Thesis (M.N.)--Memorial University of Newfoundland, 2004. / Typescript. Bibliography: leaves 141-150.

Sweden holds third place of diabetes incidence in young people after Finland and Sardinia. One fifth of the population is nowadays of foreign descent. We have a substantial number of immigrants from countries where the risk for T1D is considerably lower. Migration as a natural experiment is a concept to assess the risk for diabetes in offspring of immigrant parents and assess the interaction between genetics (genotype) and the impact of environment (phenotype). Aims: To study the risk of incurring diabetes for children of immigrant parents living in Sweden (I) and further study the risk if the child is born in Sweden or not (II); to specifically study and evaluate if children from East Africa have increased risk to develop T1D (III). To investigate if clinical and sociodemographic status at T1D onset differs between immigrant children compared to their Swedish indigenous peers (IV). Finally to study the clinical outcome and the impact of socio-demographic factors at diabetes onset after three years of treatment (V). Methods: All five studies are observational, nationwide and population based, on prospectively collected data. Statistics mainly by logistic and linear regressions. Results: Parental country of origin is a strong determinant for diabetes in the offspring. Children born to immigrant parents seem to keep their low risk compared to their Swedish peers (I). When adding the factor of being born in Sweden, the pattern changed; there was a significantly (p < 0.001) increased risk for T1D if the child was born in Sweden (II). East Africans have a substantial risk for T1D and especially if the children are born in Sweden (III). Immigrant children and adolescents have worse metabolic start at T1D onset compared to their indigenous Swedish peers (IV). After 3 years of treatment, the immigrant children had a sustained higher median HbA1c, compared to their Swedish peers (V). Conclusions: Genotype and influences during fetal life or early infancy have an important impact for the risk of T1D pointing towards epigenetics playing a substantial role. Children with an origin in East Africa have a high risk of incurring T1D. Immigrant children have worse metabolic start at T1D onset, which sustains after three years of treatment

Introduction: The management of type 1 diabetes through the use of Continuous Subcutaneous Insulin Infusion (CSII); also known as insulin pump therapy, has become an increasingly popular option for children and adolescents. A systematic review of studies that measured Quality of Life (QoL) in children associated with CSII was conducted. Eighteen studies were reviewed, and the results showed insufficient evidence to conclude that CSII improves QoL in children and adolescents with type 1 diabetes. The current study aimed to address the gap in the literature by exploring children and parents’ perspectives on the use of CSII for managing diabetes. Method: Data were gathered from five children aged 8 – 14 years (and five parents), using one to one semi-structured interviews. Interviews were recorded, transcribed and analysed using Interpretative Phenomenological Analysis (IPA). Results: Five super-ordinate themes were identified for parents: ‘Parenting a Child with Diabetes’; ‘Worth the Hard Work’; ‘Strive for Normality’; The Pump as an Enabler’; and ‘An eye on the Future’. Three Super-ordinate themes were identified for children ‘Feeling Different’; Grappling for Control’; and ‘Better…’ which were associated with a central theme of ‘Developing a Relationship with the Pump’. Children’s data is presented separately within a journal article format. Discussion: Findings suggest that parents value the insulin pump, despite acknowledging the challenges, particularly the hard work required to manage it. Children seemed to have an ambivalent but developing relationship with the insulin pump. They experience a number of benefits and drawbacks associated with the use of CSII and it seems to affect their identity and their locus of control. Conclusion: This research provides a greater insight into the lived experience of CSII for children and their parents. The benefits of CSII seemed to outweigh the challenges involved particularly for parents; and children seemed to be developing a relationship with the pump within the realms of their relationship with diabetes.

Type 1 diabetes(T1D)is a disease that only gets more common. The etiology of the disease is not known but there are many existing theories about what the cause is. These different theories have been tested in vivoin rodents or invitro. The resultsfrom experiments done in those waysarenotall realistic because rodents differnotablyfrom humans,and when studies areperformed in vitrowith human isletsof Langerhans different hormones will accumulate. The aim of this studywas to establisha dynamic ex vivosystem in which stimulation of human islets of Langerhans can be performed in a more lifelike environment. To study islets in this system couldin the future lead to increased knowledge in the etiology of T1D.The perifusion system PERI-4.2 from Biorep Technologies together with an incubator with 37°Cand5% CO2were used to arrangethe ex vivosystem. An Insulin ELISA from Mercodia was performedto analyze the insulin secretion from the islets. Fourdifferent set ups for the system were tested and the last one showed the best results.In conclusion this study has shown that it is possible to preserve human islets of Langerhans in a dynamic ex vivosystem with a constant medium exchange if it is done under conditionswhere the islets are protected from shear forces from the supplying medium,together with a medium exchange rate which replaces the whole medium in at least one hour.

Activated islet specific T cells are central to the destructive autoimmune response observed in type 1 diabetes (T1D). Not surprisingly, intense focus is placed on understanding how autoreactive T cell responses arise and contribute to disease pathology in the hope of using this information to develop novel therapeutic strategies for treatment of T1D. Here we investigate the mechanisms underlying defective c-Maf binding to the IL-4 promoter in T cells from diabetes prone mice and identify the mechanisms responsible for suppression of T cells by the inhibitory receptor Ly49A. It is not clear why development of protective Th2 cells is poor in T1D. c-Maf transactivates the IL-4 gene promoting Th2 cell development; therefore abnormalities in c-Maf may contribute to reduced IL-4 production by CD4 cells from nonobese diabetic (NOD) mice. Here we demonstrate that, despite normal expression, c-Maf binds poorly to the IL-4 promoter (IL-4p) in NOD CD4 cells. Immunoblots demonstrate that c-Maf can be modified at lysine 33 by small ubiquitin-like modifier-1 (SUMO-1). Sumoylation is facilitated by direct interaction with the E2 conjugating enzyme Ubc9 and increases following T cell stimulation. In addition, c-Maf physically interacts with p65/RelA. This interaction is dependent on the DNA binding domain of c-Maf and phosphorylation of p65 at serine 536. In transfected cells, overexpression of SUMO-1 or p65 decreases c-Maf transactivation of IL-4p-driven luciferase reporter activity, reduces c-Maf binding to the IL-4p in chromatin immunoprecipitation (ChIP) assays and enhances c-Maf localization into promyelocytic leukemia nuclear bodies (PML-NBs) or nucleoli, respectively. Sumoylation of c-Maf and phosphorylation of p65 are increased in NOD CD4 cells compared to CD4 cells from diabetes-resistant B10.D2 mice, suggesting that increased c-Maf sumoylation and interaction with p65 contribute to immune deviation in T1D by reducing c-Maf access to and transactivation of the IL-4 gene. Islet specific CD4 cells expressing inhibitory receptors may be a useful therapeutic tool for treating T1D. Engagement of transgenic Ly49A inhibits CD4 cell activation and delays onset of T1D in mice. However, in vitro studies suggest the inhibitory effect of Ly49A is incomplete. Here we report that following simultaneous T cell receptor (TCR) and Ly49A engagement, phosphorylation of Zap70, Erk1/2 and c-Jun were significantly diminished. Kinetic studies indicated that Ly49A did not simply delay activation but had a long-lasting effect. In contrast, when only costimulatory signals were provided through CD28, Ly49A engagement did not block p38 MapK or Akt phosphorylation. Likewise, expression of the downstream targets Bcl-xl and Baff were unaffected. Together these data suggest that engagement of Ly49A selectively inhibits signals downstream of the TCR but spares those unique to CD28. These results suggest that when considering its use as an immunotherapy, the potency of inhibitory receptors such as Ly49A may be further improved by pairing them with costimulatory blockade. Take together, these studies suggest that abnormal post-translational regulation of c-Maf function is a novel marker of altered T cell function in T1D and use of inhibitory receptors such as Ly49A may be optimized combining this approach with other complementary therapies.

O Diabetes Mellitus tipo 1 (DM1) está associado a uma incidência aumentada de doença micro e macrovascular. Estudos sugerem que a doença vascular no DM1 tenha como evento precursor a disfunção endotelial (DE). A hiperglicemia parece causar DE no DM1 através da geração do estresse oxidativo. O momento exato do surgimento da DE na história natural do DM1, assim como a influência do controle glicêmico de curto e longo prazo ainda não estão estabelecidos. Objetivo: O objetivo principal do presente estudo foi avaliar a função endotelial através da Dilatação Arterial Mediada por Fluxo (DMF) em indivíduos com Diabetes Mellitus tipo1. Os objetivos secundários foram analisar os fatores que possam estar envolvidos com a disfunção endotelial no DM1, como o tempo de diabetes, o controle glicêmico e a presença de complicações microvasculares, como a microalbuminúria. Métodos: Estudo prospectivo transversal com 57 pacientes com DM1 e 10 indivíduos não diabéticos, consecutivamente alocados e comparados quanto à presença de DE, através da DMF, aferida pela dilatação da artéria braquial após hiperemia reativa (dilatação endotélio-dependente) e após dilatação mediada por uso de nitrato sublingual (dilatação endotélio-independente). Considerou-se como DE quando valores de DMF foram menores ou iguais a 8% em relação ao valor basal. Os pacientes foram orientados a fazer monitorização glicêmica capilar intensiva nos 30 dias que antecederam a avaliação vascular. No 30º dia, houve coleta de exames laboratoriais e a avaliação vascular foi realizada. Dados prospectivos e históricos de hemoglobina glicosilada (HbA1c), através da técnica de imunoturbidimetria (Cobas Integra 400; Roche), foram obtidos aos 3, 6, 9,12,15,18 e 24 meses anteriores ao teste para DMF. Os critérios de exclusão foram: tabagismo, hipertensão, obesidade, hipotireoidismo, uso de estatina, gestação, história de neoplasia ou doença vascular. Resultados: Em 57 pacientes com DM1 estudados, 28 (49%) apresentaram DE. A média da dilatação endotélio-dependente foi significativamente menor nos pacientes com DM1, comparados aos indivíduos não-diabéticos (9,48±6,48% vs.14,56±5,60%, p=0,02). A dilatação endotélio-independente foi significativamente menor nos pacientes com DM1 em relação aos controles (22,26±9,2% vs. 29,31±4,2%, p=0,02, VR: acima de 8%), mas não houve diferença entre os DM1 com ou sem DE (p= 0,72). O tempo de DM1 (meses) foi maior nos pacientes com DE do que nos sem DE (105,4±74,7 vs. 66,3±48,0, p=0.02) e houve correlação linear negativa entre duração do DM e presença de DE (r-0,28, p=0,02). A média da HbA1c (%) coletada no momento da avaliação vascular foi semelhante entre pacientes com DM1 com DE e sem DE (8,97%±1.85 vs 8,23%±1.45, p=0.10) e não houve correlação significativa com a DMF (r=-0,128 p=0,34). Todavia, quando as HbA1c históricas foram avaliadas, houve correlação significativa com a HbA1c aos 15 meses (r=-0,303, p=0,02) e no período de 12-24 meses anteriores ao exame vascular (r=-0,289, p=0,03), mas não com a HbA1c média de 0-12m (r=-0,181 p=0,18). A DMF foi menor nos pacientes com microalbuminúria em relação aos normoalbuminúrcos (4,83±3,81% vs 10,35±6,50%, p=0,015). A microalbuminúria também foi mais prevalente nos DM1 com DE do que sem DE (22,2% vs 3,5%, p=0,04). Considerando apenas os pacientes com DM1 com tempo de DM menor que 5 anos, 10/28 (35,7%) apresentaram DE. Com relação a dilatação não-dependente de endotélio (%), não houve diferença em relação aos controles (p=0,16) e nem entre os DM1 com e sem DE (p=0,27). A média da HbA1c na época do exame vascular também não foi diferente nos pacientes com e sem DE (8,20±0,94% vs. 7,99±1,37%, p=0,66). As correlações de Pearson entre a DMF e as HbA1c históricas foram negativas aos 12 meses (r=-0,419, p=0,03), aos 15 meses (r=-0,437, p=0,03) e com a HbA1c média de12-24 meses (r=-0,426, p=0,027). Conclusões: Pacientes com DM1 apresentam prejuízo na função endotelial, quando comparados a controles não diabéticos. A DE é um evento precoce na história natural do DM1, e está presente nos pacientes antes dos 5 anos de doença, estando associada, ao tempo de DM1, à presença de microalbuminúria e ao controle metabólico de longo-prazo. A ausência de disfunção de músculo liso endotelial no grupo com menos de 5 anos de DM, com valores de dilatação não-endotéliodependente semelhantes aos controles, sugere ser a DE um fenômeno ainda reversível nos primeiros anos de doença. / Patients with Type 1 diabetes (T1DM) are at high-risk for developing micro and macrovascular complications. Endothelial dysfunction (ED) has been suggested to be a precursor of both complications in Type 1 diabetes. Hyperglycemia may be associated to ED through generation of oxidative stress. The exactly moment when ED occurs in T1DM is until not well established. Also we do not known if long-term rather than short term metabolic control have a greater impact in ED. Objective: The aim of this study was to assess endothelial function by Flow Mediated Dilation (FMD) in (T1DM) patients and compare with non- diabetic controls. Secondary objectives were to analyze factors that could be associated to ED: duration of T1DM, glycemic control and microvascular complications like microalbuminuria. Research design and methods: In a cross-sectional study 57 adolescents with T1DM and 10 non-diabetic controls, were recruited and compared for the presence of ED by FMD with evaluation of reactive hyperemia (endothelium-dependent dilatation) and after using sublingual nitrate spray for assessed non-endothelialdependent dilatation. ED was considered when FMD ≤ 8% in relation to basal value. Patients performed intensive self monitoring blood glucose for 30 days before vascular studies. At day 30, blood was drawn for biochemical determinations and endothelial function was carried out. Historical data from Glycated hemoglobin (HbA1c), determined by immunoturbidimetry (Cobas Integra 400; Roche) were collected at 3, 6, 9,12,15,18 and 24 months before the test for FMD. Excluding criteria were any time tobacco use, clinical hypertension, obesity, hypothyroidism, statin use, current pregnancy and any history of previous neoplasia or vascular disease. Results: Of 57 T1DM patients studied, 28 (49%) presented ED. FMD was significantly decreased in T1DM compared to controls (9.48±6.48% vs. 14.56±5.60%, p=0.02). Nitrate-mediated dilation (%) was decreased in T1DM compared to controls (22.26±9.2% vs. 29.31±4.2%, p=0.02, RV= >8%), but it was not different between T1DM with or without ED (p=0.72). The duration of T1DM was longer in ED vs. Non- ED patients: 105.4±74.7 vs. 66.3±48.0 months, p= 0.02 and presented negative linear correlation between duration of T1DM and FMD (r=-0.284, p=0.03). HbA1c at the moment of the vascular analysis did not differ between ED and Non-ED patients (8.97±1.85% vs. 8.23±1.44%, p= 0.10) and it was not associated with FMD (r=-0.128, p=0.34). However, we found significant negative correlation between HbA1c and FMD at 15 months (r=-0.303, p=0.02) and at 12-24 months before vascular study, but not with median HbA1c of 0-12m (r=-0.181 p=0.8). Microalbuminuria was more prevalent in T1DM patients with ED than Non-ED (22.2% vs. 3.5%, p=0.04). FMD was decreased in microalbuminuric compared to normoalbuminuric patients (4.83±3.81% vs 10.35±6.50%, p=0.015). In T1DM patients with less than 5 years of disease, 10 of 28 (35.7%) presented ED. Nitrate-mediated dilation, in this group, was not decreased compared to controls (p=0.16) and it was not different in T1DM patients with or without ED (p=0.27). HbA1c at the moment of vascular analysis did not significantly differ in ED compared to Non-ED patients (8.20±0.94% vs.7.99±1.37%, p=0.66). Pearson’s correlation between FMD and historical HbA1c was negative with HbA1c at 12 (r=-0.419, p=0.03), at 15 (r=-0.437, p=0.03) and 12-24 months before vascular analysis (r=- 0.426, p=0.02). Conclusions: Endothelial function is impaired in T1DM patients compared to nondiabetic controls. ED is a phenomenon that can occur quite early in the natural history of T1DM, presented before 5 years of disease and is related to duration of disease, long- term metabolic control and microalbuminúria. Vascular smooth muscle was not impaired in T1DM patients with less than 5 years of disease, with values of non-endothelial-dependent dilation similar to controls, suggesting that ED can be a reversible event in this first years of disease.

Type 1 diabetes in pregnancy is associated with higher rates of maternal and infant complications. The complications are associated with maternal hyperglycaemia. Thus, the main goal of treatment for these women is to optimise glycaemic control and thereby improve clinical outcomes for themselves and for their baby. This thesis examines glycaemic control in the mothers and infants of pregnancies affected by type 1 diabetes. I present the first home studies of closed-loop insulin delivery in this population. The aim of these studies was to assess the feasibility, efficacy, and utility of overnight and then day-and-night closed-loop insulin delivery in pregnant women with type 1 diabetes. The overnight study, which examined 16 pregnant women (mean age 34.1 years, HbA1c 6.8%, 14.4 weeks gestation), compared overnight use of the closed-loop system with sensor-augmented pump therapy in a 2x4-week randomised crossover design. We found that closed-loop therapy was associated with a 15% improvement in overnight time spent with target glucose concentration (3.5-7.8 mmol/L; 74.7% during closed-loop use vs 59.5% during sensor-augmented pump therapy use). The day-and night study also examined 16 pregnant women (mean age 32.8 years, HbA1c 8.0%, 16.4 weeks’ gestation) using a 2x4-week randomised crossover design to compare continuous day-and-night use of closed-loop insulin delivery with sensor-augmented pump therapy. This study enrolled a more diverse range of participants than the overnight study, but found that closed-loop therapy was associated with comparable glucose control and significantly less hypoglycaemia than sensor-augmented pump therapy. Chapter 4 examines women’s experiences of using the closed-loop system during pregnancy. While the system was generally well-received by participants, individual interactions and perceptions of the system varied markedly, and often did not align with biomedical measures of glycaemic response. After participation in either crossover study, participants could choose to continue using the technology until delivery (overnight study), or until 6 weeks post-partum (day and night study). Those data are presented in Chapters 2 and 3. The combined data from the women who used the closed-loop system during labour and delivery in either study are presented in Chapter 5. Tight glycaemic control during labour and delivery has traditionally been considered important for reducing rates of neonatal hypoglycaemia. However, despite very tight maternal glycaemic control in the women who used closed-loop insulin delivery, rates of neonatal hypoglycaemia were high. In order to better characterise the relationship between maternal glucose control in type 1 diabetes pregnancy and neonatal hypoglycaemia, Chapter 6 details an observational study in which continuous glucose monitoring was used to measure maternal and neonatal glycaemic control in 16 mother-infant pairs. The study found that, while neonatal hypoglycaemia was very frequent, it was generally, but not always, detected and treated effectively. Together, these studies suggest that a novel management tool, closed-loop insulin delivery, can improve overnight glycaemic control, and perhaps reduce hypoglycaemia during type 1 diabetes-affected pregnancies above what is possible with currently available treatments. However, complication rates remain high for these women and their babies. Further research is needed both to further develop treatments that can improve maternal glycaemic control, and to better understand the pathogenesis of diabetes-related pregnancy complications, with the ultimate goal of improving outcomes for women and their children. A definitive trial to assess the clinical efficacy, patient acceptability, and cost effectiveness of closed-loop is now warranted.

O Diabetes Mellitus tipo 1 (DM1) está associado a uma incidência aumentada de doença micro e macrovascular. Estudos sugerem que a doença vascular no DM1 tenha como evento precursor a disfunção endotelial (DE). A hiperglicemia parece causar DE no DM1 através da geração do estresse oxidativo. O momento exato do surgimento da DE na história natural do DM1, assim como a influência do controle glicêmico de curto e longo prazo ainda não estão estabelecidos. Objetivo: O objetivo principal do presente estudo foi avaliar a função endotelial através da Dilatação Arterial Mediada por Fluxo (DMF) em indivíduos com Diabetes Mellitus tipo1. Os objetivos secundários foram analisar os fatores que possam estar envolvidos com a disfunção endotelial no DM1, como o tempo de diabetes, o controle glicêmico e a presença de complicações microvasculares, como a microalbuminúria. Métodos: Estudo prospectivo transversal com 57 pacientes com DM1 e 10 indivíduos não diabéticos, consecutivamente alocados e comparados quanto à presença de DE, através da DMF, aferida pela dilatação da artéria braquial após hiperemia reativa (dilatação endotélio-dependente) e após dilatação mediada por uso de nitrato sublingual (dilatação endotélio-independente). Considerou-se como DE quando valores de DMF foram menores ou iguais a 8% em relação ao valor basal. Os pacientes foram orientados a fazer monitorização glicêmica capilar intensiva nos 30 dias que antecederam a avaliação vascular. No 30º dia, houve coleta de exames laboratoriais e a avaliação vascular foi realizada. Dados prospectivos e históricos de hemoglobina glicosilada (HbA1c), através da técnica de imunoturbidimetria (Cobas Integra 400; Roche), foram obtidos aos 3, 6, 9,12,15,18 e 24 meses anteriores ao teste para DMF. Os critérios de exclusão foram: tabagismo, hipertensão, obesidade, hipotireoidismo, uso de estatina, gestação, história de neoplasia ou doença vascular. Resultados: Em 57 pacientes com DM1 estudados, 28 (49%) apresentaram DE. A média da dilatação endotélio-dependente foi significativamente menor nos pacientes com DM1, comparados aos indivíduos não-diabéticos (9,48±6,48% vs.14,56±5,60%, p=0,02). A dilatação endotélio-independente foi significativamente menor nos pacientes com DM1 em relação aos controles (22,26±9,2% vs. 29,31±4,2%, p=0,02, VR: acima de 8%), mas não houve diferença entre os DM1 com ou sem DE (p= 0,72). O tempo de DM1 (meses) foi maior nos pacientes com DE do que nos sem DE (105,4±74,7 vs. 66,3±48,0, p=0.02) e houve correlação linear negativa entre duração do DM e presença de DE (r-0,28, p=0,02). A média da HbA1c (%) coletada no momento da avaliação vascular foi semelhante entre pacientes com DM1 com DE e sem DE (8,97%±1.85 vs 8,23%±1.45, p=0.10) e não houve correlação significativa com a DMF (r=-0,128 p=0,34). Todavia, quando as HbA1c históricas foram avaliadas, houve correlação significativa com a HbA1c aos 15 meses (r=-0,303, p=0,02) e no período de 12-24 meses anteriores ao exame vascular (r=-0,289, p=0,03), mas não com a HbA1c média de 0-12m (r=-0,181 p=0,18). A DMF foi menor nos pacientes com microalbuminúria em relação aos normoalbuminúrcos (4,83±3,81% vs 10,35±6,50%, p=0,015). A microalbuminúria também foi mais prevalente nos DM1 com DE do que sem DE (22,2% vs 3,5%, p=0,04). Considerando apenas os pacientes com DM1 com tempo de DM menor que 5 anos, 10/28 (35,7%) apresentaram DE. Com relação a dilatação não-dependente de endotélio (%), não houve diferença em relação aos controles (p=0,16) e nem entre os DM1 com e sem DE (p=0,27). A média da HbA1c na época do exame vascular também não foi diferente nos pacientes com e sem DE (8,20±0,94% vs. 7,99±1,37%, p=0,66). As correlações de Pearson entre a DMF e as HbA1c históricas foram negativas aos 12 meses (r=-0,419, p=0,03), aos 15 meses (r=-0,437, p=0,03) e com a HbA1c média de12-24 meses (r=-0,426, p=0,027). Conclusões: Pacientes com DM1 apresentam prejuízo na função endotelial, quando comparados a controles não diabéticos. A DE é um evento precoce na história natural do DM1, e está presente nos pacientes antes dos 5 anos de doença, estando associada, ao tempo de DM1, à presença de microalbuminúria e ao controle metabólico de longo-prazo. A ausência de disfunção de músculo liso endotelial no grupo com menos de 5 anos de DM, com valores de dilatação não-endotéliodependente semelhantes aos controles, sugere ser a DE um fenômeno ainda reversível nos primeiros anos de doença. / Patients with Type 1 diabetes (T1DM) are at high-risk for developing micro and macrovascular complications. Endothelial dysfunction (ED) has been suggested to be a precursor of both complications in Type 1 diabetes. Hyperglycemia may be associated to ED through generation of oxidative stress. The exactly moment when ED occurs in T1DM is until not well established. Also we do not known if long-term rather than short term metabolic control have a greater impact in ED. Objective: The aim of this study was to assess endothelial function by Flow Mediated Dilation (FMD) in (T1DM) patients and compare with non- diabetic controls. Secondary objectives were to analyze factors that could be associated to ED: duration of T1DM, glycemic control and microvascular complications like microalbuminuria. Research design and methods: In a cross-sectional study 57 adolescents with T1DM and 10 non-diabetic controls, were recruited and compared for the presence of ED by FMD with evaluation of reactive hyperemia (endothelium-dependent dilatation) and after using sublingual nitrate spray for assessed non-endothelialdependent dilatation. ED was considered when FMD ≤ 8% in relation to basal value. Patients performed intensive self monitoring blood glucose for 30 days before vascular studies. At day 30, blood was drawn for biochemical determinations and endothelial function was carried out. Historical data from Glycated hemoglobin (HbA1c), determined by immunoturbidimetry (Cobas Integra 400; Roche) were collected at 3, 6, 9,12,15,18 and 24 months before the test for FMD. Excluding criteria were any time tobacco use, clinical hypertension, obesity, hypothyroidism, statin use, current pregnancy and any history of previous neoplasia or vascular disease. Results: Of 57 T1DM patients studied, 28 (49%) presented ED. FMD was significantly decreased in T1DM compared to controls (9.48±6.48% vs. 14.56±5.60%, p=0.02). Nitrate-mediated dilation (%) was decreased in T1DM compared to controls (22.26±9.2% vs. 29.31±4.2%, p=0.02, RV= >8%), but it was not different between T1DM with or without ED (p=0.72). The duration of T1DM was longer in ED vs. Non- ED patients: 105.4±74.7 vs. 66.3±48.0 months, p= 0.02 and presented negative linear correlation between duration of T1DM and FMD (r=-0.284, p=0.03). HbA1c at the moment of the vascular analysis did not differ between ED and Non-ED patients (8.97±1.85% vs. 8.23±1.44%, p= 0.10) and it was not associated with FMD (r=-0.128, p=0.34). However, we found significant negative correlation between HbA1c and FMD at 15 months (r=-0.303, p=0.02) and at 12-24 months before vascular study, but not with median HbA1c of 0-12m (r=-0.181 p=0.8). Microalbuminuria was more prevalent in T1DM patients with ED than Non-ED (22.2% vs. 3.5%, p=0.04). FMD was decreased in microalbuminuric compared to normoalbuminuric patients (4.83±3.81% vs 10.35±6.50%, p=0.015). In T1DM patients with less than 5 years of disease, 10 of 28 (35.7%) presented ED. Nitrate-mediated dilation, in this group, was not decreased compared to controls (p=0.16) and it was not different in T1DM patients with or without ED (p=0.27). HbA1c at the moment of vascular analysis did not significantly differ in ED compared to Non-ED patients (8.20±0.94% vs.7.99±1.37%, p=0.66). Pearson’s correlation between FMD and historical HbA1c was negative with HbA1c at 12 (r=-0.419, p=0.03), at 15 (r=-0.437, p=0.03) and 12-24 months before vascular analysis (r=- 0.426, p=0.02). Conclusions: Endothelial function is impaired in T1DM patients compared to nondiabetic controls. ED is a phenomenon that can occur quite early in the natural history of T1DM, presented before 5 years of disease and is related to duration of disease, long- term metabolic control and microalbuminúria. Vascular smooth muscle was not impaired in T1DM patients with less than 5 years of disease, with values of non-endothelial-dependent dilation similar to controls, suggesting that ED can be a reversible event in this first years of disease.

abstract: Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by progressive autoimmune destruction of insulin-producing pancreatic β-cells. Genetic, immunological and environmental factors contribute to T1D development. The focus of this dissertation is to track the humoral immune response in T1D by profiling autoantibodies (AAbs) and anti-viral antibodies using an innovative protein array platform called Nucleic Acid Programmable Protein Array (NAPPA). AAbs provide value in identifying individuals at risk, stratifying patients with different clinical courses, improving our understanding of autoimmune destructions, identifying antigens for cellular immune response and providing candidates for prevention trials in T1D. A two-stage serological AAb screening against 6,000 human proteins was performed. A dual specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) was validated with 36% sensitivity at 98% specificity by an orthogonal immunoassay. This is the first systematic screening for novel AAbs against large number of human proteins by protein arrays in T1D. A more comprehensive search for novel AAbs was performed using a knowledge-based approach by ELISA and a screening-based approach against 10,000 human proteins by NAPPA. Six AAbs were identified and validated with sensitivities ranged from 16% to 27% at 95% specificity. These two studies enriched the T1D “autoantigenome” and provided insights into T1D pathophysiology in an unprecedented breadth and width. The rapid rise of T1D incidence suggests the potential involvement of environmental factors including viral infections. Sero-reactivity to 646 viral antigens was assessed in new-onset T1D patients. Antibody positive rate of EBV was significantly higher in cases than controls that suggested a potential role of EBV in T1D development. A high density-NAPPA platform was demonstrated with high reproducibility and sensitivity in profiling anti-viral antibodies. This dissertation shows the power of a protein-array based immunoproteomics approach to characterize humoral immunoprofile against human and viral proteomes. The identification of novel T1D-specific AAbs and T1D-associated viruses will help to connect the nodes in T1D etiology and provide better understanding of T1D pathophysiology. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2015

Achieving tight glucose control safely in type 1 diabetes with currently available methods of insulin delivery is challenging. Aggressive regimens carry an increased risk of hypoglycaemia, particularly overnight. Both alcohol consumption and exercise predispose further to low glucose levels. The demands are even greater in pregnancy where, in addition to limiting hypoglycaemia, avoidance of postprandial hyperglycaemia is critical to minimising adverse obstetric outcomes. The aim of my studies was to evaluate feasibility and safety of a closed-loop or ’artificial pancreas’ system linking insulin delivery with continuous glucose monitoring (CGM), in adults with type 1 diabetes in a controlled setting. Three randomised crossover studies compared closed-loop insulin delivery with conventional insulin pump therapy on two separate occasions, matched in meals and activities. During closed-loop visits, CGM values were entered into a computer containing a model predictive control algorithm which advised on basal insulin infusion for subcutaneous delivery, every 15 minutes. During control visits, usual insulin pump regimen was continued. The feasibility study evaluated overnight closed-loop in 12 adults (seven females, mean age 37.7 years, HbA1c 7.8%) following 60g- carbohydrate evening meal. A follow-up study assessed overnight closed-loop in 12 further adults (seven females, mean age 37.2 years, HbA1c 7.8%) following 100g-carbohydrate meal and (mean 564 ml) white wine. The third study evaluated 24 hours of closed-loop in 12 pregnant women (mean age 32.9 years, 19 to 23 weeks gestation, HbA1c 6.4%) during normal daily activities, including low and moderate intensity exercise. Activity and glucose levels were also measured during free-living. CGM performance during exercise was evaluated. Overnight closed-loop insulin delivery in adults, compared with conventional pump therapy, increased time spent with plasma glucose in target range (3.9−8.0 mmol/l) following both standard meal (81% versus 57%; p = 0.012) and large meal accompanied by alcohol (70% versus 46%; p = 0.012). Glycaemic variability, and time spent in hypo- and hyper- glycaemia were lowered. In pregnant women, day and night closed-loop insulin delivery was as effective as usual pump regimen (81% versus 81% time spent with plasma glucose 3.5−7.8 mmol/l; p = 0.754). Hypoglycaemia occurred following exercise, although closed-loop prevented nocturnal episodes. Glycaemic control during free-living was suboptimal, compared with controlled diet and exercise conditions. Accuracy of CGM was lower during exercise. In conclusion, these studies confirm the feasibility and efficacy of overnight closed-loop insulin delivery in adults with type 1 diabetes. Closed-loop is safe during pregnancy and may be beneficial in women with suboptimal glycaemic control. Meals and physical activity currently limit optimal daytime use of closed-loop.