Made available in DSpace on 2014-12-17T14:16:36Z (GMT). No. of bitstreams: 1 YonaraMCO_DISSERT.pdf: 1678750 bytes, checksum: 32e7b234c6a83f6881e86eb536e38bec (MD5) Previous issue date: 2012-02-27 / Studies report that the pathophysiological mechanism of diabetes complications is associated with increased production of Reactive Oxygen Species (ROS)-induced by hyperglycemia and changes in the capacity the antioxidant defense system. In this sense, the aim of this study was to evaluate changes in the capacity of antioxidant defense system, by evaluating antioxidant status, gene expression and polymorphisms in the genes of GPx1, SOD1 and SOD2 in children, adolescents and young adults with type 1 diabetes. We studied 101 individuals with type 1 diabetes (T1D) and 106 normoglycemic individuals (NG) aged between 6 and 20 years. Individuals with type 1 diabetes were evaluated as a whole group and subdivided according to glycemic control in DM1G good glycemic control and DM1P poor glycemic control. Glycemic and metabolic control was evaluate by serum glucose, glycated hemoglobin, triglycerides, total cholesterol and fractions (HDL and LDL). Renal function was assessed by measurement of serum urea and creatinine and albumin-to-creatinine ratio (ACR) in spot urine. Antioxidant status was evaluate by content of reduced glutathione (GSH) in whole blood and the activity of erythrocyte enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD). We also analyzed gene expression and gene polymorphisms of GPx1 (rs1050450), SOD1 (rs17881135) and SOD2 (rs4880) by the technique of real-time PCR (Taqman?). Most individuals with DM1 (70.3%) had poor glycemic control (glycated hemoglobin> 8%). Regarding the lipid profile, individuals with type 1 diabetes had significantly elevated total cholesterol (p <0.001) and LDL (p <0.000) compared to NG; for triglycerides only DM1NC group showed significant increase compared to NG. There was an increase in serum urea and RAC of individuals with DM1 compared to NG. Nine individuals with type 1 diabetes showed microalbuminuria (ACR> 30 mg / mg). There was a decrease in GSH content (p = 0.006) and increased erythrocyte GPx activity (p <0.001) and SOD (p <0.001) in DM1 group compared to NG. There was no significant difference in the expression of GPx1 (p = 0.305), SOD1 (.365) and SOD2 (0.385) between NG and DM1. The allele and genotype frequencies of the polymorphisms studied showed no statistically significant difference between the groups DM1 and NG. However, the GPx1 polymorphism showed the influence of erythrocyte enzyme activity. There was a decrease in GPx activity in individuals with type 1 diabetes who had a polymorphic variant T (p = 0.012). DM1 patients with the polymorphic variant G (AG + GG) for polymorphism of SOD2 (rs4880) showed an increase in the RAC (p <0.05). The combined data suggest that glucose control seems to be the predominant factor for the emergence of changes in lipid profile, renal function and antioxidant system, but the presence of the polymorphisms studied may partly contribute to the onset of complications / Estudos relatam que o mecanismo fisiopatol?gico das complica??es do diabetes est? associado ao aumento na produ??o de Esp?cies Reativas de Oxig?nio (ERO) induzido pela hiperglicemia persistente e altera??es na capacidade de defesa do sistema antioxidante. Nesse sentido, o presente estudo teve como objetivo avaliar altera??es na capacidade de defesa do sistema antioxidante, atrav?s da avalia??o do status antioxidante, express?o g?nica e pesquisa de polimorfismos nos genes da GPx1, SOD1 e SOD2 de crian?as, adolescentes e adultos jovens com Diabetes Mellitus tipo 1 (DM1). Foram estudados 101 indiv?duos com diabetes tipo 1 (DM1) e 106 indiv?duos normoglic?micos (NG) com idade entre 6 e 20 anos. Os indiv?duos com DM1 foram avaliados como um grupo total e subdivididos de acordo com o controle glic?mico em DM1NC diab?tico n?o-compensado e DM1C diab?ticos compensados. Para avaliar o controle glic?mico e metab?lico foram realizadas as dosagens de glicose s?rica, hemoglobina glicada, triglicer?deos, colesterol total e fra??es (HDL e LDL). A fun??o renal foi avaliada pelas dosagens de ureia e creatinina s?ricas e a rela??o albumina/creatinina (RAC) urin?ria. Os par?metros antioxidantes avaliados foram o conte?do da glutationa reduzida (GSH) em sangue total e a atividade eritrocit?ria das enzimas glutationa peroxidase (GPx) e super?xido dismutase (SOD). Tamb?m foi avaliada a express?o g?nica e a pesquisa dos polimorfismos dos genes GPx1 (rs1050450), SOD1(rs17881135) e SOD2 (rs4880) pela t?cnica da PCR em tempo real (Taqman?). A maioria dos indiv?duos com DM1 (70,3%) apresentou controle glic?mico insatisfat?rio (hemoglobina glicada >8%). Em rela??o ao perfil lip?dico, indiv?duos com DM1 apresentaram valores significativamente elevados de colesterol total (p<0,001) e LDL (p<0,000) em rela??o ao NG; para os triglicer?deos s? o grupo DM1NC apresentou aumento significante em rela??o ao NG. Observou-se o aumento na ur?ia s?rica e na RAC dos indiv?duos com DM1 em rela??o ao NG. Nove dos indiv?duos com DM1 apresentaram microalbumin?ria (RAC> 30 &#956;g/mg). Houve diminui??o no conte?do de GSH (p=0,006) e aumento na atividade eritrocit?ria da GPx (p<0,001) e SOD (p<0,001) do grupo DM1 em rela??o ao NG. N?o foi observada diferen?a significante na express?o de GPx1 (p=0,305), SOD1 (0,365) e SOD2 (0,385) entre NG e DM1. As freq??ncias genot?picas e al?licas dos polimorfismos estudados n?o mostraram diferen?a estatisticamente significante entre os grupos DM1 e NG. Por?m o polimorfismo da GPx1 mostrou influ?ncia na atividade eritrocit?ria da enzima, observando-se diminui??o da atividade nos indiv?duos com DM1 que possu?am a variante polim?rfica T (p=0,012). J? para o polimorfismo Ala16Val da SOD2 observou-se eleva??o da RAC para aqueles indiv?duos diab?ticos que possu?am o alelo G (p<0,05). O conjunto dos dados sugere que o controle glic?mico parece ser o fator predominante para o surgimento de altera??es no perfil lip?dico, fun??o renal e no sistema antioxidante, por?m a presen?a dos polimorfismos estudados possam, pelo menos em parte, contribuir para o aparecimento de complica??es

A Neuropatia autonômica cardiovascular (NAC), apesar de ter sido apontada como fator de risco independente para doença cardiovascular (DCV) em pacientes com diabetes tipo 1 (DM1), permanece subdiagnosticada. Os objetivos do trababalho foram determinar a prevalência de NAC e seus indicadores clínicos e laboratoriais em pacientes com DM1 e a associação com outras complicações crônicas do diabetes, além de avaliar a concordância entre os critérios diagnósticos da NAC determinados pelos parâmetros da análise espectral e pelos testes reflexos cardiovasculares. Pacientes com DM1, duração da doença &#8805; 5 anos e com idade &#8805; 13 anos foram submetidos a um questionário clínico-epidemiológico, a coleta de sangue e de urina para determinação da concentração urinária de albumina, ao mapeamento de retina, e exame clínico para pesquisa de neuropatia diabética sensitivo motora além da realização de testes reflexos cardiovasculares. Cento e cinquenta e um pacientes com DM1, 53.6 % do sexo feminino, 45.7% brancos, com média de idade de 33.4 13 anos, idade ao diagnóstico de 17.2 9.8 anos, duração de DM1 de 16.3 9.5 anos, índice de massa corporal (IMC) de 23.4 (13.7-37.9) Kg/m2 e níveis de hemoglobina glicada de 9.1 2% foram avaliados. Após realização dos testes para rastreamento das complicações microvasculares, encontramos neuropatia diabética sensitivo motora, retinopatia diabética, nefropatia diabética e NAC em 44 (29.1%), 54 (38%), 35 (24.1%) e 46 (30.5%) dos pacientes avaliados, respectivamente. A presença de NAC foi associada com idade (p=0.01), duração do DM (p=0.036), HAS (p=0.001), frequência cardíaca em repouso (p=0.000), HbA1c (p=0.048), uréia (p=0.000), creatinina (p=0.008), taxa de filtração glomerular (p=0.000), concentração urinária de albumina (p=0.000), níveis séricos de LDL-colesterol (p=0.048), T4 livre (p=0.023) e hemoglobina (p=0.01) e a presença de retinopatia (p=0.000), nefropatia (p=0.000) e neuropatia diabética sensitivo motora (p=0.000), além dos seguintes sintomas; lipotimia (p=0.000), náuseas pós alimentares (p=0.042), saciedade precoce (p=0.031), disfunção sexual (p=0.049) e sudorese gustatória (p=0.018). No modelo de regressão logística binária, avaliando o diagnóstico de NAC como variável dependente, foi observado que apenas a FC em repouso, presença de neuropatia diabética sensitivo motora e retinopatia diabética foram consideradas variáveis independentes significativamente. A NAC é uma complicação crônica comum do DM1, atingindo cerca de 30% dos pacientes estudados e encontra-se associada à presença de outras complicações da doença. Indicadores da presença de NAC nos pacientes avaliados incluíram a idade, duração do diabetes, presença de HAS, frequência cardíaca de repouso e presença de sintomas sugestivos de neuropatia autonômica. O presente estudo ratifica a importância do rastreamento sistemático e precoce desta complicação. / The cardiovascular autonomic neuropathy (CAN), although considered as an independent risk factor for cardiovascular disease (CVD) in both patients with type 1 diabetes (T1D), remains underdiagnosed. The objective were to determine the prevalence, clinical and laboratorial indicators of CAN in patients with T1D and its association with other chronic complications of diabetes and evaluate the concordance between the diagnostic criteria for CAN diagnosis determined by the parameters of spectral analysis and the cardiovascular reflex tests. Patients with T1D aged &#8805; 13 years and diabetes duration &#8805; 5 years underwent a clinical-epidemiological survey, had blood samples collected, urinary samples for the determination of urinary albumin concentration, ophtalmoscopic exam, clinical neurological examination for diabetic neuropathy screeening and cardiovascular reflex tests. One hundred and fifty one patients with T1D, 53.6 % female, 45.7% Caucasian, mean age of 33.4 13 years, age at diagnosis of 17.2 9.8 years, diabetes duration of 16.3 9.5 years, body mass index (BMI) of 23.4 (13.7-37.9) kg/m2, glycated hemoglonin levels of 9.1 2% were evaluated. After performing the tests for screening for microvascular complications, we found diabetic sensory motor neuropathy, diabetic retinopathy, diabetic nephropathy and CAN in 44 (29.1%), 54 (38%), 35 (24.1%) and 46 (30.5%) of the patients, respectively. CAN was associated with age (p=0.01), diabetes duration (p=0.036), hypertension (p=0.001), resting heart rate (p=0.000), HbA1c (p=0.048), urea (p=0.000), creatinine (p=0.008), glomerular filtration rate (p=0.000), urinary albumin concentration (p=0.000), LDL-cholesterol (p=0.048), free T4 (p=0.023), hemoglobin (p=0.01) and presence of retinopathy (p=0.000), nephropathy (p=0.000) and diabetic neuropathy (p=0.000), the following symptons syncope (p=0.000), post prandial nausea (p=0.042), early saciety (p=0.031), sexual dysfunction (p=0.049) and gustatory sweating (p=0.018). In binary logistic regression model evaluating the diagnosis of CAN as a dependent variable, it was observed that only resting heart rate, presence of diabetic neuropathy and retinopathy were considered independent variables significantly. CAN is a common chronic complication of T1D affecting about 30% of the studied population and is associated with the presence of other chronic complications of T1D. Indicators of the presence of CAN included age, duration of diabetes, presence of hypertension, resting heart rate and symptoms suggestive of autonomic neuropathy. This study confirms the importance of systematic and early screening for this complication.

Made available in DSpace on 2014-12-17T15:16:19Z (GMT). No. of bitstreams: 1 FlavioSS_DISSERT.pdf: 1009224 bytes, checksum: 11ff4662d4d8985817935b34117dbf5f (MD5) Previous issue date: 2014-02-03 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Our aim was to investigate the effects of an aerobic training program on adverse and early left ventricle (LV) remodeling, using an experimental model of short-term type 1 diabetes (T1D). Wistar rats were divided in 4 groups: sedentary control (SC), trained control (TC), sedentary diabetic (SD) and trained diabetic (TD). T1D was induced by streptozotocin (45 mg/kg). The training program consisted of 4 weeks running on a treadmill (13 m/min, 60 min/day, 5 days/week). At the end of the experiments, hearts were collected for analysis of morphology and transcriptional profile of LV, by focusing on its remodeling. Deaths were recorded during the 4-week period. We verified high mortality among animals of DS group, whereas it was significantly reduced in DT group. DS group also showed an increase in cross-sectional area of cardiomyocytes and fibrosis. TD group exhibited reduction in measures of cardiac trophism, but with respect to collagen content, it was similar to CS group. Analysis of gene expression related to cardiac remodeling revealed decreased expression of collagen I and III, as well as low expression of MMP-2 in DS group. TD group showed decreased levels of mRNA for MMP-9, and unchanged gene expression of MMP-2 when compared with the CS group. The expression of MMP-2 and TGF-&#61538;1 were increased in CT group. The ratio between gene expression of collagen I and III was increased in the CT group and decreased in diabetic groups. These results establish early changes of the structure and transcriptional profile of LV myocardium. Moreover, they indicate that aerobic exercise training plays specific protection against mechanisms responsible for cardiac damage observed in T1D / Nosso objetivo foi investigar os efeitos de um programa de treinamento aer?bio sobre o remodelamento adverso e precoce do ventr?culo esquerdo (VE), utilizando modelo experimental de curto prazo de diabetes tipo 1 (DM1). Ratos Wistar foram divididos em 4 grupos: controle sedent?rio (CS), controle treinado (CT), diab?tico sedent?rio (DS) e diab?tico treinado (DT). O DM1 foi induzido por estreptozotocina (45 mg/kg). O programa de treinamento consistiu em 4 semanas de corrida em esteira (13 m/min, 60 min/dia, 5 dias/semana). Ao fim dos experimentos, os cora??es foram coletados para analise da morfologia e do perfil transcricional do VE, com foco em seu remodelamento. Os ?bitos foram registrados durante as 4 semanas. Verificamos alta mortalidade entre os animais do grupo DS, enquanto que esta foi significativamente reduzida no grupo DT. O grupo DS apresentou aumento na ?rea de sec??o transversa dos cardiomi?citos e fibrose. O grupo DT exibiu redu??o das medidas de trofismo card?aco, mas com rela??o ao conte?do col?geno, foi similar ao grupo CS. As an?lises de express?o de genes ligados ao remodelamento card?aco revelaram redu??o na express?o dos col?genos I e III, al?m de baixa express?o da MMP-2, no grupo DS. O grupo DT apresentou diminui??o dos n?veis de mRNA para MMP-9, e express?o g?nica de MMP-2 inalterada, se comparado ao grupo CS. As express?es da MMP-2 e do TGF-&#61538;1 foram aumentadas no grupo CT. A raz?o entre express?o g?nica dos col?genos I e III mostrou-se elevada no grupo CT e reduzida nos grupos diab?ticos. Esses resultados estabelecem altera??es precoces da estrutura e do perfil transcricional do VE. Ainda, indicam que o treinamento aer?bio exerce prote??o espec?fica contra mecanismos respons?veis pelo dano card?aco observado no DM1

Background: The medical literature shows that alcohol consumption is common among diabetic individuals and is associated with poor adherence to treatment, resulting in increased morbidity and mortality. However, no study has assessed the association between high-risk alcohol consumption and the presence of anxiety and depression in individuals with type 1 diabetes mellitus (1DM). Objectives: To know the degree of alcohol use, the prevalence at risk alcohol use and symptoms of anxiety and depression, the level of glycemic control and the association between these variables in adolescents and adults diagnosed with type 1 diabetes treated at the Clinic of Endocrinology Outpatient, Federal University of Uberlândia (UFU) and the Municipal Diabetic Care Center of Uberlândia. Methods: The present cross-sectional study assessed 209 outpatients in regards to alcohol consumption and the presence of anxiety and depression symptoms, using the Alcohol Use Disorders Identification Test (AUDIT), the Hospital Anxiety and Depression (HAD) scale, and glycemic control, according to the levels of glycated hemoglobin (HbA1c). The chi-square test and logistic regression analysis were used to investigate the association between the investigated variables. Results: The prevalence of high-risk alcohol consumption (AUDIT &#8805; 8) among individuals with 1DM was high, specifically 24.88% among the entire group of subjects, 12.9% among the adolescents, 14.7% among the females, and 34.6% among the males. Upon comparisons based on gender and age, the odds of high-risk drinking were higher among males and participants aged 30 to 40 years (33.93%). The frequency of high-risk alcohol consumption did not differ as a function of gender among adolescents (females: 9.09%, males: 16.21%; p=0.374). Neither the frequency of anxiety (total: 29.66%, females: 37.25%, males: 22.43%) nor depression (total: 11%, females: 17.65%, males: 4.7%) symptoms exhibited associations with high-risk alcohol consumption. Moreover, the odds of female subjects exhibiting anxiety or depression symptoms were higher (odds ratio OR: 4.37 and OR: 7.4, respectively). Glycemic control was inadequate in most of the sample and did not exhibit an association with high-risk alcohol consumption or the presence of anxiety and depression symptoms. Conclusions: The prevalence of high-risk alcohol consumption and the presence of anxiety and depression symptoms were high in this patient sample. The frequency of high-risk drinking increased together with age and was greater among males; however, this frequency did not exhibit differences in terms of gender among adolescents. Glycemic control was inadequate in most of the sample independent of alcohol consumption and the presence of anxiety and depression symptoms. / Introdução: A literatura médica mostra que o uso de álcool é frequente em pacientes com diabetes e está associado à má adesão ao tratamento, o que pode levar a uma maior morbidade e mortalidade. Entretanto, faltam pesquisas que avaliem a associação entre o uso de risco de álcool e a presença de sintomas de ansiedade e de depressão em pacientes com diabetes tipo 1 (DM1). Objetivos: verificar o grau de utilização de álcool, a prevalência do uso de risco de álcool e de sintomas de ansiedade e de depressão, o nível de controle glicêmico e a associação entre essas variáveis em adolescentes e adultos com diagnóstico de DM1, atendidos no Ambulatório de Endocrinologia do Hospital de Clínicas da Universidade Federal de Uberlândia (UFU) e no Centro Municipal de Atenção ao Diabético de Uberlândia (CMAD). Métodos: Este estudo transversal avaliou 209 pacientes em tratamento ambulatorial, quanto ao consumo de álcool, à presença de sintomas de ansiedade e de depressão por meio dos testes de rastreamento Alcohol Use Disorders Identification Test (AUDIT) e Hospital Anxiety and Depression (HAD) e o controle glicêmico por meio da hemoglobina glicada (HbA1c). Utilizaram-se os testes estatísticos de qui-quadrado e regressão logística para verificar a associação entre as variáveis. Resultados: A prevalência do consumo de risco de álcool em pacientes com DM1 foi de 24,8%, sendo 14,7% em mulheres e 34,6% em homens. A chance de uso de risco foi maior nos homens (OR=2,79), em pessoas com faixa etária mais elevada (OR=1,07), naqueles com história familiar de uso de álcool (OR=7,05), nos que referiram consumo de drogas ilícitas (OR=5,89), cigarro (OR=8,25) e na faixa etária de 30 a 40 anos (OR = 1,07). Quanto ao consumo de risco na adolescência, não houve diferença entre os gêneros (mulheres=9,09% e homens=16.21% e p=0,374). As frequências de sintomas de ansiedade (11% no total, 17,65% nas mulheres e 4,7% nos homens) e de depressão (29,66% no total, 37,25% nas mulheres e 22,43% nos homens) não mostraram relação com o consumo de bebidas alcoólicas. As mulheres apresentaram mais sintomas de ansiedade (OR=2,05) e de depressão (OR=4,37) do que os homens, assim como os adultos com mais de 30 anos em relação aos mais jovens (OR=2,34 para ansiedade e OR= 7,44 para depressão). O controle glicêmico foi predominantemente inadequado e não se verificou associação com o uso de risco de álcool e com a presença de sintomas de ansiedade e de depressão. Não houve associação do nível de HbA1c com o uso de risco de álcool, provavelmente devido ao número pequeno de pacientes com bom controle glicêmico. Conclusões: A prevalência do consumo de risco de álcool e a presença de sintomas de ansiedade e de depressão em adolescentes e adultos com DM1 foram elevadas. O uso de risco de álcool aumentou proporcionalmente ao aumento da faixa etária e foi mais prevalente entre os homens; somente entre os adolescentes não houve diferença entre os gêneros. Na amostra estudada, o controle glicêmico foi inadequado na maioria dos pacientes,independentemente do consumo de álcool e da presença de sintomas de ansiedade e de depressão. / Mestre em Ciências da Saúde

Esta dissertação está composta de três estudos que investigaram aspectos de resiliência, qualidade de vida e suporte social em adolescentes com diabete melito tipo 1 (DM1). O primeiro estudo consiste de uma revisão sistemática da literatura sobre resiliência e suporte social em adolescentes com DM1. Em geral os estudos revisados relacionam o suporte social com a melhora no controle glicêmico, manejo da doença e adesão ao tratamento. No segundo estudo buscou-se identificar possíveis correlações entre as variáveis qualidade de vida e suporte social em adolescentes com DM1. Participaram 102 adolescentes, 46 meninos e 56 meninas, entre 12 e 17 anos, pacientes de um serviço de atendimento da cidade de Porto Alegre. Os participantes responderam aos instrumentos KIDSCREEN-52 e Versão Brasileira do Social Support Appraisals. Foi verificado que de forma geral os adolescentes avaliam bem sua qualidade de vida e o suporte social recebido, sendo que as variáveis apresentaram correlação positiva moderada. O terceiro estudo investigou processos de resiliência em adolescentes com DM1, identificando fatores de risco e proteção, através de estudos de caso múltiplos. Participaram três adolescentes, uma menina e dois meninos, entre 13 e 14 anos, também pacientes do serviço de atendimento, e suas mães. Os instrumentos utilizados foram entrevistas semiestruturadas e o Mapa dos Cinco Campos. Constatou-se que fatores de proteção como suporte social, vinculação afetiva e características pessoais de autoestima, otimismo e altruísmo, contribuem para a manifestação de processos de resiliência. Destaca-se a importância de fortalecer a rede de apoio como fator de proteção para o enfrentamento do DM1, principalmente através da integração entre os diferentes contextos nos quais o adolescente está inserido. / This dissertation is composed of three studies that investigated aspects of resilience, quality of life and social support of adolescents with type 1 diabetes. The first study consists of a systematic review of literature about resilience and social support in adolescents with type 1 diabetes. Most of the studies reviewed demonstrated that social support is related with the improvement of glycemic control, disease management and treatment adherence. The second study investigated correlations between quality of life and social support in adolescents with type 1 diabetes. In total, 102 adolescents, 46 boys and 56 girls, between 12 and 17 years old, participated in the study. Participants were patients of a healthcare program in the city of Porto Alegre. Two questionnaires, the KIDSCREEN-52 and the Brazilian version of Social Support Appraisals were used to evaluate quality of life and social support. Results showed positive moderate correlation between the variables, and good assessment of quality of life and social support. In the third part of the research, study case method was used to identify risk and protective factors and to investigate processes of resilience in adolescents with type 1 diabetes. Three adolescents, one girl and two boys, between 13 e 14 years old, and their mothers participated in the study. Semi-structured interviews and the Five Field Map were used as instruments. It was found that social support, close bonds and personal characteristics such as self-esteem, optimism and altruism, contribute to the development of resilience processes. The research highlights the importance of strengthening the support network as a protective factor for coping with T1D, particularly through the integration between the different contexts in which the adolescent is inserted.

The main objective of this study was research on biomarkers used in both diagnosis and therapy of diabetic complications. The main focus of our work came to be on one of these biomarkers - glycemic variability (GV). High GV is linked with more frequent occurance of hypoglycemia. There are even indications it might contribute to development of diabetic complications. With modern technology - continuous glucose monitoring (CGM), we are now able to reliably describe, calculate and reduce GV. So far it is unclear whether increased GV can contribute to the development of microvascular complications (MVC) in type 1 diabetes (T1D). Studies published so far have assessed GV primarily from routine self-monitoring of blood glucose (SMBG) using glucometers. In the light of this uncertaity, the first part of this work compares GV calculated from CGM with the presence of MVC in T1D patients. GV calculated from CGM, but not from SMBG, proved to be significantly higher in T1D patients with MVC, even though there was no significant difference in glycated hemoglobin (HbA1c). This finding supports the hypothesis that higher GV is related to higher risk of MVC and that HbA1c does not describe diabetes control completely. Moreover, it was shown that GV calculated from SMBG is insufficient. There is still no fully...

Introduction: Physical activity should be part of our everyday life. However, for people with Type 1 Diabetes Mellitus it is the most common cause of hypoglycemia. To control diabetes in the right way, it is therefore necessary to follow certain rules and recommendations that help preventing hypoglycemia while the physical activity remains beneficial at the same time. Aim of the work: The main aim of this study is to clarify the influence of physical activity in connection to long-term control of Type 1 Diabetes Mellitus. Methods: 102 respondents with diagnosed Type 1 Diabetes Mellitus in the age of 19-69 years participated in a quantitative analysis. This research was conducted in a form of multicentric examination at two independent medical centers. The data collection was done through questionnaires focused on physical activity and daily regime. The data from questionnaires were then compared to the values of glycated hemoglobin (HbA1C), HDL cholesterol and the total daily dose of insulin of certain patients. For statistical evaluation, analytical tools of Microsoft Office program were used (F-test and t-test). Results: We found correlation between HbA1C values in patients physically active for less than 2 hours/week compared to those who are physically active for more than 2 hours/week (62,72...

No description available.

Background Regulatory T cells (Treg) are a subpopulation of CD4+ T cells that play an important role in the peripheral tolerance mechanisms of the immune system. Their suppressive function on autoreactive T cells can prevent autoimmunity. In type 1 diabetes (T1D), Treg have been inconsistently reported to be impaired in their capability to suppress autoreactive T cells (Tan, et al., 2014; Zhang, et al., 2012). Treg can be thymus derived (tTreg) or generated from naïve CD4+ CD25- T cells in the periphery (pTreg), which exhibit similar suppressive qualities as tTreg. They have also been reported to be actively induced (iTreg) under tolerogenic conditions (Kleijwegt, et al., 2010; Yuan and Malek, 2012). Although several Treg subpopulations have been described, the archetypical Treg express the major markers CD4, CD25 and FOXP3, while CD127 is heavily downregulated. However, activated conventional T cells (Tconv) show a similar phenotype, at least transiently (Miyara, et al., 2009). Since Treg and Tconv have opposing functions and therapeutic indications, it is important to obtain markers that confidently identify bona fide Treg. Scientific aim The aim of my thesis is to define the heterogeneity of human T cells with a specific emphasis to identify bona fide Treg. I examined heterogeneity of this population in healthy controls and T1D patients, as my model disease, and examined how T cells that are exposed to antigen can be defined as Treg or Tconv. Material and Methods For marker phenotyping I used samples from new onset T1D patients (age 7-11 years), autoantibody positive (Aab+) patients and age-matched healthy controls, which were tested by flow cytometry with an array of Treg-associated markers. Separately, freshly isolated CD4+CD25+CD127lo Treg and CD+CD25- Tconv were used for transcriptomic analysis, which was done by RNAseq on isolated whole RNA. For functional analysis of antigen specific gene expression patterns I developed a multi-dye proliferation assay. Treg (CD4+CD25+CD127lo) and Tconv (CD4+CD25-CD127+/lo) were sorted from isolated peripheral blood mononuclear cells (PBMC). I recombined the sorted and proliferation dye stained subsets with CD4- cells to simulate whole PBMC assays and stimulated them with tetanus-, influenza- or auto-antigens (GAD65, proinsulin). Cells were incubated for 5 days and responding proliferating cells as well as non-responding cells were single cell sorted and analyzed by multiplex qPCR. In investigating therapeutic approaches to expand or generate Treg, I examined in vitro approaches for de novo induction of Tregs with tolerogenic dendritic cells (tDCs). The tDCs were differentiated from monocytes either in the presence of 1α,25-OH(2)Vitamin D3 and/or Dexamethasone and matured with lipopolysaccharide. In a multistep assay, naïve T cells were incubated with DCs for two rounds and functional suppression assays were performed. The resultant T cells were analyzed at the DNA, protein, and functional level. Results Substantial phenotypic heterogeneity of peripheral blood CD4+ T cells was observed and documented for three major populations: resting Tconv (CD25-CD127+/lo), activated Tconv (CD25+CD127+) and Treg (CD25+CD127lo) in healthy controls. Despite this, I observed no differences between the Treg subpopulations from new onset T1D patients, Aab+ patients and healthy controls. In addition, there were no differences in the Treg transcriptome of T1D patients and healthy controls by RNAseq. I was, however, able to identify a small set of differentially expressed genes was discovered in Tconv suggesting a role of neutrophils in the onset of T1D. Heterogeneity of antigen-responsive Tconv and Treg was identified by gene expression profiling. I was able to define Treg specific as well as activation specific profiles, and found different expression profiles if T cells are foreign antigen or autoantigen activated and if the responding cells are Treg or Tconv. Genes that define the specific profiles include FOXP3, CD127, several cytokines, transcription factors and activation markers. The manipulation of naïve CD4+CD25- T cells by tDCs led to an unstable CD25+CD127loFOXP3+ phenotype of the generated cells. However, none of the subsequently performed functional assays could confirm that the resultant cells were iTreg or exhausted activated Tconv. In particular, methylation status of the Treg-specific demethylated region (TSDR) was inconsistent with stable Treg, suggesting that so-called tolerogenic protocols may not lead to a long-lived Treg phenotype. Conclusion CD4+CD25+ T cells are heterogeneous. I defined marker combinations that will help distinguish Treg from ex vivo and in vitro activated Tconv cells. With these tools, I was able to show that healthy controls and patients with type 1 diabetes cannot be distinguished by Treg phenotype. Comprehensive single cell analysis of antigen activated T cells provided the most promising avenue for identifying antigen-specific Treg and opens new possibilities to analyze immune therapeutic approaches, particularly when Treg expansion is the therapeutic objective. The findings will be used for monitoring children participating in antigen-based prevention studies in children at risk for T1D. / Hintergrund Regulatorische T Zellen (Treg) sind eine Subpopulation der CD4+ T Zellen, welche eine wichtige Rolle in den peripheren Toleranzmechanismen des Immunsystems spielen. Ihre suppressive Funktion auf autoreaktive T Zellen kann Autoimmunität verhindern. Verschiedene Studien berichteten widersprüchlich, dass Treg in Typ 1 Diabetes (T1D) in ihrer Fähigkeit beeinträchtigt sind autoreaktive T Zellen zu supprimieren (Tan et al., 2014; Zhang et al., 2012). Treg können im Thymus differenzieren (tTreg) oder aus peripheren naïven CD4+CD25- T Zellen generiert werden (pTreg), welche ähnliche suppressive Eigenschaften wie tTreg besitzen. Es wurde außerdem berichtet, dass Treg aktiv unter tolerisierenden Konditionen induziert werden können (iTreg) (Kleijwegt et al., 2010; Yuan and Malek, 2012). Obwohl verschiedene Treg Subpopulationen beschrieben wurden, exprimieren die archetypischen humanen Treg die Hauptmarker CD4, CD25 und FOXP3 exprimieren, während CD127 herunterreguliert ist. Jedoch zeigen auch aktivierte konventionelle T Zellen (Tconv) diesen Phänotyp (Miyara et al., 2009). Da Treg und Tconv gegensätzliche Funktionen und therapeutische Indikationen aufweisen, ist es wichtig Marker zu erhalten, die sicher bona fide Treg identifizieren. Fragestellung Das Ziel meiner Arbeit ist es, die Heterogenität von humanen T Zellen zu definieren mit einen spezifischen Fokus bona fide Treg zu identifizieren. Dafür untersuchte ich die Heterogenität dieser Zellpopulation in gesunden Individuen und T1D Patienten, als Krankheitsmodell, und wie T Zellen als Treg oder Tconv definiert werden können wenn sie einem Antigen ausgesetzt sind. Material und Methoden Für das Phänotypisieren habe ich Proben von Patienten mit beginnendem T1D (Alter 7-11 Jahre), Autoantikörper positiven Patienten (Aab+) und gesunden Individuen mittels Durchflusszytometrie auf eine Reihe von Treg-assoziierten Markern getestet. Des Weiteren wurden frisch isolierte CD4+CD25+CD127lo Treg und CD+CD25- Tconv für die Transkriptomanalyse (RNAseq) genutzt, welche mit der Gesamt-RNA durchgeführt wurden. Für die funktionelle Analyse von Antigen-spezifischen Genexpressionsmustern habe ich ein Multifarbenproliferationstest entwickelt. Treg (CD4+CD25+CD127lo) und Tconv (CD4+CD25-CD127+/lo) wurden aus isolierten mononukleären Zellen des peripheren Blutes (PBMC) sortiert. Ich habe die sortierten und gefärbten Zellen mit CD4- Zellen zusammengefügt, um einen Gesamt-PBMC-Test zu simulieren und habe die Zellen mit Tetanus-, Influenza- oder Auto-antigen (GAD65, Proinsulin) stimuliert. Die Zellen wurden für 5 Tage inkubiert und die Antigen-reagierenden und -proliferierenden Zellen sowie die nicht-reagierenden Zellen Einzelzell sortiert und mittels Multiplex qPCR analysiert. Um therapeutische Ansätze zum Expandieren oder Generieren von Treg zu untersuchen, habe ich in vitro Ansätze für die de novo Induktion von Treg durch die Nutzung von tolerisierenden dendritischen Zellen (tDCs) untersucht. Die tDCs wurden von Monozyten in Anwesenheit von 1α,25-OH(2)Vitamin D3 und/oder Dexamethason differenziert und mit Lipoploysaccharid maturiert. Naïve T Zellen wurden in einem Mehrschrittverfahren mit DCs inkubiert. Die resultierenden T Zellen wurden auf DNA, Protein und funktioneller Ebene analysiert. Ergebnisse Substantielle phänotypische Heterogenität von peripheren Blut CD4+ T Zellen wurde in drei Hauptpopulationen in gesunden Individuen beobachtet und dokumentiert: ruhende Tconv (CD25-CD127+/lo), aktivierte Tconv (CD25+CD127+) und Treg (CD25+CD127lo). Weiterführend ergab der phänotypische Vergleich von Patienten mit beginnender T1D, Aab+ Patienten und gesunden Individuen keine Unterschiede in den Treg Subpopulationen. Außerdem zeigten sich keine Unterschiede in den durch RNAseq gemessenen Treg Transkriptomen von T1D Patienten und gesunden Individuen. Jedoch wurde ein kleine Gruppe von differentiell exprimierten Genen in Tconv entdeckt, welche eine mögliche Rolle von Neutrophilen in T1D andeuten. Heterogenität von Antigen-spezifischen Tconv und Treg Antworten wurde durch Genexpressionsanalysen identifiziert. Ich konnte Treg- sowie Aktivierungs-spezifische Muster definieren und verschiedene Expressionsprofile finden, wenn T Zellen durch Fremd- oder Autoantigen aktiviert wurden und ob sie die reagierenden Zellen Treg oder Tconv sind. Folgende Gene waren hauptsächlich in die Profilbildung involviert: FOXP3, CD127, mehrere Zytokine, Transkriptionsfaktoren und Aktivierungsmarker. Die Manipulation von naïven CD4+CD25- T Zellen durch tDCs führte zu einem instabilen CD25+CD127loFOXP3+ Phänotyp der generierten Zellen. Jedoch konnte keiner der weiterführenden funktionellen Analysen unterscheiden, ob die resultierenden Zellen iTreg oder aktivierte erschöpfte T Zellen waren. Insbesondere war der Methylierungsstatus der Treg-spezifisch demethylierten Region (TSDR) nicht konsistent mit einen stabilen Treg Phänotyp, was darauf hinweist, dass sogenannte tolerisiernde Protokolle nicht zu einem langlebigen Treg Phänotyp führen. Schlussfolgerungen CD4+CD25+ T Zellen sind heterogen. Ich habe Markerkombinationen definiert die helfen werden Treg von ex vivo und in vitro aktivierten Tconv Zellen zu unterscheiden. Mit diesen Mitteln war ich in der Lage zu zeigen, dass gesunde Individuen und Patienten mit Typ 1 Diabetes nicht anhand ihres Treg Phänotyps unterschieden werden können. Umfassende Einzelzell-Analysen von Antigen aktivierten T Zellen lieferten den vielversprechendsten Ansatz für die Identifizierung von Antigen-spezifischen Treg und eröffnen neue Möglichkeiten um immuntherapeutische Ansätze zu analysieren, insbesondere wenn Treg Expansion das therapeutische Ziel ist. Diese Erkenntnisse werden zukünftig für das Monitoring von Kindern, mit einem hohen T1D Risiko, genutzt die an Antigen-basierten Präventionsstudien teilnehmen.

INTRODUÇÃO: portadores de diabetes mellitus tipo 1 (DM1) apresentam, progressivamente, complicações vásculo-neurais. Os fatores que aumentam o risco de coronariopatia - hipertensão, dislipidemia e idade avançada - explicam, em parte, a alta mortalidade cardiovascular, entretanto diabéticos tipo 1 podem morrer de coronariopatia precoce e não apresentar os fatores de risco clássicos para aterosclerose. Modificações estruturais e funcionais nas lipoproteínas, alterando a sua composição e trocas lipídicas poderiam justificar o aumento de eventos vasculares, entretanto estas alterações podem não ser detectadas através das dosagens rotineiras de lípides plasmáticos. OBJETIVOS: através de nanoemulsão lipídica artificial (LDE) que simula a estrutura lipídica da LDL avaliamos, em portadores de DM1, normolipidêmicos, intensivamente tratados e sem complicações significativas da doença, a taxa de esterificação do colesterol, a remoção da nanoemulsão da circulação, o tamanho da partícula HDL e as transferências de lípides entre a nanoemulsão e as partículas HDL. Secundariamente, determinamos a influência do controle glicêmico, resistência à insulina (RI) e insulinização no metabolismo lipídico. MÉTODOS: estudamos 36 indivíduos diabéticos e 37 controles não-diabéticos pareados para idade, sexo e índice de massa corpórea. Nanoemulsão lipídica artificial com marcação radioativa nos lípides éster de colesterol (CE), colesterol livre (CL), triglicérides (TG) e fosfolípides (PL) foi utilizada para os estudos. Nanoemulsão com marcação 14C-CE e 3H-CL foi injetada nos participantes e amostras de sangue foram coletadas durante 24 horas para mensuração da radioatividade. Remoção dos lípides da circulação foi calculada por análise compartimental. A taxa da esterificação do colesterol livre foi calculada após extração e separação de lípides do plasma por cromatografia em camada delgada. Para estudo da transferência de lípides, nanoemulsões com marcação 14C-CE e 3H-CL ou 14C-PL e 3H-TG foram incubadas com plasma e a radioatividade dos lípides transferidos para as HDL foi contada após a precipitação de lipoproteínas contendo apoB. O diâmetro da HDL foi mensurado por método de dispersão da luz. A RI nos diabéticos foi mensurada por fórmula que estima a taxa de captação da glicose. RESULTADOS: hemoglobina glicada foi de 8,8±1,3 mg/dl e concentrações de LDLc foram menores nos diabéticos (85±22 vs. 98±26 mg/dl), p=0, 035. Não houve diferenças em relação às taxas de esterificação, transferências de lípides da nanoemulsão para as HDL e tamanho da partícula HDL entre os grupos. Não encontramos relação entre as análises cinéticas e HbA1c, glicemia, índices de RI e dose de insulina. A taxa de remoção do 14C-CE foi mais rápida em diabéticos tipo 1 que nos controles (0, 059±0, 022 vs.0, 039±0, 022 h-1), p=0, 019. 16 CONCLUSÕES: apesar de controle glicêmico ruim nos DM1, as transferências de lípides da nanoemulsão para as HDL, a taxa de esterificação e a remoção da 3H-CL são semelhantes às dos controles. O controle glicêmico, perfil lipídico, índices de RI e dose de insulina não influenciaram nas transferências de lípides e na taxa de esterificação. A remoção do 14C-CE é mais rápida em indivíduos diabéticos, o que poderia justificar as concentrações de LDLc mais baixas encontradas nesta população. Acreditamos que a terapia insulínica intensiva pode justificar estes achados / INTRODUTION: people with type 1 diabetes mellitus (DM1) have progressively neuro-vascular complications. Factors that increase the risk of coronary artery disease hypertension, dislipidemia and advanced age explains part of increased cardiovascular mortality, however some DM1 died of early coronary artery disease and often do not have atherosclerosis classical risk factors. Structural and functional changes in lipoproteins, altering their composition and activities of lipid exchange could justify the increase in vascular events but these changes are generally not detected by routine clinical laboratory plasma lipid exams. OBJETIVES: in normolipidemic DM1, intensively treated and without significant complications of disease we evaluated, by an artificial lipid nanoemulsion that resembles the lipid structure of LDL, rates of cholesterol esterification, nanoemulsion removal of the circulation, HDL particle size and lipid transfer from nanoemulsion to HDL. Secondarily, we determine the influence of glycemic control, insulin resistance (IR) and insulinization on lipid metabolism. METHODS: we studied 36 diabetics and 37 non-diabetic controls paired by age, sex and body mass index. Artificial lipid nanoemulsion labeled with radioactive lipids cholesterol ester (CE), cholesterol (CL), phospholipids (PL) and triglycerides (TG) was used for studies. Intravenous infusion of nanoemulsion 14C-CE e 3H-CL was injected in participants and blood was sampled over 24 hours for radioactivity measurement. Circulation lipid removal was calculated through compartmental analysis. Rate of cholesterol esterification was calculated after lipid extraction and separation by thin-layer chromatography. Nanoemulsion was incubated with plasma and radioactivity of lipids 14C-EC, 3H-CL, 14C-PL and 3H-TG transferred to the HDL was quantified after the precipitation of other apoB lipoproteins. The HDL diameter was measured by laser light scattering. The insulin resistance in diabetic patients was measured by formula that estimates the rate of uptake of glucose. RESULTS: glycated hemoglobin was 8,8±1,3 mg/dl and LDL concentrations were lower in diabetic patients (85 ± 22 vs. 98 ± 26 mg / dl), p = 0035. There were no differences between groups regarding rates of cholesterol esterification, lipids transfer from nanoemulsion to HDL and HDL particle size. We found no relationship between the kinetic analyses and HbA1c, blood glucose, measures of IR and dose of insulin. The rate of removal of 14C-EC was faster in diabetics type 1 than controls (0.059 ± 0.022 vs.0.039 ± 0.022 h- 1), p = 0.019. CONCLUSIONS: despite suboptimal glycemic control in diabetics, lipids transfer from nanoemulsion to HDL, rate of cholesterol esterification and removal of 3H-CL are similar to those of non-diabetic individuals. Glycemic control, lipid profile, measures of IR and dose of insulin did not influence lipids transfer and rate of cholesterol esterification. Removal of 14C-EC from diabetic circulation is faster than controls which could justify the 18 lower LDL concentration found in this population. We believe that intensive insulin therapy could explain these findings