Дисертації з теми "MicroRNAs - biomarkers - type 1 diabetes"

Type 1 diabetes (T1D) results from a T cell mediated destruction due to a breach in immune tolerance towards insulin producing β-cells, leading to an absolute insulin deficiency. To date, the pathogenesis of the disease is not fully understood and exogenous insulin still represents the gold standard as therapy. It is believed that in some genetically predisposed individuals environmental factors can lead to a breakdown in immunological tolerance. T regulatory cells (Tregs), a specialized subpopulation of T cell, are the major components of peripheral tolerance mechanisms due to their ability to restrain autoreactive effector T cells. Interestingly, microRNAs, small non-coding RNAs that negatively regulate gene expression, are emerging as crucial modulators of immune cell functions and their deregulation has been associated with autoimmune disease like T1D, thus representing major players in the regulation of immune homeostasis. Against this background, characterizing microRNA expression in Tregs deriving from tissues close to target organ in T1D patients will be crucial to gain a deeper insight into immune deregulation. Interestingly, microRNAs have also been detected extracellularly in biological fluids, thus representing new potential disease biomarkers. The need of new biomarkers in the field is underlined by the complexity and heterogeneity of T1D with the clinical presentation being preceded by a long asymptomatic phase in which most β-cells are destroyed. Moreover, immunomodulatory trials in T1D, although having failed their primary endpoints, have identified through post-hoc analysis subgroups of patients for whom interventions were beneficial. Therefore, the identification of new biomarkers would help to specifically select patients that would benefit from specific immunotherapies. Thus, the main purpose of this work was to gain insight into the microRNA regulation occurring in Tregs residing in the pancreatic draining lymph nodes (PLN) of T1D patients and to establish circulating microRNAs as new biomarkers in diabetes autoimmune prone mice in order to monitor the lymphocyte mediated inflammatory state in the pancreas and to predict therapeutic responses in an Ag-specific combination therapy. Tregs circulating in the peripheral blood are likely to differ from those residing in the LN draining the organ targeted by an autoimmune response, indeed a previous finding showed that only Tregs deriving from PLN of T1D patients have an impaired regulatory activity in vitro. Therefore, in the first project (Chapter 4) we investigated the microRNA expression profile in Tregs isolated from blood and PLN of patients with T1D and non-diabetic subjects. Among the 72 microRNAs detected in purified cells, miR-125a was highly expressed only in Treg cells purified from PLN of patients with T1D. Bioinformatic analysis of the target genes revealed CCR2 as a possible gene modulated by miR-125a. Accordingly, elevated miR-125a levels were detected in Tregs isolated from the PLN but not from the peripheral blood of donors with T1D and it was associated with a reduced CCR2 expression. A specific β-cell expression of the CCR2 ligand CCL2 (MCP1) was also observed in the pancreata of multi-organ donors, suggesting that β-cells are prone to attract CCR2+ Tregs. These data showed a reduced expression of CCR2 on Tregs deriving from PLN of T1D patients; this Treg status may limit their migration and eventual function in the pancreas. In the second project (Chapter 5) we aimed to investigate whether circulating microRNA alterations could reflect the pathologic process within the specific diseased tissue. Indeed, we identified miR-409-3p, a miRNA that negatively regulates the expression of IFNγ, as downregulated in both plasma and pancreatic lymphocytic infiltrates of recent-onset diabetic vs. normoglycemic NOD mice. Furthermore, we showed that circulating miR-409-3p plasma levels negatively correlated with in situ IFNγ expression in islet-infiltrating cells both in basal condition and following a therapeutic intervention with anti-CD3 treatment, thus highlighting the potential role of miR-409-3p as a biomarker of islet inflammation. Finally, we confirmed miR-409-3p downregulation in plasma samples derived from newly-diagnosed T1D patients vs. age-matched controls, indicating a possible application of circulating miR-409-3p as biomarker in order to monitor IFNγ mediated islet inflammation. The host lab previously showed that a combination therapy consisting of a 5 day course of anti-CD3 antibodies at disease onset along with a 6 weeks oral administration of live genetically modified Lactococcus lactis (L. lactis) producing human proinsulin and IL-10, restored durable normoglycemia in approximately 60% of NOD mice. Even though L. lactis therapy was successful in the reversal of autoimmune diabetes in around 60% of NOD mice, 40% of them were nonresponsive to therapy. The route to bring this successful antigen-based therapy from preclinical models to clinic will depend on implementation of biomarkers and profound understanding of diverse mechanisms underlining therapeutic success. Therefore, the aim of the third project (Chapter 6) was to establish whether circulating microRNAs could be used as predictor biomarkers for therapeutic response. Indeed, we found a circulating microRNA signature consisting of 6 microRNAs that were specifically upregulated at disease onset in non-responders mice. Moreover, the combination of 2 microRNAs were able to distinguish with good specificity and sensitivity. responder and non-responders at diabetes onset, thus emerging as a valuable tool to tailor this intervention therapy towards a more effective clinical study. Moreover, bioinformatics target analysis of the circulating microRNAs differentially expressed between responders and nonresponders in the plasma after treatment course, highlighted pathways related to the metabolic status of T cells, suggesting the induction of a T cell exhausted phenotype in responder mice. To conclude, this PhD project demonstrated the importance of assessing microRNA regulation in Tregs deriving from tissue sites close to the organ targeted by the autoimmune attack, as it differs from circulating Tregs. Moreover, plasma circulating microRNAs appear to be promising novel biomarkers in order to: i) monitor IFNγ mediated islet inflammation ii) predict therapeutic response and understand the tolerance induction mechanisms induced by L. lactis-combined therapy. Molecular understanding of Tregs function in the site of the autoimmune attack together with the discovery of new circulating biomarkers will surely help to monitor T1D progression and enhance the effective translation of this Ag-specific L.lactis intervention therapy in future clinical trials.

INTRODUÇÃO: O diabetes tipo 1 autoimune (DM1A) está associado a alterações na imunidade inata e adaptativa. A agressão autoimune, órgão específica, determina a destruição das células beta do pâncreas e a deficiência da produção de insulina. O infiltrado inflamatório do tipo linfomononuclear, configurando a insulite, e a escassez ou a ausência das células ?, definem o quadro histológico do DM1A. Os autoanticorpos contra antígenos das células beta, que geralmente se desenvolvem na fase pré-clínica, conferem predisposição para DM1A. No entanto, é difícil definir quando e quais indivíduos progredirão para o diabetes manifesto, justificando a busca de outros biomarcadores que auxiliem nas indicações de tratamentos preventivos. Nesse contexto, sabe-se que os microRNAs (miRNAs), pequenos RNAs que atuam pós transcrição, desempenham papel crucial na regulação de genes, integrando fatores genéticos e ambientais e influenciando o funcionamento de órgãos e tecidos de maneira pontual ou sistêmica. OBJETIVOS: avaliar o envolvimento biológico e a relevância da expressão de miRNAs na resposta imunológica e na função das células ? na patogênese do DM1A. MÉTODOS: analisamos o perfil dos miRNAs séricos em 4 grupos, a saber: pacientes portadores de DM1A, até 6 meses do diagnóstico (DM1A recente), (n=30); pacientes portadores de DM1A com duração de 2-5 anos (DM1A 2-5)(n=26) e indivíduos com autoanticorpos pancreáticos positivos sem diabetes (AcP) (n=25), os quais foram comparados aos indivíduos controles saudáveis(n= 29). A expressão dos microRNAs foi obtida com ensaios individuais TaqMan® MicroRNA Assays 5x primers e TaqMan MicroRNA Human Array Card A, (Applied Biosystems- Forster City CA, USA) constituído por 377 alvos e 4 endógenos. Os dados de expressão foram analisados no Software Cloud, (Thermo Fisher Scientific) e no programa Limma (Linear Models for Microarray and RNA-Seq Data). RESULTADOS: Não houve diferença nas características demográficas, como idade, cor auto referida e sexo entre os grupos (p > 0,05). Pacientes portadores de DM1A (recente e com duração de 2-5 anos), diferiram do grupo controle pelos valores elevados de glicose, hemoglobina glicada, títulos de autoanticorpos pancreáticos, e menores de peptídeo C (p < 0,05) e foram semelhantes entre si. Os portadores de autoanticorpos (AcP) tinham características intermediárias entre os grupos: menores valores de HbA1c e de anticorpo anti-tirosina-fosfatase (anti-IA2) e maiores de peptídeo C em relação aos dois grupos com diabetes. Diferiram dos controles apenas pelos maiores títulos de anticorpo anti-insulina (IAA) e anti-descarboxilase do ácido glutâmico 65 (anti-GAD65). A frequência dos alelos HLA de risco para diabetes (-DR3 ou -DR4 e -DQ2 ou DQ-8) decresceu dos grupos DM1A recente e DM 2-5 para AcP e controles. Foram avaliados 135 miRNAs que estavam expressos em 20% ou mais das amostras dos quatro grupos analisados. Maior expressão foi observada em 13, 4 e 33 miRNAs dos grupos AcP, DM1A recente e DM1A 2-5 respectivamente e menor em 11, 7 e 31 miRNAs destes grupos. Destes, 4 miRNAs foram diferencialmente expressos nos grupos AcP, DM1A recente e DM1A 2-5 em relação ao grupo controle. Os miRNAs: miR -16, miR-195 e miR-454, relacionados com regeneração endócrina do pâncreas, efeito anti-inflamatório e resposta à injúria da célula ? estavam diminuídos nestes 3 grupos. O miR-200a, implicado em apoptose das células beta, estava aumentado nos grupos AcP e DM1A recente e diminuído nos pacientes com maior duração do diabetes (DM1A 2-5), possivelmente devido à escassez destas células. Outros 8 miRNAs apresentaram expressão diferente da do grupo controle em dois dos grupos avaliados, e tendência semelhante no terceiro grupo, sendo 4 deles elevados (miR-193a-5p, miR- 323-3p, miR-423-5p, e miR-92a) e 4, diminuídos (miR-191, miR-19a, miR- 376a, miR-590-5p) ou neutralidade no 3º grupo (miR-15b, miR-100, miR-181a e miR-483-5p) Resposta antagônica foi observada para o miR-25 e miR-485- 3p, diminuídos no grupo AcP e aumentados no DM1A 2- 5. Tais miRNAs estão relacionados com resposta imunológica, secreção de insulina, lesão de células ? e glicotoxicidade, à semelhança do observado para o miR-101-3p, validado por ensaios individuais numa casuística maior. CONCLUSÃO: nossos dados sugerem que miRNAs circulantes podem estar envolvidos na patogênese do DM1A
INTRODUCTION: Autoimmune type 1 diabetes (T1D) is associated with changes in innate and adaptive immunity. The organ-specific autoimmune aggression determines the destruction of beta-cells in the pancreas and the deficient insulin production. The inflammatory infiltration of the lymphomononuclear type, configuring the insulite, and the scarcity or the absence of the beta cells, define the histological picture of T1D. Autoantibodies against beta-cell antigens, which usually develop in the preclinical phase, confer predisposition to T1D. However, it is difficult to define when and which individuals will progress to overt diabetes, justifying the search for other biomarkers that could be indicative of preventive treatments. In this context, it is known that the microRNAs (miRNAs) - small RNAs that act post transcription - play a crucial role in regulating genes and in integrating genetic and environmental factors, influencing the function of organs and tissues in a punctual or systemic way. OBJECTIVES: to evaluate the biological involvement and relevance of miRNA expression in the immune response and ?-cell function in the pathogenesis of T1D. METHODS: we analyzed the profile of serum miRNAs of 4 groups, namely: patients with T1D up to 6 months after diagnosis (recent T1D), (n = 30); patients with T1D lasting 2-5 years (T1D 2- 5) (n = 26) and individuals expressing pancreatic autoantibodies without diabetes (AbP) (n = 25), which were compared to healthy controls (n = 29). Expression of the microRNAs was obtained with individual assays TaqMan® MicroRNA Assays 5x primers and TaqMan MicroRNA Human Array Card A (Applied Biosystems-Forster City CA, USA), consisting of 377 targets and 4 endogenous. The expression data was analyzed in the Cloud Software (Thermo Fisher Scientific) and Limma (Linear Models for Microarray and RNASeq Data) program. RESULTS: There was no difference in demographic characteristics, such as age, self-reported color, and sex among groups (p > 0.05). Patients with T1D (both recent and 2-5 years), similar to each other, differed from the control group by high glucose, glycated hemoglobin levels, pancreatic autoantibody titers, and lower C peptide values (p < 0.05) . Pancreatic autoantibodies (AbP) carriers had intermediate characteristics among the groups: lower HbA1c and anti-tyrosine phosphatase antibody (anti- IA2) values and higher C-peptide levels than the two groups with diabetes. They differed from controls only by the higher titers of anti-insulin (IAA) and anti-decarboxylase of glutamic acid 65 (anti-GAD65) autoantibodies. The frequency of high risk HLA alleles for diabetes (-DR3 or -DR4 and -DQ2 or DQ- 8) decreased from the recent T1D and T1D 2-5 groups to the AbP and controls. We evaluated 135 miRNAs that were expressed in 20% or more of the samples from the four groups analyzed. Higher expression was observed in 13, 4 and 33 miRNAs of the Abp, recent T1D and T1D 2-5 groups respectively and lower in 11, 7 and 31 miRNAs of these groups. Of these, 4 miRNAs were differentially expressed in the AbP, recent T1D and T1D 2-5 groups in relation to the control group.The miRNAs: miR -16, miR-195 and miR-454, related to endocrine regeneration of the pancreas, anti-inflammatory effect and response to beta-cell injury were decreased in these 3 groups. miR-200a, implicated in beta-cell apoptosis, was increased in the recent and decreased AbP and T1D groups in patients with longer duration of diabetes (T1D 2-5y), possibly due to the shortage of these cells. Another eight miRNAs showed different expression of the control group in two of the evaluated groups, and a similar trend in the third group, four of them high (miR-193a-5p, miR-323-3p, miR-423-5p, and miR- 92a ) and four, decreased (miR-191, miR-19a, miR-376a, miR-590-5p) or neutrality in the 3rd group (miR-15b, miR-100, miR-181a and miR-483-5p) was observed for miR-25 and miR-485-3p, decreased in the AbP group and increased in T1D 2-5y. Such miRNAs are related to immune response, insulin secretion, ?-cell damage and glycotoxicity, similar to that observed for the miR- 101-3p, validated by individual trials in a larger cohort. CONCLUSION Our data suggests that circulating miRNAs may be involved in the pathogenesis of T1D

Abstract Loss of insulin-producing β-cells is central to the development of Type 1 diabetes (T1D). Currently, we lack diagnostic tools to quantitate this β-cell loss. Non-protein coding RNAs called microRNAs (miRNAs/miRs) play an important role in islet development and function. Recent detection of miRNAs in peripheral circulation, has renewed interest in microRNA biomarkers of diabetes. Comparably, circulating insulin cell-free (cf)DNA has been proposed as a direct biomarker of β-cell death. DNA methylation studies have identified specific sites within DNA that are unmethylated in β-cells but methylated in other cell types, thus providing a handle to discriminate between cfDNA from β-/non-β-cells. Previous research carried out in the Hardikar lab identified a signature of 20 miRNAs (the ‘RAPID’ signature) with potential as a biomarker of β-cell death. The RAPID signature was revised to accommodate other microRNAs finally constituting a panel of 50 microRNAs (PREDICT T1D panel). An analysis of these 50 miRNAs, as well as insulin cfDNA in serum/plasma from individuals before, during and after clinical diagnosis of T1D is presented. Human islet cell death assays using sodium nitroprusside exposure identified a subset of 27 miRNAs and insulin cfDNA associated with islet cell stress/death. Non-obese diabetic mice (N=32) were found to have elevated candidate miRNAs prior to immune infiltration and glycaemic dysfunction. This trend was also noted in the human progression to T1D; 26 miRNAs were elevated in (N=19) high-risk individuals and those at diagnosis (N=199) but decreased within 6-weeks after diagnosis. Furthermore, candidate miRNAs exhibited differential abundance with disease duration, residual C-peptide, and microvascular complications in 180 subjects with prolonged T1D. At diagnosis, miRNAs and cfDNA associated with GAD III autoantibody titres (N=167 P-values range from 0.044 to <0.0001) and HbA1c levels (N=187, P-values range from 0.047 to 0.00095). Such biomarkers may inform medical researchers as to how to predict the development of T1D, monitor response to interventions such as islet transplantation, vaccines & drugs aiming to retard β-cell loss. In basic research, such an assay may help to select treatments to block β-cell death and guide the development of new treatments to lessen the burden of diabetes.

>Magister Scientiae - MSc
Type 2 diabetes mellitus (T2DM), a metabolic disease characterized by chronic hyperglycemia, is the most prevalent form of diabetes globally, affecting approximately 95 % of the total number of people with diabetes i.e. approximately 366 million. Furthermore, it is also the most prevalent form in South Africa (SA), affecting approximately 3.5 million individuals. This disease and its adverse complications can be delayed or prevented if detected early. Standardized diagnostic tests for T2DM have a few limitations which include the inability to predict the future risk of normal glucose tolerance individuals developing T2DM, they are dependent on blood glucose concentration, its invasiveness, and they cannot specify between T1DM and T2DM. Therefore, there is a need for biomarkers which could be used as a tool for the early and specific detection of T2DM. MicroRNAs are small non-coding RNA molecules which play a key role in controlling gene expression and certain biological processes. Studies show that dysregulation of microRNAs may lead to various diseases including T2DM, and thus, may be useful biomarkers for disease detection. Therefore, identifying biomarkers like microRNAs as a tool for the early and specific detection of T2DM, have great potential for diagnostic purposes. The main focus of this investigation, therefore, is the early detection of T2DM by the identification and validation of novel biomarkers. Furthermore, based on previous studies, the aim of the investigation was to identify differentially expressed miRNAs as well as identify their potential target genes associated with the onset and progression of T2DM. An in silico approach was used to identify miRNAs found to be differentially expressed in the serum/plasma of T2DM individuals. Three publically available target prediction software were used for target gene prediction of the identified miRNA. The target genes were subjected to functional analysis using a web-based software, namely DAVID. Functions which were clustered with an enrichment score > 1.3 were considered significant. The ranked target genes mostly had gene ontologies linked with “transcription regulation”, “neuron signalling, and “metal ion binding”. The ranked target genes were then split into two lists – an up-regulated (ur) miRNA targeted gene list and a down-regulated (dr) miRNA targeted gene list. The in silico method used in this investigation produced a final total of 4 miRNAs: miR-dr-1, miR-ur-1, miR-ur-2, and miR-ur-3. Based on the bioinformatics results, miR-dr-1 and its target genes LDLR, PPARA and CAMTA1, seemed the most promising miRNA for biomarker validation, due to the function of the target genes being associated with T2DM onset and progression. The expression levels of the miRNAs were then profiled in kidney tissue of male Wistar rats that were on a high fat diet (HFD), streptozotocin (STZ)-induced T1DM, and non-diabetic control rats via qRT-PCR analysis. The hypothesis was that similar miRNA expression would be found in the HFD kidney samples compared to serum expression levels of the miRNA obtained from the two databases, since kidneys are involved in cleansing the blood from impurities. This hypothesis proved to be true for all miRNAs except for miR-ur-2. Additionally, miR-ur-1 seemed the most significant miRNA due to it having different expression ratios for T1DM and T2DM (i.e. -7.65 and 4.2 fold, respectively). Future work, therefore, include validation of the predicted target genes to the miRNAs of interest i.e. miR-dr-1: PPARA and LDLR and miR-ur-1: CACNB2, using molecular approaches such as the luciferase assays and western blots.

Type 1 diabetes (T1D) is a chronic multifactorial autoimmune disease that includes 5-10% of diabetes cases and is characterized by the immune-mediated insulin secreting β cell destruction with consequent loss of β cell mass and hyperglycemia. In autoimmune diabetes, the selective β cell destruction is the result of a series of mechanisms including, but not limiting to autoreactive T cells direct β cell destruction and inflammatory stress, resulting in a dramatic loss of functional β cell mass. MicroRNAs are small non coding 19-24 nucleotides RNAs that negatively regulate gene expression, binding selectively their mRNAs target and leading to their translational repression and/or degradation. It was also remarked that miRNAs are involved in T1D pathogenesis, by regulating a series of processes both in β cells and lymphocytes that ranged from cell metabolic dysfunction and apoptosis to dedifferentiation and immune molecule secretion. Additionally, it has been suggested that secreted microRNAs can take part in the communication between immune system and pancreatic endocrine cells; therefore, studying such dialogue could help to better understand T1D pathogenesis and to the identification of biomarkers that reflect the diseased status of the target organ. In order to investigate the role of miRNAs as biomarkers of T1D pathogenesis both at endocrine and immune interface, the study can be subdivided into the following sections: 1) MiRNAs as circulating biomarkers of diseased-target tissue- In this section, miR-409-3p was found downregulated in murine and human plasma of T1D subjects, highlight the importance to find a lymphocyte deriving circulating biomarker that reflect the target organ pathological status. 2) MiRNAs as modulators of β cell function- miR-183-5p was downregulated in pancreatic endocrine tissue of mouse and human diabetic patients, contributing to β cell protection from apoptosis through the modulation of anti-apoptotic factor Bach2 and through the potential induction of a dedifferentiation phenomenon. This study demonstrated that β cells are able to react to stress and to cell damage through a miRNA-mediated protective phenomenon. 3) Secreted/Immune-derived miRNAs as mediators of β cell apoptosis- Exosomes enriched in miR-142-3p/5p and miR-155 and secreted by T autoreactive lymphocytes, induced selective β cell apoptosis, through the modulation of proinflammatory chemokine expression. The selective AAV “Sponge” inactivation of miR-142-3p/5p, miR-150 and miR-155 induced β cell protection of prediabetic NOD mice from the autoimmune attack that occurs in T1D, inducing the reduction of the proinflammatory chemokine CXCL10 specifically in β cells than in α cells. Such study remarked the exosome-mediated communication dynamics between immune system components and endocrine cells in the center of this dialogue, miRNAs give their fundamental contribution and more importantly, as it has already been reported, it’s possible to actively and therapeutically interfere in this dialogue in order to positively change the β cell fate.

The principal aim of the study was to develop methods for the measurement of potential urinary biomarkers of oxidative stress using liquid chromatography/tandem mass spectrometry with minimum sample preparation to avoid artefact formation. Initially the development of an assay to measure the urinary concentrations of isoprostanes (8- isoPGF2α) was attempted but this did not prove to be sufficiently sensitive and gave nonreproducible results. An assay to measure the intact sulphate and glucuronide conjugates of urinary metabolites of vitamin E [α-tocopheronolactone (α-TLHQ) and α-carboxy-ethylhydroxychroman (α-CEHC)] was then developed, as it has been suggested that α-TLHQ with an oxidised chroman ring might be an indicator of oxidative stress. A novel method was also developed to quantitate urinary amino acids associated with NO• metabolism (Larginine - precursor, L-citrulline - product, L-ADMA –inhibitor of nitric oxide synthase and L-homocysteine – reduces bioavailability of nitric oxide). This method was extended to quantitate seven additional amino acids. The latter two methods were applied to 32 children with type 1 diabetes and compared with age and sex matched controls. The mean concentrations of all the α-THLQ conjugates were highly significantly increased in the diabetic subjects (p<0.002). The concentrations of the α-CEHC conjugates were also increased but not to the same degree of significance (p<0.05). When the diabetic children were divided into those who were poorly (n=24) and adequately (n=8) controlled, the α- THLQ conjugates remained highly significantly increased (p<0.002) in the poorly controlled group compared to controls. However, the concentrations of the α-CEHC conjugates were not significantly different. The diabetic subjects had a highly significantly increased concentration (p<0.0001) of all the urinary amino acids studied compared to controls. These results suggest that the measurement of urinary α-TLHQ conjugates may provide a useful biomarker of oxidative stress. The clinical relevance of the increased concentrations of urinary amino acids in children with type 1 diabetes requires further investigation.

Primary prevention of type 2 diabetes (T2DM) is an important strategy for curbing its rising global burden. Though lifestyle modification has provided an effective method of preventing/delaying incidence of diabetes in high-risk individuals, it has not been widely implemented even in developed countries due to its high-cost, need for expertise and difficulties in translating the benefits of lifestyle intervention to the community at large. Hence, there is an urgent need to identify an alternate mode of delivery to transmit healthy lifestyle information to high-risk individuals. In this trial, we sought to determine whether lifestyle advice through mobile phone text messaging could reduce incident diabetes compared to standard lifestyle advice in Asian Indian men with prediabetes. The study showed for the first time that mobile phone messaging is an effective and acceptable method to deliver advice and support towards lifestyle modification to prevent T2DM in men at high risk. The identification of novel predictors for T2DM is an arduous task. The glycaemic markers of diabetes (fasting plasma glucose, 2hr post glucose load and HbA1c) are, in fact, risk factors for microvascular complications of diabetes and it was on this basis that diagnostic cut-offs for diabetes were arrived at. Nevertheless, elevated levels of glycaemic markers in the sub-clinical, or pre-diabetic, range are associated with increased risk of progression to diabetes. However, there is already considerable deterioration of beta cell function by the time diabetic dysglycaemia occurs. The way forward is clearly to identify biomarkers that serve as reliable predictors of progression to diabetes rather than simply reflecting accompanying levels of glycaemia. In this thesis using the database of the above mentioned trial it was aimed to identify the predictors of T2DM in Asian Indian cohort with prediabetes at baseline. The classical risk factors studied here are: 1) increased prevalence of the hypertriglyceridemic waist phenotype, 2) a combination of HbA1c and gamma glutamyl transferase and 3) a measure of beta cell compensation (disposition index) predicted incident diabetes. Among these, the disposition index was the most powerful predictor in the cohort. In addition to these classical risk factors mentioned above, in a small nested-case control, cross sectional study, the association of adipokines (adiponectin, leptin, interleukin-6 (IL-6), retinol-binding protein4 (RBP4)) and vitamin D3 were assessed to study the mechanistic link of novel biomarkers with diabetes. In this cohort, lower levels of baseline adiponectin, and higher IL-6 and RBP4 were associated with diabetes. Though, many of these provided a novel mechanistic pathogenic link with diabetes they did not improve prediction over and above that of glycaemic measures in identifying individuals with diabetes. However, the non-glycaemic biomarkers appear to have a role in the underlying pathogenesis of diabetes.

O timo é um orgão linfóide primário, no qual ocorre a indução da tolerância imunológica central aos antígenos do próprio que são expressos pelos tecidos periféricos (PTAs). A medula tímica é formada por células tímicas medulares epiteliais (mTECs) que expressam centenas desses PTAs que representam virtualmente todos os órgãos e tecidos do corpo. Esse fenômeno foi denominado de expressão gênica promíscua (PGE) a qual é parcialmente regulada pelo modulador da transcrição Autoimmune regulator (Aire). Os precursores de células T oriundos da medula óssea migram para o timo (agora são denominados de timócitos) e na medula desse órgão, passam pela seleção negativa mediada pelas mTECs. As células sobreviventes evoluem para células T maduras e funcionais que migram para a periferia com capacidade de reconhecimento das moléculas de MHC e tolerantes aos PTAs. Além de controlar a transcrição de genes PTAs, Aire também controla a expressão de microRNAs (miRNAs), relacionados com a integridade e funcionalidade do microambiente tímico. A seleção negativa no timo é um processo essencial para a manutenção da autotolerância imunológica e o desbalanço desse processo está associado com o desenvolvimento de doenças autoimunes como, por exemplo, o diabetes mellitus do tipo 1 (DM1).Tendo em vista essas premissas, nosso trabalho se fundamentou em duas hipóteses: 1) Variações na expressão do gene Aire podem perturbar a expressão de genes PTAs e miRNAs no timo, causando alterações na PGE, 2) A expressão balanceada de genes como Aire e/ou PTAs nas mTECs, é fundamental para a integridade da tolerância central. O desbalanço na expressão desses genes, está associado com a emergência do diabetes mellitus tipo 1 no camundongo. Para testar nossa primeira hipótese efetuamos o silenciamento de Aire (Aire knockdown) por meio de eletrotransfeção de RNA interferente (siRNA) anti-Aire in vivo no timo de camundongos BALB/c. Análises do transcriptoma (mRNAs) e miRNoma (miRNAs) das mTECs, revelaram que silenciamento parcial e transitório de Aire foi suficiente para afetar a expressão de PTAs Aire dependentes bem como a de miRNAs. Redes de interação miRNA-mRNA, revelaram que o controle pós-transcricional da PGE também é afetado pelo silenciamento de Aire. Os resultados encontrados revelam que Aire e miRNAs podem formar uma via essencial durante a indução da tolerância central. Para testar nossa segunda hipótese comparamos o transcriptoma de mTECs de camundongos BALB/c (linhagem não-autoimune) com mTECs de camundongos non-obese diabetic NOD (modelo animal utilizado nos estudos de DM1 autoimune). Nossos resultados revelaram que a expressão transcricional de autoantígenos relacionados ao DM1 está desbalanceada em camundongos NOD já numa fase precoce, quando esses animais ainda não apresentavam a doença clínica (fase pré-diabética). Inesperadamente, os níveis transcrionais de Aire apresentaram-se equivalentes no timo dessas duas linhagens, porém os níveis da proteína AIRE estavam reduzidos no timo da linhagem NOD. Esses resultados sugerem a participação de algum mecanismo de atenuação póstrascricional de Aire nessa linhagem provavelmente envolvendo atuação de miRNAs. Isso poderia explicar o desbalanço de PTAs Aire-dependentes e a repressão autoantígenos relacionados ao DM1. Concluímos que nossos resultados, além de abrir novas perspectivas para pesquisas nesta área, contribuem com melhor compreensão dos mecanismos moleculares desencadeados por Aire e por miRNAs no controle da expressão de autoantígenos no timo o que é importante para a tolerância imunológica central.
The thymus is a primary lymphoid organ, in which occurs in the induction of central immune tolerance to self peripheral tissue antigens (PTAs). The thymic medulla is formed by medullary thymic epithelial cells (mTECs) expressing hundreds of such PTAs representing virtually all organs and tissues of the body. This phenomenon has been termed promiscuous gene expression (PGE), which is partially regulated by the Autoimmune regulator (Aire) gene. The T cell precursors derived from the bone marrow migrate to the thymus (now termed thymocytes). A part of these thymocytes are eliminated by negative selection mediated mTEC cells. The surviving cells to evolve and functional mature T cells that migrate to the periphery and are capable of recognizing MHC molecules and are tolerant to PTAs. In addition to controlling the transcription of PTA genes, Aire also controls the expression of microRNAs (miRNAs). The negative selection in the thymus is a process essential to the maintenance of immunologic self-tolerance and imbalance of this process is associated with the development of autoimmune diseases such as type 1 diabetes mellitus (DM1) . Given these assumptions, our work was based on two hypothesis: 1) Changes in the expression of the Aire gene can disrupt the expression of PTA genes and miRNAs in the thymus, causing changes in PGE, 2) The balanced expression of Aire / or PTA genes in mTECs is fundamental for central tolerance. The imbalance in the expression of these genes is associated with the emergence of type 1 diabetes in mice. To test our first hypothesis we made Aire silencing (Aire knockdown) through electrotransfection of anti - Aire interfering RNA (siRNA) in vivo in the thymus of BALB/c mice. Analysis of the transcriptome (mRNAs) and miRNome (miRNAs) of mTECs revealed that partial and transient silencing of Aire was enough to affect the expression of Aire - dependent PTAs as well as miRNAs. miRNA -mRNA interaction networks revealed that the posttranscriptional control of PGE is also affected by the silencing of Aire. The results show that Aire and can form an miRNA pathway essential for the induction of central tolerance. To test our second hypothesis we compared the transcriptome of mTECs of BALB/c mice (non-autoimmune strain) with mTECs from non - obese diabetic NOD (animal model used in studies of autoimmune DM1) . Our results indicate that the transcriptional expression of DM1-related autoantigens are unbalanced in NOD mice in an very early stage, when these animals have not had clinical disease (pre-diabetic period). Unexpectedly, the transcriptional levels of Aire in the thymus was equivalent in these two strains, but the AIRE protein levels were reduced in thymus of NOD strain. These results suggest that some mechanism of post-transcriptional attenuation of Aire is acting in this lineage probably involving action of miRNAs . This could explain the imbalance of Aire - dependent PTAs and repression autoantigens related to DM1. Our results open perspectives for research in this area, contributing to better understanding the molecular mechanisms triggered by Aire and miRNAs in control of the expression of autoantigens in the thymus, which is important for the central immune tolerance.

INTRODUÇÃO: Fatores associados à etiopatogenia das complicações diabéticas, incluindo hiperglicemia e estresse oxidativo, podem causar alterações epigenéticas que modificam a expressão de genes em células-alvo, sem alterar sua sequência de DNA. São considerados mecanismos epigenéticos: (1) as modificações pós-traducionais das histonas; (2) a metilação do DNA e (3) a ação dos micro-RNAs (miRNAs); todos já foram reconhecidos na patogênese da \"memória metabólica\", situação na qual a hiperglicemia continua a exercer efeitos deletérios prolongados mesmo depois de ser normalizada. A sirtuína-1 é uma enzima que causa modificações pós-traducionais das histonas por sua atividade de histona desacetilase, silenciando a transcrição gênica. O silenciamento gênico também pode ocorrer pela ação da DNA metiltransferase 1 (DNMT1), enzima que adiciona um grupamento metil (CH3) na posição 5 de resíduos de citosina localizadas em ilhas CpG presentes nas regiões promotoras dos genes. Os miRNAs constituem uma classe de pequenos RNAs não codificadores com cerca de 19 a 25 nucleotídeos que controlam a expressão gênica por meio da repressão da tradução ou da degradação do RNA mensageiro-alvo. As hipóteses do presente estudo são (1) que exista um perfil sérico de miRNAs associado à presença ou ausência de complicações crônicas e (2) que existam variantes em genes relacionados à desacetilação das histonas e à metilação de citosinas que poderiam predispor ao aparecimento das complicações diabéticas, o que se constituiria na \"genética da epigenética\". OBJETIVOS: (1) caracterizar e comparar o perfil de miRNAs sérico de pacientes com diabetes mellitus tipo 1 (DM1) sem nenhuma complicação microvascular versus aqueles com três complicações microvasculares: retinopatia diabética (RD), doença renal diabética (DRD) e neuropatia diabética, para identificar vias epigeneticamente moduladas nesses dois grupos de pacientes e (2) avaliar a frequência de polimorfismos de um único nucleotídeo nos genes que codificam as enzimas DNMT1 e sirtuína-1 e suas associações com cada uma das complicações microvasculares em pacientes com DM1. MÉTODOS: O perfil sérico de 381 miRNAs foi avaliado com o uso do estojo comercial Taqman® Human MicroRNA Array A em 10 pacientes bem caracterizados clínica e laboratorialmente divididos em dois grupos: Pacientes com DM1 sem complicações [sem DRD (Clearance de creatinina > 90 ml/min/1,73 m2 e excreção urinária de albumina < 20 mg/g de creatinina), sem polineuropatia sensitivo-motora distal (ausência de sintomas sugestivos de neuropatia, sensibilidade térmica e dolorosa e reflexo aquileu normais), sem neuropatia autonômica cardiovascular (NAC) e sem RD] e Pacientes com DM1 com complicações [DRD (Clearance de creatinina < 60 ml/min/1,73 m2 e excreção urinária de albumina > 200 mg/g de creatinina), com polineuropatia sensitiva-motora distal, com NAC instalada e RD moderada ou grave]. Os cinco miRNAs mais diferencialmente expressos foram validados em uma casuística bem caracterizada de 20 pacientes com DM1 sem nenhuma complicação e 27 com todas as complicações microvasculares, com o emprego do estojo comercial TaqMan(TM) Advanced miRNA cDNA Synthesis. A avaliação da frequência de polimorfismos de um único nucleotídeo nos genes que codificam as enzimas DNMT1 (rs8112895, rs7254567, rs11085721, rs17291414, rs10854076) e sirtuína-1 (rs10997870; rs12766485) foi realizada após a genotipagem por reação em cadeia da polimerase em tempo real, em uma casuística composta por 466 pacientes com DM1. RESULTADOS: Do total de 377 miRNAs-alvo avaliados no soro dos pacientes com DM1, um total de 21 miRNAs estava superexpresso no grupo com complicações. Dos 5 miRNAs para os quais foi realizada a validação na casuística de 47 pacientes com DM1, dois foram confirmados como superexpressos na população com complicações (hsa-miR-518d-3p e hsa-miR-618). O polimorfismo rs11085721 no gene que codifica a DNMT1 associou-se à presença de NAC no sexo feminino, sendo o alelo raro C considerado de risco e conferindo um odds ratio (intervalo de confiança de 95%) de 2,44 (1,26-5,28). Nenhum polimorfismo da sirtuína-1 associou-se às complicações microvasculares avaliadas. CONCLUSÃO: o perfil de miRNAs séricos difere entre pacientes com DM1 com e sem complicações. O achado de uma variante em um gene que codifica a enzima de uma via epigenética conferir suscetibilidade a uma complicação crônica sugere que também exista a \"genética da epigenética\" modulando o desenvolvimento das complicações
INTRODUCTION: Factors associated with the etiopathogenesis of diabetic complications, including hyperglycemia and oxidative stress, may cause epigenetic changes that modify the expression of genes in target cells without altering their DNA sequence. The following mechanisms are considered epigenetics: (1) post-translational modifications of histones; (2) methylation of DNA and (3) action of micro-RNAs (miRNAs); all have already been recognized in the pathogenesis of \"metabolic memory\", a situation in which hyperglycemia exerts prolonged deleterious effects even after its normalization. Sirtuin-1 is an enzyme that causes post-translational modifications of histones by their histone deacetylase activity, silencing gene transcription. Gene silencing may also occur through the action of DNA methyltransferase 1 (DNMT1), an enzyme that adds a methyl group (CH3) at position 5 of cytosine residues located in CpG islands from gene-promoter regions. miRNAs are a class of small non-coding RNAs with about 19 to 25 nucleotides that control gene expression by promoting translation repression or degradation of target messenger RNAs. The hypotheses of the present study are (1) there is a serum profile of miRNAs associated with the presence or absence of chronic complications and (2) there are variants in genes related to histone deacetylation and cytosine methylation that could predispose to diabetes complications, which would constitute the \"genetics of epigenetics\". OBJECTIVES: (1) to characterize and compare the serum miRNA profile of patients with type 1 diabetes mellitus (T1D) without any microvascular complications versus those with three microvascular complications: diabetic retinopathy (DR), diabetic kidney disease (DKD) and diabetic neuropathy to identify signaling pathways epigenetically modulated in these two groups of patients and (2) to assess the frequency of single nucleotide polymorphisms in the genes encoding DNMT1 and sirtuin-1 and their associations with each of the microvascular complications in T1D patients. METHODS: The serum profile of 381 miRNAs was evaluated using the Taqman® Human MicroRNA Array A kit in 10 clinical and laboratory well-characterized patients divided into two groups: Patients without microvascular complications: without DKD (creatinine clearance> 90 ml/min/1.73 m2 and urinary albumin excretion < 20 mg / g creatinine), without distal sensory-motor polyneuropathy (absence of symptoms suggestive of neuropathy and normal thermal and pain sensitivity and Achilles reflex), without cardiovascular autonomic neuropathy (CAN) and without DR; and T1D patients with complications: with DKD (creatinine clearance < 60 ml / min / 1.73 m2 and urinary albumin excretion> 200 mg / g creatinine), with distal sensory-motor polyneuropathy, with CAN and with DR moderate or severe. The five most differentially expressed miRNAs were validated in a well-characterized case series of 20 patients with no complications and 27 patients with all microvascular complications using the TaqMan (TM) Advanced miRNA cDNA Synthesis kit. The evaluation of the frequency of single nucleotide polymorphisms in genes encoding the DNMT1 (rs8112895, rs7254567, rs11085721, rs1729414, rs10854076) and sirtuin-1 (rs10997870; rs12766485) was performed by genotyping using real-time polymerase chain reaction in a sample of 466 T1D patients. RESULTS: Of the total of 377 target miRNAs evaluated in the serum of T1D patients, 21 miRNAs were overexpressed in the group with complications. Of the 5 miRNAs for which validation was performed in 47 patients, two were confirmed as overexpressed in the group with complications (hsa-miR-518d-3p and hsa-miR-618). The polymorphism rs11085721 in the gene encoding DNMT1 was associated with the presence of CAN in female patients, with the minor allele C being considered of risk and conferring an odds ratio (95% confidence interval) of 2.44 (1.26 - 5.28). Polymorphisms in the gene encoding Sirtuin-1 did not associate with microvascular complications. CONCLUSION: the serum miRNA profile differs between patients with and without microvascular complications. A variant in a gene encoding a enzyme of an epigenetic pathway conferring susceptibility to a chronic complication suggests that there is also the \"genetics of epigenetics\" modulating the development of complications

O Diabetes Mellitus tipo 1 (DM1) resulta de um ataque autoimune contra as células pancreáticas, extinguindo a produção de insulina e levando à hiperglicemia. Evidências indicam uma associação entre o estresse oxidativo (que pode causar danos no DNA) e o DM1, sendo que apenas alguns trabalhos da literatura relataram a expressão de genes relacionados à respostas ao estresse oxidativo e reparo do DNA em DM1. Ainda, os microRNAs (reguladores pós-transcricionais da expressão gênica) estão envolvidos em vários processos biológicos e condições patológicas, mas informação sobre a expressão dos microRNAs em DM1 ainda é escassa. A fim de proporcionar um melhor entendimento sobre as vias de regulação de genes participantes de processos biológicos relevantes para o DM1, o presente estudo consistiu em analisar os perfis de expressão gênica (método de microarranjos) de microRNAs e de mRNAs (bem como de algumas proteínas) provenientes de células mononucleares do sangue periférico (PBMCs, do inglês peripheral blood mononuclear cells) de pacientes DM1 (n=19) em comparação com indivíduos sadios não diabéticos (n=11), dando maior enfoque a genes associados à resposta ao estresse oxidativo e reparo do DNA. Os resultados de expressão obtidos pelo método de microarranjos apontaram 44 microRNAs diferencialmente expressos (35 induzidos e nove reprimidos) nos pacientes DM1 e esses microRNAs apresentaram grande especificidade ao estratificar pacientes DM1 dos controles, incluindo hsa-miR-101, hsa-miR148a, hsa-miR-27b e hsa-miR-424, cujos dados de expressão foram confirmados por qRT-PCR. A análise funcional dos genes-alvo dos microRNAs, tanto dos induzidos quanto dos reprimidos, apontou 22 e 12 vias KEGG significativamente enriquecidas, respectivamente, incluindo vias relacionadas ao câncer. Com relação à análise de expressão de mRNAS, 277 genes diferencialmente expressos foram identificados nos pacientes DM1, sendo que 52% deles são potenciais alvos dos microRNAs diferencialmente expressos nos pacientes DM1. Dentre esses alvos foram encontrados genes candidatos ao desenvolvimento da doença, assim como genes implicados nos processos biológicos resposta ao estresse oxidativo e reparo do DNA, como UCP3, PTGS2, ATF3, FOSB, DUSP1 e TNFAIP3, cujos dados de expressão foram confirmados por qRT-PCR. Já a análise de grupos gênicos identificou 49 e 55 grupos gênicos significativamente expressos e enriquecidos em pacientes DM1, respectivamente, destacando-se vias relacionadas à sinalização apoptótica, resposta ao hidroperóxido, reparo do DNA por recombinação homóloga e resposta ao estresse do retículo endoplasmático. Quanto aos dados de expressão proteica (western blotting), PTGS2 e ATF3 não apresentaram níveis de expressão detectáveis em nenhum dos dois grupos estudados, enquanto que para DUSP1 não foi observada diferença estatisticamente significativa entre os grupos, apesar de os três genes se apresentarem induzidos em pacientes DM1. Os resultados do ensaio do gene repórter da luciferase demonstraram a ocorrência da interação entre hsa-miR-148a e DUSP1 em meio celular. Essa evidência aliada aos dados de western blotting, sugerem a possibilidade de hsa-miR-148a atuar na repressão traducional de DUSP1. Em conjunto, os resultados do presente estudo indicaram perfis distintos de expressão de microRNAs e mRNAs em PBMCs de pacientes DM1 comparados a indivíduos sadios, sendo que adicionalmente, dados inéditos relacionados à interação microRNAs-mRNAs em DM1 foram obtidos, principalmente associados à resposta ao estresse oxidativo e reparo do DNA, sugerindo um distúrbio na rede microRNA-alvo em pacientes DM1.
Type 1 Diabetes Mellitus (T1DM) results from an autoimmune attack against the pancreatic cells, ceasing insulin production, which causes hyperglycemia. Although associations between oxidative stress, which can cause DNA damage, and T1DM have been demonstrated, only a few studies have reported differential expression of genes associated with response to oxidative stress and DNA repair in T1DM patients. Moreover, microRNAs (post-transcriptional regulators of gene expression) are implicated in many biological processes and pathological conditions; however, only scarce information is available in the literature concerning the expression of microRNAs in T1DM. In order to better understand the regulatory pathways involved in biological processes that are relevant to T1DM, we aimed to investigate the microRNA and mRNA transcriptional expression profiles by microarray analysis (as well as expression of selected proteins) in peripheral blood mononuclear cells (PBMCs) from T1DM patients (n=19) compared with healthy non-diabetic individuals (n=11), emphasizing genes related to response to oxidative stress and DNA repair. Microarray expression results indicated 44 differentially expressed microRNAs (35 up- and nine down-regulated) in T1DM patients, with those microRNAs possessing a discriminatory power to clearly stratify the patients from the controls, including hsa-miR-101, hsa-miR148a, hsa-miR-27b, and hsa-miR-424, whose expression data were confirmed by qRT-PCR. Functional annotation analysis performed on the predicted targets of the differentially expressed microRNAs pointed 22 and 12 annotated KEGG pathways for the overexpressed and repressed microRNAs, respectively, many of them related to cancer. Regarding mRNA microarray results, we detected 277 differentially expressed genes in T1DM patients, with 52% of them being potential targets of the differentially expressed microRNAs in T1DM patients. Among these targets, we identified candidate genes for T1DM as well as genes involved in the biological processes response to oxidative stress and DNA repair, such as UCP3, PTGS2, ATF3, FOSB, DUSP1 and TNFAIP3, whose expression data were confirmed by qRT-PCR. Furthermore, out of the 49 and 55 significantly expressed/enriched gene sets in T1DM patients, respectively, five pathways related to apoptotic signaling, response to hydroperoxide, DNA repair via homologous recombination, and response to endoplasmic reticulum stress were of interest for the present work. Concerning protein expression results (western blotting), PTGS2 and ATF3 expression was not detected for either the patient or the control group, while significant difference in DUSP1 expression was not observed between the two groups, although the corresponding mRNAs of those genes were found induced. Regarding the luciferase assay, our results demonstrated that the interaction between hsa-miR-148a and DUSP1 occurs in the cellular milieu. Therefore, these findings together with those western blotting results suggest that hsa-miR-148a could play a role in DUSP1 translational repression. Altogether, our results indicate distinctive microRNA and mRNA expression profiles in PBMCs from T1DM patients relative to healthy non-diabetic individuals. Furthermore, we have provided novel data regarding microRNA-mRNA interactions in T1DM, in particular involving genes associated with response to oxidative stress and DNA repair, suggesting a perturbation in the microRNA-target network in T1DM patients.

Indiana University-Purdue University Indianapolis (IUPUI)
The role of beta cell miR-21 in Type 1 Diabetes (T1D) pathophysiology has been controversial. Here, we sought to define the context of beta cell miR-21 upregulation in T1D and the phenotype of beta cell miR-21 overexpression through target identification. Furthermore, we sought to identify whether circulating extracellular vesicle (EV) beta cell-derived miR-21 may reflect inflammatory stress within the islet during T1D development.. Results suggest that beta cell miR-21 is increased in in-vivo models of T1D and cytokine-treated cells/islets. miR-21 overexpression decreased cell count and viability, and increased cleaved caspase-3 levels, suggesting increased cell death. In silico prediction tools identified the anti-apoptotic mRNA B Cell Lymphoma 2 (BCL2) as a conserved miR-21 target. Consistent with this, miR-21 overexpression decreased BCL2 transcript and protein expression, while miR-21 inhibition increased BCL2 protein levels and reduced cleaved caspase-3 levels following cytokine-treatment. miR-21-mediated cell death was abrogated in 828/33 cells, which constitutively overexpress BCL-2. Luciferase assays suggested a direct interaction between miR-21 and the BCL2 3’untranslated region. With miR-21 overexpression, PRP revealed a shift of BCL-2 message toward monosome-associated fractions, indicating inhibition of BCL2 translation. Finally, overexpression in dispersed human islets confirmed a reduction in BCL2 transcripts and increased cleaved caspase 3 production. Analysis of EVs from human beta cells and islets exposed to cytokines revealed a 3-5-fold increase in miR-21. Nanoparticle tracking analysis showed no changes in EV quantity in response to cytokines, implicating specific changes within EV cargo as responsible for the miR-21 increase. Circulating EVs from diabetic non-obese diabetic (NOD) mice displayed progressive increases in miR-21 that preceded diabetes onset. To validate relevance to human T1D, we assayed serum samples collected from 19 pediatric T1D subjects at the time of diagnosis and 16 healthy controls. Consistent with our NOD data, EV miR-21 was increased 5-fold in T1D samples. In conclusion, in contrast to the pro-survival role reported in other systems, our results demonstrate that miR-21 increases beta cell death via BCL2 transcript degradation and inhibition of BCL2 translation. Furthermore, we propose that EV miR-21 may be a promising marker of developing T1D.

Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de Farmácia
O presente documento foi elaborado no âmbito da unidade curricular “Estágio Curricular” integrada no Mestrado Integrado em Ciências Farmacêuticas. O mesmo inclui os relatórios relativos ao período de estágio na Direção de Produtos de Saúde (DPS) do INFARMED I.P. e ao período de estágio na Farmácia Oudinot, em Aveiro. Cada um dos relatórios de estágio tem por base uma análise SWOT (Strengths, Weaknesses, Opportunities and Threats), tendo sido elaborada uma lista de pontos positivos e negativos, de âmbito quer interno, quer externo ao estágio (Oportunidades e Ameaças). O documento inclui, ainda, a monografia intitulada “Papel dos microRNAs no diagnóstico e tratamento da Diabetes Mellitus tipo 2 e suas complicações”. A Diabetes Mellitus tipo 2 (DMT2) é caraterizada por hiperglicemia causada essencialmente por resistência periférica à insulina. Atualmente, não existe nenhum biomarcador aceitável para a deteção precoce da Diabetes Mellitus tipo 2 e simultaneamente continua a ser necessário identificar estratégias terapêuticas mais vantajosas que as existentes. Os microRNAs controlam a expressão génica. A sua desregulação pode estar na origem de alterações em vários mecanismos fisiológicos. Estes têm demonstrado potencial na terapêutica de várias doenças,entre as quais a Diabetes Mellitus tipo 2. Alguns microRNAs apresentam níveis plasmáticos que variam com a progressão da Diabetes Mellitus tipo 2 e/ou suas complicações, podendo, no futuro, ser usados como biomarcadores desta doença e suas complicações.
This document includes reports of two internships accomplished under the scope of the course “Curricular Internships” which is part of the integrated master's degree in Pharmaceutical Sciences. These internships took place in Infarmed I.P.'s Direção DeProdutos de Saúde (DPS) and in Farmácia Oudinot, located, the latter, in the city of Aveiro.Each one of the reports is based upon a SWOT (Strengths, Weaknesses, Opportunities andThreats) analysis, under which it has been made a list of positive and negative aspects, both in and out of the internship's reach.This document also includes a monography named “Papel dos microRNAs nodiagnóstico e tratamento da Diabetes Mellitus tipo 2 e suas complicações” (Role microRNAs play in the diagnosis and treatment of Type 2 Diabetes Mellitus and its complications). Type 2 Diabetes Mellitus (T2DM) is characterized by hyperglycemia caused as a result of peripheral resistance to insulin. Currently, there is not any acceptable biomarker for a before hand detection of T2DM, while it is still necessary to identify improving therapeutical strategies. MicroRNAs control gene expression. Their deregulation may cause changes in a multitude of physiological mechanisms. These have been showing to be potentially useful for the therapeutics of various diseases, including the T2DM. Some microRNAs display plasmatic levels which vary as T2DM and/or its complications progress in time, which opens the possibility for them to be used as biomarkers for this disease in the future.

Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin-secreting pancreatic β-cells in genetically susceptible individuals, whose incidence is increasing worldwide. So far, our ability to curb this pathogenic immune response is limited by the poor understanding of the exact nature and kinetics of the immunological mechanisms leading to T1D. Indeed, triggers of islet autoimmunity and the precise mechanisms responsible for the progressive β-cell failure are not completely understood. Several scientific evidence supports a crucial role for the environment, which is also suggested by the rapid increase of incidence of this autoimmune disease. The search for a single cause of T1D has been vain, more probably there are a number of different factors that may contribute either to the initiating of the disease process or precipitating the manifest disease in subjects on their way to get diabetes. High living standard, good parent education, few siblings, pronounced hygiene, are all factors with possible important effects on immune system and autoimmune risk. In addition, the recent escalation of obesity in affluent countries has been suggested to contribute to the increased incidence of T1D, as a precipitating factor. Understanding the link between metabolism and immune tolerance could lead to the identification of new markers for the monitoring of disease onset and progression. We studied several immune cell subsets and factors with high metabolic impact as markers associated with disease progression in type 1 diabetes patients at onset, at 12 and at 24 months after diagnosis, in high-risk subjects and in non-diabetic controls. A multiple correlation matrix among different parameters was evaluated statistically and assessed visually on two-dimensional graphs. The meta-immunological profile changed significantly among the three studied groups and in patients over time, and a specific signature that associated with worsening disease was identified. Finally, markers to predict residual β-cell function up to one year after diagnosis were identified in a multivariate logistic regression model. This latter model, measuring age, body mass index (BMI), fasting C-peptide (C-pep), number of circulating CD3+CD16+CD56+ cells and percentage of CD1c+CD19-CD14-CD303- type 1 myeloid dendritic cells (mDC1s) at disease onset had a significant predictive value. The identification of a specific meta-immunological profile associated to disease status may contribute to understand the basis of diabetes and its progression, and provide novel biomarkers that could help clinicians in disease monitoring and in the choose of the most appropriate therapeutic approach according to disease status and aggressiveness.

INTRODUÇÃO: Na transição da infância para a adultez, ocorrem mudanças significativas que se manifestam a nível bio-psico-socio-espiritual. O adolescente, protagonista da sua própria história, no caminho da construção da sua identidade, vai aprendendo a gerir as múltiplas mudanças de que está a ser alvo, desenvolvendo estratégias de coping, umas mais positivas, outras menos. Quando as mudanças inerentes à adolescência confluem com um diagnóstico de diabetes mellitus tipo 1 (DM 1), as exigências aumentam. Consideram-se resilientes os adolescentes que ultrapassam o processo adverso, saindo dele fortalecidos. Com este estudo, pretendemos contribuir para a excelência dos cuidados de enfermagem, sugerindo instrumentos sensíveis e fidedignos que permitam o desenho de intervenções personalizadas, porque direcionadas para os problemas detetados, tendo em vista um melhor controlo da patologia e uma vida com mais qualidade. OBJETIVO: Perceber se os adolescentes com um nível mais elevado de resiliência evidenciam melhor resultado adaptativo e melhor controlo da DM 1. MATERIAL E MÉTODOS: Realizámos um estudo de paradigma quantitativo, do tipo correlacional e transversal, desenvolvido em três fases: (i) adaptação e validação da Escala Toulousiana de Coping (ETC), numa amostra de 291 adolescentes portugueses a frequentar o 3º Ciclo dos Ensinos Básico e Secundário; (ii) estudo junto duma amostra de 112 adolescentes com DM 1 em Consultas de Endocrinologia Pediátrica de cinco hospitais, para analisar relações entre as variáveis em estudo, a identificação dos fatores preditores da resiliência e a seleção da amostra, para a 3ª fase, em função dos scores de resiliência; e (iii) colheitas de saliva e estudo das relações entre Cortisol, Dehidroepiandrosterona-sulfato, resiliência e controlo metabólico, numa amostra de 50 adolescentes. Foram respeitados os princípios éticos para a investigação, em grupos vulneráveis. RESULTADOS: A ETC apresenta características psicométricas adequadas para adolescentes (alfa de Cronbach de .85). A resiliência dos adolescentes, medida pela Healthy Kids Resilience Assessment Module (HKRAM), é influenciada pelo sexo, idade, retenção escolar, situação laboral do pai/padrasto, escolaridade da mãe/madrasta, estratégias de controlo e de suporte social. Globalmente, os Acontecimentos Vitais Stressantes (AVS) interferem negativamente na resiliência. O controlo metabólico apresentou médias mais baixas, nos adolescentes provenientes de famílias tradicionais, nos que não sofreram retenção escolar, nos que identificaram menor numero de AVS e/ou menor impacto emocional. Foram ainda identificados como preditores positivos da resiliência, as estratégias de Controlo e de Suporte Social, e, como preditores negativos, o Retraimento, Conversão e Aditividade; a Recusa, e os AVS Impacto. Identificaram-se três modelos de perfil de stresse, dois com curvas atípicas de Cortisol. O modelo 2, é o que mais se aproxima dos valores de cortisol de referência, revelando os valores médios, que estes adolescentes apresentam recursos de resiliência intermédios e melhor controlo metabólico. CONCLUSÕES: A ETC, a AVS e a HKRAM são instrumentos fiáveis e úteis para os enfermeiros, com vista à implementação de cuidados preferencialmente proativos e personalizados. Dos resultados desta investigação, decorre a necessidade do desenvolvimento de estudos longitudinais, no âmbito da resiliência, a fim de perceber a possível transitoriedade das mudanças; de estudos em amostras representativas, com grupo controlo, preferencialmente multinível e desenvolvidos por equipas transprofissionais.
Introduction: In the transition from childhood to adulthood, there are significant changes that manifest themselves at the bio-psycho-socio-spiritual level. The teenager, protagonist of his own history, in the process of building his identity, learns how to manage the multiple changes he has been suffering, developing coping strategies, some more positive, some less. When the changes of adolescence come together with a diagnosis of type 1 diabetes mellitus (DM 1), the requirements increase. Adolescents, who overcome the adverse process, are considered resilient coming out from it stronger. With this study we intend to contribute to the excellence of nursing care, suggesting sensitive and reliable instruments that allow the design of personalized interventions, because they are directed towards the problems detected, in order to better control of the pathology and a life with more quality. Objective: To understand if adolescents with a higher level of resilience show better adaptive outcome and better control of DM 1. Material and methods: We carried out a quantitative, correlational and transverse paradigm study, developed in three phases: (i) adaptation and validation of the Toulousiana Coping Scale (ETC) in a sample of 291 Portuguese adolescents attending the 3rd Cycle of Basic and Secondary Education; (ii) study of a sample of 112 adolescents with DM 1 in Pediatric Endocrinology Consultations from 5 hospitals to analyze the relationships between the variables in study, the identification of the predictive factors of resilience and the selection of the sample for the 3rd phase depending of the scores of the resilience; and (iii) saliva harvests and study of the relationships between cortisol, dehydroepiandrosterone sulphate, resilience and metabolic control in a sample of 50 adolescents. Ethical principles for research in vulnerable groups were respected. Results: ETC presents adequate psychometric characteristics for adolescents (Cronbach's alpha of .85). Adolescent resilience, measured by the Healthy Kids Resilience Assessment Module (HKRAM), is influenced by gender, age, school retention, work status of the father/stepfather, schooling of the mother/step mother, strategies of control and social support. Globally, stressful life events (AVS) negatively affect resilience. Metabolic control presented averages means in adolescents from traditional families, in those who did not have school retention, in those that identified a lower number of AVS and/or less emotional impact. We also identified positive predictors of resilience, Control and Social Support strategies, and, as negative predictors, Retraction, Conversion and Addiction, Refusal, and Impact AVS. Three stress profile models were identified, two with atypical cortisol curves. Model 2, is the closest to the reference cortisol values, revealing the average values that these adolescents present, intermediate resilience resources and better metabolic control. Conclusions: ETC, AVS and HKRAM are reliable and useful tools for nurses to implement preferentially proactive and personalized care. From the results of this investigation, the development of longitudinal studies in the context of resilience is necessary in order to perceive the possible transitoriness of the changes; of studies in representative samples, with a control group, preferably multilevel and developed by cross-professional teams.