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.

Le patient atteint de diabète de type 1 s’ajuste à la maladie afin de maintenir un équilibre glycémique vital. La présente thèse explore, dans le cadre des modèles théoriques d’autorégulation, les déterminants psychosociaux associés à cet ajustement. A cette fin, l’étude en ligne intitulée MONDIAB a été développée et mise en oeuvre. Les patients atteints de diabète de type 1 ont complété un questionnaire évaluant leur vécu à deux reprises. Au total, 321 patients âgés de 16 à 65 ans ont répondu au premier questionnaire, dont 120 patients ont participé à la deuxième évaluation six mois plus tard. Les résultats sont présentés sous forme de trois études empiriques identifiant les corrélats de l’ajustement comportemental, physiologique et émotionnel du patient. Une première étude montre que le contrôle perçu, la motivation autonome, ainsi que l’interaction de ces deux facteurs sont des prédicteurs de l’autocontrôle glycémique et de la diète diabétique. Une deuxième étude indique que le soutien de l’équipe soignante influence indirectement le taux d’hémoglobine glyquée du patient à six mois. Cet effet indirect est médiatisé par un renforcement de la motivation autonome ainsi que de la compétence perçue du patient. Une troisième étude met en évidence l’impact de la perturbation des buts de vie, des styles de coping ainsi que du sentiment d’autoefficacité et du soutien social perçu sur le bien-être psychologique du patient. En conclusion, les résultats de la présente thèse soulignent l’importance de la prise en compte des déterminants psychosociaux dans la compréhension et l’amélioration de l’ajustement au diabète de type 1 / Patients with type 1 diabetes need to adjust to the disease in order to maintain an adequate glycemic control. Based on the assumptions of self-regulation theory, the present thesis explores the role of psychosocial factors in adjustment to type 1 diabetes. For this purpose the online study MONDIAB has been developed and implemented. Patients who met the criteria for participation and who agreed to the study completed a questionnaire related to their personal experiences at two time intervals. Altogether, 321 patients aged between 16 and 65 years filled in the first questionnaire, thereof 120 patients participated in the second survey six months later. Results are presented by a means of three empirical studies that identify correlates of patients’ behavioral, physiological and emotional adjustment. The first study shows that perceived control, autonomous motivation, as well as the interaction of these factors serve as predictors for gylcemic control and diabetes diet. A second study points out the indirect effect of autonomy supportiveness of health care providers on patients’ glycosylated hemoglobin six months later. This indirect effect is mediated by a reinforcement of both patients’ autonomous motivation and perceived competence. The third study underlines the impact of goal disturbance, coping styles and self-efficacy, as well as perceived social support on patients’ well-being. In conclusion, the results of the present thesis elucidate the importance of considering psychosocial factors for understanding and improvingadjustment to type 1 diabetes

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.

Introdução: A presença de anticorpos (Ac) anti-HLA específicos contra o doador representam um potencial risco para o desenvolvimento de rejeição do enxerto pulmonar. Apesar de existirem evidências sobre a importância de anticorpos anti-receptor de tipo 1 da angiotensina II (AT1R) para a sobrevida do enxerto a longo prazo em transplante de rim e coração, não existe qualquer informação da sua frequência e relação com bronquiolite obliterante no transplante de pulmão. Objetivos: Descrever a freqüência e o impacto da presença de Ac anti-AT1R para o desenvolvimento de bronquiolite obliterante (BO) após o transplante pulmonar. Pacientes e Métodos: Foram alocados 50 pacientes com mais de seis meses de transplante, em acompanhamento ambulatorial. Uma alíquota de soro pré-transplante e outra colhida no momento da alocação foram testadas para a presença de Ac anti-HLA e anti-MICA em plataforma Luminex® e anti-AT1R (AT1R) por ELISA,. O acompanhamento médio foi de 78,3 meses. Foram avaliadas as características e desfechos clínicos, bem como o tempo de sobrevida para o desenvolvimento de BO. Os pacientes que nunca desenvolveram anti-AT1R foram comparados com aqueles que tinham o Ac pré-formado ou que o desenvolveram após o transplante. Foi utilizada a curva de sobrevida de Kaplan-Meier, reverse censoring method, log-rank e regressão de Cox (anti-HLA, anti-MICA, infecção por CMV e rejeição aguda). Os resultados foram descritos como risco relativo (RR) e intervalos de confiança (IC) de 95%, sendo significativos os valores de P<0,05. Resultados: A prevalência de anti-AT1R pré-formado foi de 22% e de novo 15,3%. Não se demonstrou associação entre anti-AT1R e anti-HLA pré-formados (P=0,279;1,1[0,8 a 1,7]), com tendência de associação com anti-HLA positivo pós transplante (P=0,063;1,3[0,9 a 1,8]). Cinquenta por cento (4/8) dos receptores por bronquiolite viral tinham anti-AT1R pré-formado, comparados a 16,7% (7/42) dos transplantados por outra patologia (P=0,037; 1,7 [0,8 a 3,4]), sem associação com anti-HLA (P=0,716) e anti-MICA (P=0,659) pré-formados. Nenhum paciente com linfangioliomiomatose (LAM) apresentou anti-AT1R pré ou pós-transplante. O RR para o desenvolvimento de BO comparando-se pacientes com anti-AT1R àqueles que nunca manifestaram o Ac foi de 1,50 (0,72 a 3,14) [P = 0,282]. Conclusão: Há uma tendência mostrando a associação entre a presença de anti-AT1R e o desenvolvimento de BO após o transplante pulmonar. A doença pulmonar de base parece ter implicação no desenvolvimento de anti-AT1R, sendo a que bronquiolite obliterante viral mostra maior risco para seu aparecimento enquanto a LAM risco mínimo. / Introduction: The presence of specific anti-HLA antibodies (Ab) against organ donors represent a potential risk for the development of rejection in lung grafts. There are consistent evidences about the importance of the anti-receptor for angiotensin II type 1 (AT1R) for long-term graft survival in kidney and heart transplantation. However, there is no information about AT1R in lung transplantation and its relationship with bronchiolitis obliterans. Objectives: The aim of this study was to describe the frequency and impact of the presence of anti-Ac AT1R for the development of bronchiolitis obliterans (BO) after lung transplantation. Patients and Methods: We studied fifty patients after six months of transplantation. All patients had an aliquot of pre-transplantation serum and other sample at the time of their selection for the study. The serum was tested for the presence of Ab anti-HLA and anti-MICA in a Luminex platform® and anti-AT1R (AT1R) by ELISA,. The mean followup was 78.3 months. We analyzed the clinical characteristics and outcomes and also survival time for the development of BO. We compared the patients who never developed anti-AT1R with those who had the preformed antibody or developed it after transplantation. We applied the survival curve of Kaplan-Meier, reverse censoring method, log-rank and Cox regression (anti-HLA anti-MICA, CMV infection and acute rejection). The results were described as relative risk (RR) and confidence intervals (CI) of 95%, with significant P values <0.05. Results: The prevalence of preformed anti-AT1R was 22% and 15,3% de novo. There were no association between preformed anti-AT1R and anti-HLA (P = 0.279, 1.1 [0.8 to 1.7]), with a trend of association with positive anti-HLA post transplantation (P = 0.063; 1.3 [0.9 to 1.8]). Fifty percent (4/8) of the receptors for viral bronchiolitis (BOV) had preformed anti-AT1R, compared to 16.7% (7/42) of the all transplanted patientes for other diseases (P = 0.037, 1.7 [0 8 to 3.4]). There were no association with preformed anti-HLA (P = 0.716) and anti-MICA (P = 0.659). No patients with lymphangioleiomyomatosis (LAM) had anti-AT1R pre-or post-transplant. The relative risk for developing BO when comparing patients with anti-AT1R to those who never expressed antibodies was 1.50 (0.72 to 3.14) [P = 0.282]. Conclusion: There is a trend showing the association between the presence of anti-AT1R and developing BO after lung transplantation. The underlying lung disease seems to have implications in developing anti-AT1R, and viral bronchiolitis obliterans that shows higher risk for onset while LAM minimum risk.

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.

Made available in DSpace on 2014-06-11T19:32:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-08-07Bitstream added on 2014-06-13T18:43:12Z : No. of bitstreams: 1 nunes_vs_dr_botfm.pdf: 1297313 bytes, checksum: 470ba33e35f9a75851e361e40639894a (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A Neoplasia Endócrina Múltipla tipo 1 (MEN1; OMIM 131100) é uma doença genética, herdada de forma autossômica dominante, caracterizada pela presença de tumores em pelo menos dois dos seguintes tecidos endócrinos: paratireóide, enteropancreático e adenohipófise. Além destes componentes maiores, tumores adrenocorticais, carcinóides, lipomatose, angiofibroma e colagenoma facial têm sido associados. Trata-se de uma síndrome rara com uma prevalência estimada de 2-3/100000 indivíduos, causada por mutações inativadoras no gene MEN1. Este, por sua vez, codifica uma proteína chamada menin, que tem demonstrado interagir com diversas proteínas envolvidas em processos celulares essenciais, como controle do crescimento e ciclo celular, reparo de DNA, regulação da transcrição gênica, regulação estabilidade genômica, e controle da apoptose. A identificação do gene MEN1 possibilitou a detecção de mutações causadoras da doença e, com isto, a confirmação do diagnóstico clínico em pacientes acometidos, bem como o diagnóstico precoce em familiares assintomáticos. É preconizada a pesquisa dos principais tumores associados a MEN1 em pacientes já com o diagnóstico da síndrome ou nos portadores da mutação, mas a abordagem inversa que é a investigação da MEN1 em pacientes com diagnóstico inicial apenas de um dos principais tumores associados tem sido pouco explorada. Por isto, esta pesquisa consistiu em... / Multiple endocrine neoplasia type 1 (MEN1; OMIM 131100) is a genetic disease, inherited in the dominant autosomal form and characterized by the presence of tumors in at least two of the following endocrine tissues: parathyroid, enteropancreatic and adenopituitary. Besides the aforementioned major components, adrenocortical and carcinoid tumors, lipomas, collagenomas and facial angiofibromas have been associated with the disease. MEN1 is a rare disease, with an estimated prevalence of 2-3/100000 individuals; it is caused by inactivated mutations of the MEN1 gene. This gene encodes one protein called menin, which has been shown to interact with a number of proteins that are involved in essential cell processes such as cell division and proliferation, DNA repair, transcriptional regulation, genome stability, and apoptosis control. The MEN1 gene identification has enabled the detection of MEN1 mutations and the confirmation of the disease’s clinical diagnosis as well as its early diagnosis in asymptomatic relatives. The screening of these principal tumors associated with MEN1 has been recommended in patients with MEN1 syndrome or MEN1 mutation. However, the inverse approach (i.e., the investigation of MEN1 in patients with an initial diagnosis of only one of the principal tumors) has been little explored. For this reason, the present study aimed to evaluate the frequency of MEN1 in a group the patients with pituitary adenoma (PA), and to identify, in these individuals, variables... (Complete abstract click electronic access below)

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

Sabe-se que um dos principais problemas odontológicos na clínica de felinos é a gengivite crônica e intratável. Tal afecção pode ser iniciada e/ou exacerbada por agentes virais, como o vírus da imunodeficiência dos felinos (FIV), o Herpesvírus tipo 1 e o Calicivírus. Os gatos da raça Maine Coon apresentam grande predisposição ao desenvolvimento de gengivite-estomatite juvenil e intratável. A depleção de linfócitos T CD4+ e T CD8+ pode exercer papel determinante na iniciação e manutenção das doenças inflamatórias da gengiva. O escopo do presente estudo foi verificar se os animais da raça Maine Coon são mais predispostos à calicivirose, bem como avaliar quantitativamente a resposta imunológica celular, mediada por linfócitos TCD4+ e TCD8+, visando a correlacionar à influência do número de linfócitos na presença e curso da gengivite nesta determinada raça, utilizando-se como controle gatos de outras raças com e sem gengivite. Os valores absolutos médios de linfócitos totais em Maine Coons com gengivite crônica mostraram-se inferiores aos de gatos da raça Maine Coon sem doença oral e de gatos de outras raças com gengivite crônica (p<0,05); os valores médios de linfócitos TCD4+ em Maine Coons com gengivite crônica mostraram-se inferiores quando comparados aos valores de animais da mesma raça, sem doença oral instalada (p<0,05); animais da raça Maine Coon possuem menor relação CD4+:CD8+ quando comparados a animais de outras raças com gengivite crônica e também quando comparados a Maine Coons sem doença oral (p<0,05). O calicivírus está altamente relacionado à ocorrência da gengivite, independentemente da raça estudada, não havendo maior prevalência na raça Maine Coon. O efeito do calicivírus não foi significativo nas alterações de nenhuma das variáveis celulares estudadas. Tais fatos apontam para uma possível predisposição racial ao quadroinflamatório gengival, com alteração de alguns componentes celulares relacionados à imunidade celular. Isto tem como fator importante alertar o clínico frente ao uso de glicocorticóides no tratamento da gengivite crônica nesta raça, visando a evitar maior comprometimento da imunidade celular destes animais. / Chronic untreatable feline gingivitis is widely recognized as one of the major oral diseases seen in feline patients. It can be either triggered or exacerbated by virus such as feline immunodeficiency virus, feline herpesvirus type 1 and calicivirus. One may therefore propose that lymphocytes T CD4+ and T CD8+ depletion can play an important role in initiating and maintaining the inflammatory gingival disease. Maine Coon cats are highly predisposed to juvenile untreatable gingivitis. The purpose of this study was to evaluate whether Maine Coon cats are more predisposed to calicivirus infection and to verify, quantitatively, their immunological cellular response mediated by lymphocytes T CD4+ and TCD8+. The main idea was to investigate the influence imposed by lymphocyte counts in gingivitis development and progression within this breed; for this, we selected non-Maine Coon cats (with and without gingivitis) to serve as controls. Mean absolute values of total lymphocytes in Maine Coon cats presented with gingivitis were inferior than the same values taken for both Maine Coon cats free of oral disease and non-Maine Coon cats with chronic gingivitis (p<0,05); lymphocytes TCD4+ average values in Maine Coon cats with chronic gingivitis were also lower than the ones taken from cats of the same breed but without oral disease (p<0,05). Maine Coon cats have lower CD4+:CD8+ ratio when compared to non-Maine Coon cats with chronic gingivitis as well as with Maine Coon cats without oral disease (p<0,05). The calicivirus is highly involved with the occurrence of gingivitis, no matter the breed being evaluated. The action virus imposes in changing cellular immunology was not significant, at least considering the cellular variables studied. All these lead us to point out a possible breed predisposition to the gingival inflammation, with modification of some cellular components related with cellular immunity. Furthermore, concerning practical terms, these results serve as a relevant alert to the clinicians regarding the use of glucocorticoids for treating chronic gingivitis in this breed, in order to prevent further impairment of cellular immunity of these animals.