessa tese, foi utilizado para analisar o ciclo estral, desenvolvimento fetal e organização placentária. O diabetes foi induzido por aloxana e estudado em dois períodos 30-50D (curto prazo) e 90-110D (longo prazo). Placentas e fetos foram coletados, pesados, e submetidos a técnicas moleculares, bioquímicas e morfológicas. Detectaram-se alterações no perfil temporal do ciclo estral. O grupo 30-50D apresentou altas taxas de perdas embrionárias e IUGR, e o 90-110D malformações, mortes fetais, IUGR e aumento no peso placentário. As placentas diabéticas apresentaram aumento e desorganização da zona juncional, redução do labirinto e vasodilatação. A expressão dos colágenos I e III aumentou e a do V diminuiu em 30-50D, porém, a deposição destes aumentou concomitante com a redução da atividade da MMP9. A deposição dos colágenos III e V e a atividade da MMP2 aumentaram em 90-110D. Nossos resultados reiteram a importância do fator temporal nas complicações do diabetes sobre a gestação. / Gestational loss, malformations and intrauterine growth restriction (IUGR) are often associated with pregnancies. To increase the knowledge about this topic, our group has developed a model of pregnancy complicated by type 1 diabetes in mice. In this study, was analyzed the estrous cycle and the fetal and placental development. For this, diabetes was induced by alloxan and studied in two time-periods 30-50D (short term) and 90-110D (long term). Placentas and fetuses were collected, weighed and analyzed by biochemical, morphological and molecular procedures. We detected changes in the temporal profile of the estrous cycle. The 30-50D group showed high rates of embryonic loss and IUGR whereas malformations, fetal death, IUGR and increased placental weight was detected in 90-110D. Increase and disorganization of junctional zone, reducing labirinth and vasodilation characterize diabetic placentas. The expression of collagen I and III was increased whereas collagen V decreased in the 30-50D. The deposition of this collagen, however increased concomitant with the reduction of MMP9 activity. In 90-110D deposition of collagen III and V and the MMP2 activity was increased. Together, our results reinforce the relevance of the time factor in the complications of diabetes on pregnancy.