Bakgrund: Diabetes typ 1 är en autoimmun kronisk sjukdom som har ökat i antal drabbade de senaste 20 åren. Kontroll av plasmaglukosvärdet är centralt för att kunna utföra behandlingsåtgärder samt minska risken för komplikationer. En del av sjuksköterskans roll är att bidra med information, stödja och ge råd. Hantering av diabetes typ 1 bygger på egenvård där sjuksköterskan genom individanpassning kan skapa motivation till förändring hos personer med diabetes typ 1. Syfte: Syftet var att beskriva hur personer med diabetes typ 1 upplever sitt dagliga liv Metod: Litteraturöversikt med induktiv ansats där 10 artiklar analyserades enligt Fribergs femstegsmodell. Resultat: Resultatet utmynnade i två huvudkategorier. Dagliga resurser, som efterföljs av underkategorierna behov av stöd från sjukvården, behov av socialt stöd och behov av kunskap. En diagnos som förändrar livet följt av underkategorierna vardagen struktureras och mental påverkan av egenvården. Resurser som stöd och kunskap beskrevs i relation till egenvård och det dagliga livet. Det framkom även att faktorer som struktur, ansvar och rädsla förändrades vid diabetes typ 1. Slutsats: Rädsla för hypoglykemi och bristande individualiserad vård var utmärkande bland deltagarna. Stöd och information kan utveckla möjligheten för sjuksköterskan att stötta personer med diabetes typ 1 i deras dagliga liv relaterat till egenvård. / Background: Diabetes type 1 is an autoimmune chronic disease wich has increased in number over the past 20 years. Control of the plasma glucose value is central to being able to perform treatment measures and reduce the risk of complications. Part of the nurse's role is to contribute information, support and advise. Management of diabetes type 1 is based on self-care where the nurse can, through individual adaptation, create motivation for change in people with type 1 diabetes. Aim: The aim was to describe how people with Type 1 Diabetes experiencing their daily life. Method: A literature review with an inductive onset, 10 articles was analyzed through Friberg’s five-step model. Results: The result culminated in two main categories. Daily resources, which are followed by the subcategories of need for healthcare support, the need for social support and the need for knowledge. A diagnosis that changes life followed by the subcategories everyday life is structured and mental impact of self-care. Resources like support and knowledge was described in relation to self-care and daily life. Factors of structure, responsibility and fear emerged to be changing in relation to diabetes type 1. Conclusion: Fear of hypoglycemia and lack of individualized care was characteristic throughout the result. Support and information can develop the opportunity for nurses to support people with type 1 diabetes in their daily lives related to self-care.

O diabetes mellitus tipo 1 é uma doença metabólica, caracterizada pela desregulação glicêmica, que ocorre devido a um ataque autoimune. A insulinoterapia é o tratamento clássico para o DM1. Contudo, alguns pacientes que apresentam essa doença não respondem de forma eficiente a este tratamento e apresentam episódios frequentes de hipoglicemia severa e despercebida (pacientes hiperlábeis). Essas complicações comprometem de forma significativa a qualidade de vida dessas pessoas. O transplante de ilhotas é uma importante alternativa para o tratamento de pacientes hiperlábeis com DM1. No entanto, essa terapia apresenta restrições como a necessidade de mais de um doador por transplante e significativa morte das ilhotas devido ao estresse provocado pelo procedimento de isolamento, além da morte promovida pelo sistema imune do paciente nos primeiros momentos pós-transplante. A autofagia é um mecanismo de reciclagem de componentes citoplasmáticos que é fundamental para a homeostase celular. Em condições de estresse, este mecanismo é ativado acima do seu nível basal, promovendo a degradação de agregados proteicos e organelas defeituosas, evitando assim, danos celulares que comprometam a viabilidade da célula. Trabalhos realizados por nosso grupo têm mostrado a citoproteção que PRL promove em células-beta, reduzindo a apoptose induzida por citocinas pró-inflamatórias. Também demonstramos o papel essencial de HSPB1 na inibição de apoptose induzida por PRL após o tratamento com citocinas. Além disso, resultados recentes de nosso laboratório mostraram um aumento nos níveis de autofagia em células-beta após sua exposição a citocinas, bem como uma restauração a níveis normais na presença de PRL. Visando um melhor entendimento do papel da PRL na modulação da autofagia em células-beta, o objetivo desse projeto foi estudar se HSPB1 também é essencial no mecanismo de regulação da autofagia induzido por PRL.Para tal, fizemos experimentos em modelos de células-beta MIN6, MIN6 silenciadas para HSPB1 (MIN6-shHSPB1) e MIN6 com sequencia short hairpin aleatória (MIN6- SsC), medindo a morte celular através de ensaios de viabilidade, e ensaios de western blot para avaliar os níveis de marcadores de autofagia e fluxo autofágico (degradação de autofagossomos), tratando as células com citocinas, prolactina e indutores ou inibidores de autofagia. Os resultados mostraram que a modulação da autofagia ocasionada pela prolactina em células-beta se dá, em parte, através de HSPB1. O tratamento com prolactina foi capaz de inibir a morte celular induzida por citocinas, mesmo na presença de cloroquina, um bloqueador de autofagia, o que nos levou a concluir que a autofagia não é uma via envolvida na citoproteção de células beta induzida por PRL. Os resultados gerados nesse estudo contribuíram para uma melhor compreensão dos eventos moleculares induzidos por PRL em células-beta, e poderão permitir a inferência de novas abordagens que melhorem a citoproteção, cultura e transplante dessas células em pacientes com diabetes tipo 1. / Type 1 diabetes mellitus is a metabolic disease characterized by glycemic dysregulation, which occurs due to an autoimmune destruction of beta-cells. Insulin therapy is the gold standard treatment for DM1. However, some DM1 patients do not respond efficiently to this treatment and suffer frequent episodes of severe hypoglycemia unawareness. Since this complication jeopardizes the quality of life of these people, Islet transplantation is a therapeutic alternative indicated to treat these patients. However, besides the lack of enough organ donors, the loss of beta cells during both the isolation as well as the infusion of islets into the recipient induce a great estresse and thus a significant cell death is one of the drawbacks of this procedure. Autophagy is a mechanism of recycling cytoplasmic components and is essential for cellular homeostasis. Under estresse conditions, this mechanism is activated above basal levels, promoting the degradation of protein aggregates and defective organelles, thus avoiding cell damage that could compromise cell viability. Studies carried out by our group have shown not only that PRL promotes cytoprotection in beta-cells, reducing pro-inflammatory cytokines-induced apoptosis, but also that HSPB1 plays an essential role in this inhibition of apoptosis mediated by PRL after treatment with cytokines. Moreover, recent results from our laboratory showed an increase in autophagy levels in beta-cells after exposure to cytokines, as well as a restauration to normal levels in the presence of PRL. In order to better understand the role of PRL in the modulation of autophagy in these cells, the aim of this project is to study whether HSPB1 is also essential in the mechanism of autophagy regulation induced by PRL. Using MIN6 beta cell models where HSPB1 was silenced (MIN6-shHSPB1) or not (MIN6-SsC), we studied cell death by viability assays. Moreover, western blot assays were performed in order to assess levels of autophagy and autophagic flux markers in the cells.Our results showed that HSPB1 in one of the mediators of PRL-induced modulation of autophagy. Nevertheless, since hormonal treatment was still able to inhibit cytokinesinduced cell death even in the presence of chloroquin, an autophagy blocker, we conclude that autophagy is not a signaling pathway involved in PRl-induced beta-cell cytoprotection. Altogether, the results shown in this study may help to increase the knowledge of the molecular events induced by PRL in beta-cells, and may allow to infer new approaches to improve cytoprotection, culture and transplantation of these cells into type 1 diabetic patients.

Um problema para profissionais que atendem crianças com doenças crônicas é o de encontrar técnicas de atendimento que motivem o tratamento, evite as complicações da doença e mantenham a qualidade de vida dos pacientes. Psicólogos, inseridos no âmbito hospitalar, que participam de programas de atendimento para crianças com Diabetes Mellitus tipo 1 (DM1), se deparam com o sofrimento das mesmas, angústia dos pais e preocupação das equipes de saúde, principalmente quando há baixa adesão ao tratamento. Diante dos riscos de complicações graves que rodeiam essa patologia; das dificuldades no atendimento psicológico de crianças com doenças crônicas e da tendência ao uso de jogos na área da saúde; desenvolveu-se o Gamellito Adventures, um jogo digital para crianças e adolescentes com DM1. Este jogo (game) propõe aos jogadores a realização de cuidados de automonitorização, aplicação de insulina, alimentação e atividade física em um personagem com DM1. Ao considerar esse contexto, temse como objetivo apresentar Gamellito Adventures, como um dispositivo mediador na intervenção da psicologia em crianças com DM1 e realizar um estudo clínico para avaliar os alcances de sua utilização jogo, a partir de conceitos winnicottianos que enfocam o brincar e os objetos transicionais. Trata-se de uma pesquisa clínicoqualitativa que se dotou do método clínico e teve como coleta de dados entrevistas com os pais, consulta de prontuário médico e a técnica do Desenho-Estória com Tema e a utilização do próprio game como mediador e instrumento de coleta no atendimento. Participaram deste estudo cinco crianças com diagnóstico de DM1 na faixa etária de sete a onze anos, sendo quatro meninas e um menino, atendidos no Ambulatório de Especialidades do Hospital Universitário da Universidade Estadual de Londrina. Foram realizadas duas sessões com os responsáveis, entrevista inicial e devolutiva, e quatro sessões com cada criança, sendo que a primeira e última foram aplicados os Desenhos-Estórias com Tema. As demais sessões foram explorados os recursos do game, dos cuidados básicos, minigames e quizzes. Os resultados evidenciaram que a utilização do Gamellito Adventures o faz um potente recurso terapêutico em sessão de atendimento psicológico de crianças com DM1. Os aspectos clínicos comuns percebidos nos estudos de caso foram: as questões maternas que envolvem os pacientes com DM1, o sentimento de se perceber diferente em relação a outras crianças em relação ao seu estado crônico, a dificuldade de simbolização, as passagens ao ato e a negação da doença. Em relação aos aspectos do jogo, o estudo dos casos possibilitou entender que o Gamellito Adventures se fez um disparador para a fala da criança em relação à sua doença e na medida em que favorece a simbolização, é mediador para estabelecer a transferência, a aproximação do conhecimento e a responsabilização, elementos essenciais para melhorar a adesão ao tratamento. O Gamellito Adventures dá subsídios para que a criança se sinta identificada ao personagem e possa, então, falar sobre ela e sua relação com o DM1 e passar da posição de alguém sempre cuidada para um agente ativo: e cuidador / A problem for professionals who treat children with chronic diseases is to find care techniques that motivate treatment, avoid complications of the disease and maintain the quality of life of patients. Psychologists in the hospital who participate in care programs for children with Type 1 Diabetes Mellitus (T1DM) are faced with their suffering, parents\' anguish and concern of health teams, especially when there is low adherence to treatment. Faced with the risks of serious complications surrounding this pathology; the difficulties in the psychological care of children with chronic diseases and the tendency to use games in the health area; developed the Gamellito Adventures, a digital game for children and adolescents with T1DM. In considering this context, we aim to present Gamellito Adventures as a mediator device in the intervention of psychology in children with T1DM and conduct a clinical study to evaluate the scope of their game use, based on Winnicottian concepts that focus on playing and the transitional objects. It is a clinical-qualitative research that was endowed with the clinical method and had as data collection interviews with the parents, consultation of medical records and the technique of Drawing-Story with Theme and the use of Gamellito Adventures itself as mediator and instrument collection. Five children with DM1 diagnosed in the seven to eleven years age group, four girls and one boy, attended the Specialized Ambulatory of the University Hospital of the State University of Londrina (AEHU / UEL). Two sessions were carried out with those responsible, initial and devolution interview, and four sessions with each child, the first and last one being applied the Theme-Story Drawings. The other sessions explored the features of Gamellito Adventures, basic care, minigames and quizzes. The results showed that the use of Gamellito Adventures makes it a powerful therapeutic resource in the psychological treatment session of children with T1DM. The common clinical aspects perceived in the case studies were: the maternal issues involving patients with T1DM, the feeling of perceiving themselves differently in relation to other children in relation to their chronic state, the difficulty of symbolization, the passage to the act and the denial of the disease. Regarding the aspects of the game, the study of the cases made it possible to understand that the Gamellito Adventures was a trigger for the speech of the child in relation to its disease and insofar as it favors symbolization, it mediates to establish the transference, the approximation of the knowledge and accountability, essential elements to improve adherence to treatment. Gamellito Adventures gives the child the ability to feel identified and can then talk about her and her relationship with T1DM and move from the position of someone always \"cared for\" to an active agent: and caregiver

Introdução: Diabetes tipo 1A(DM1A) é uma doença causada pela destruição autoimune das células beta. Em adição aos linfócitos T helper 1(Th1) e Th2, um subtipo específico de células T helper recentemente descrito, Th17, caracterizado pela produção da interleucina 17(IL-17A), IL-17F e IL-22, está também envolvido na imunidade adaptativa e autoimunidade, incluindo DM1A. A IL-23 tem função fundamental na expansão e sobrevivência das células Th17. A mesma é composta por 2 subunidades: a p19-específica (IL-23A) e a p40. Variantes dos genes IL-23A e de seu receptor (IL-23R) ou o aumento das concentrações séricas da IL-23 estão associados a várias doenças autoimunes, mas seus efeitos no DM1A não estão definidos. Com o intuito de avaliar a importância da IL-23 na patogênese do DM1A, as variantes dos genes IL23A e IL23R foram analisadas. Metodologia: A região codificadora e os regiões intrônicas proximais do gene IL23A, incluindo a região 5 proximal foram sequenciadas. Duas variantes do gene IL23A (rs2066808 e rs11171806) e duas do gene IL-23R (rs11209026 e rs10889677) foram também genotipadas. A amostra contou com 370 pacientes com DM1A e 314 indivíduos controles saudáveis. As medidas das concentrações séricas da IL-23 e os autoanticorpos pancreáticos e extra-pancreáticos foram determinados. Resultados: Nós observamos somente uma das seis variantes da IL-23 descritas nos bancos de dados (rs11171806 G>A localizada no exon 3) e descrevemos uma nova variante no gene IL-23A, que consistiu na substituição da citosina por timina na posição c.-403 (C>T) na região 5 proximal deste gene (encontrada em heterozigose em apenas uma paciente com DM1A, do sexo feminino, com 28 anos ao diagnóstico).Os alelos G dessas duas variantes estiveram em forte desequilíbrio de ligação (D\' = -0,825 para controles, p<2,0X10-6 e D\' = -0,902, p<2,0X10-17 para pacientes). Em consequência, a análise dos haplótipos destas variantes foi realizada. O haplótipo GG foi mais frequente nos controles (16.7%) do que nos pacientes com DM1A (9.5%), conferindo proteção à doença (pc = 0,0009, OR = 0,53) . A presença do haplótipo GG diferiu de acordo com a etnia no conjunto de pacientes e controles, sendo menor naqueles de etnia caucasóide (18%) em relação aos outros grupos (39%); p<0.0001. Entretanto, o efeito protetor da haplótipo GG foi independente da etnia. As duas variantes do gene IL23R (rs10889677 e rs11209026) tinham frequência alélica e genotípica semelhante entre pacientes com DM1A e controles. Não foi observada diferença significante nas concentrações da IL-23 entre 135 pacientes com DM1A (5,65 ± 14,0 pg/mL) e 112 indivíduos controles (9,06 ± 23,7pg/mL) (p =0,18). , mesmo quando analisamos apenas o pacientes com duração do diabetes inferior a dois anos, nos quais a resposta imune contra as células beta ainda está presente, (4.65 ± 6.94 pg/mL e 9.07 ± 23.62 pg/mL, p = 0.076). Não foi encontrada associação entre as variantes do gene IL23A com a idade diagnóstica, presença do peptídeo C residual e auto-anticorpo anti-descarboxilase do ácido glutâmico em pacientes com diagnóstico recente de DM1A. Estas variantes também não influenciaram na freqüência dos auto anticorpos extrapancreáticos: anti-tireoglobulina, anti-peroxidase, anti-21 hidroxilase, fator anti-núcleo, fator reumatóide e anti-citoplasma de neutrófilos. Conclusões: O haplótipo GG das variantes do gene lL23A (rs11171806 e rs2066808) foi associado a proteção ao DM1A. As variantes do gene IL23R (rs11209026 e rs10889677) não foram associadas ao DM1A. As concentrações séricas da IL-23 foram semelhantes entre os grupos. / Introduction: Type 1 diabetes mellitus (T1D) is a disorder caused by the immune-mediated destruction of insulin-secreting pancreatic beta cells. In addition to T helper 1 (Th1) and Th2 cells, a recently discovered subset of T helper cells, Th17, characterized by the production of interleukin 17 A (IL-17A), IL-17F, and IL-22 is also involved in adaptive immunity and autoimmunity, including T1D. The Interleukin IL-23 has a central role in the expansion and survival of Th 17 cells. It is composed of two subunits: p19-specific (IL-23A) and p40. Single nucleotide polymorphisms (SNPs) of IL-23A and IL-23 receptor (IL-23R) genes or increased IL-23 serum concentrations were associated with several autoimmune diseases, but their role in T1D has not been defined. We therefore searched for variants of IL-23A and IL-23R genes that could predispose to T1D. Methods:The coding regions and boundary intron sequences of IL-23A gene, including the 5 proximal region were sequenced. Two variants (rs2066808 and rs 11171806) of IL-23A and two of IL-23R (rs11209026 and rs10889677) genes were also genotyped. IL-23 serum levels and pancreatic and extra-pancreatic auto-antibodies were also determined. The cohort involved 370 patients with T1D and 314 healthy control subjects.Results: We observed only 1 out of 6 IL-23A coding variants (rs11171806 G>A localized in exon 3) described in a database repository . A new allelic variant of the IL-23A gene, consisting of the substitution of a cytosine by a thymine at position c.-403 (C>T) in the 5 proximal region of the IL-23A gene (found in heterozygosis in only 1 female patient with T1D) was described. The G alleles of rs11171806 and rs2066808 variants of IL-23A gene were in strong linkage disequilibrium (D\' = -0,825 for controls, p<2,0X10-6 and D\' = -0,902, p<2,0X10-17 for patients). So, further analyses were performed with the haplotypes instead of separated SNPs. The GG haplotype was more frequent in controls (16,7%) than in T1D patients (9,5%), conferring a protection to the disease (pc= 0,0009, OR = 0.53). The presence of haplotype GG was also different according to the ethnic group in the overall sample (patients+controls), when we pooled the Caucasians (18%) against the other groups (39%); p<0.0001. However, the lower susceptibility to T1D conferred by GG haplotype was independent of the ethnic group. Two IL-23R gene variants (rs10889677 and rs11209026) were also analyzed. The allelic and genotypic frequency of the variants did not differ between patients with T1D and control subjects. No significant differences were observed between the plasma IL-23 concentrations of 135 T1D patients (5.65 ± 14.0) and 112 control subjects (9.06 ± 23.7) (p = 0.18), even when we only the patients with less than 2 years disease duration (n = 43), when the immune attack to beta cells is still present, were included (4.65 ± 6.94 pg/mL and 9.07 ± 23.62 pg/mL, p = 0.076). No association was found between IL-23A variants with age at diagnosis of diabetes, presence of residual C-peptide levels or frequency of glutamic acid anti-decarboxilase antibody in patients with recent-onset T1D. Furthermore, these variants were not related to the presence of the extrapancreatic autoantibodies such as thyroid peroxidase (TPO) Ab, thyroglobulin (TG) Ab, 21-Hydroxilase (21OH) Ab, Anti nuclear factor (ANA) Ab, rheumatoid factor (FR) Ab and Neutrophil cytoplasmic (ANCA) Ab. Conclusions : The GG haplotype of lL23A gene variants( rs11171806 and rs2066808) was protective against T1D. The IL23R variants (rs11209026 and rs10889677) were not associated with susceptibility toT1D . IL-23 serum concentrations did not differ between T1D patients and controls.

O objetivo geral deste trabalho foi avaliar a presença de sintomas depressivos e as possíveis influências do Diabetes Mellitus tipo I (DM1) na qualidade de vida de adolescentes com esta doença. Para alcançar tal objetivo foram avaliados adolescentes com DM1 (58) e sem DM1 (61) completando um total de 119 adolescentes avaliados; de ambos os sexos. Os locais de coleta de dados foram o Ambulatório de Endocrinologia da Criança e do Adolescente (ECA) do Hospital das Clínicas da Faculdade de Medicina da USP (HCRP-USP) para os adolescentes diabéticos e para os não diabéticos o colégio público regular Escola Municipal de Ensino Fundamental Prof. Raul Machado, no município de Ribeirão Preto-SP. Foram utilizados dois instrumentos para coleta de dados, o The World Health Organization Quality of Life-Bref (Whoqol-bref); instrumento de Qualidade de Vida; e o Inventário de Depressão de Beck; instrumento que avalia sintomas depressivos. Os dados coletados foram organizadas em banco de dados no programa estatístico Stata, versão 12.0, e analisadas a partir de técnicas de estatística descritiva e inferencial. Os resultados desta pesquisa mostraram que, nas avaliações de qualidade de vida, quando não há presença de sintomas depressivos em comparação com o grupo sem diabetes, o grupo de diabéticos apresenta pior qualidade de vida nos domínios físico, social, ambiente e satisfação, todavia, quando há presença destes sintomas, a qualidade de vida é pior nos dois grupos. Conclui-se que o DM1 e a presença de sintomas depressivos, ambos interferem na qualidade de vida dos indivíduos em que acomete, nem sempre de forma igual. Desta maneira, foi possível compreender a importância do olhar ao adolescente com e sem a doença, afim de observar as peculiaridades dos indivíduos nesta fase da vida / The aim of this study was to assess the presence of depressive symptoms and the possible influences of type I diabetes mellitus (DM1) the quality of life of adolescents with this disease. To achieve this goal were evaluated adolescents with type 1 diabetes (58) and without DM1 (61) for a total of 119 adolescents evaluated; of both sexes. Data collection sites were the Endocrinology Clinic of Child and Adolescent (ECA) of the Hospital of the Faculty of Medicine, USP (HCRP-USP) for diabetic patients and nondiabetic patients regular public school Municipal School Elementary Education Prof. Raul Machado, in Ribeirão Preto-SP. Two instruments for data collection were used, The World Health Organization Quality of Life-Bref (WHOQOL-BREF); Quality of Life instrument; and the Beck Depression Inventory; instrument that assesses depressive symptoms. The collected data were organized in a database using Stata, version 12.0, and analyzed from descriptive and inferential statistical techniques. Our results show that in life quality assessments where there is presence of depressive symptoms compared with the group without diabates, the group of diabetics has worse quality of life in the physical, social, environment and satisfaction, however, when there is presence of these symptoms, quality of life is worse in both groups. It is concluded that the presence of DM1 and depressive symptoms, both interfere with the quality of life of individuals in that affects not always equal. In this way it was possible to understand the importance of looking adolescents with and without the disease in order to observe the peculiarities of individuals at this stage of life.

La dystrophie myotonique de type 1 (DM1) est une maladie génétique caractérisée par une dégénérescence des muscles squelettiques accompagnée d’une myotonie. Cette maladie est due à une expansion instable de triplets CTG dans la région 3’ non traduite du gène DMPK. L’accumulation des ARNm DMPK mutés au sein de foci nucléaires conduit à la séquestration du facteur d’épissage MBNL1 et à des altérations de l’épissage alternatif de nombreux ARNm. En particulier, l’inclusion de l’exon 5 au sein du pré-ARNm de la troponine T cardiaque (hcTNT) est renforcée chez les patients DM1. Cette inclusion anormale participe aux anomalies cardiaques présentées par les patients. Les travaux de l’équipe, menés en collaboration avec l’équipe de Nicolas Sergeant à Lille, sur la régulation de l’épissage de l’ARNm hcTNT avaient établi l’existence de 8 sites MBNL1, dont 6 nouveaux, situés de part et d’autre de l’exon 5 et la présence de régions activatrices et inhibitrices de l’inclusion fixant des facteurs d’épissage dont l’identité n’était pas connue. L’un des objectifs de ma thèse était d’étudier l’importance fonctionnelle in cellulo de chacun des 8 sites MBNL1. J’ai ainsi pu montrer que chacun des 6 nouveaux sites participe à l’inhibition de l’inclusion de l’exon 5 par MBNL1. Les données obtenues nous ont amené à proposer un modèle dans lequel MBNL1 s’associe avec les triplets de sites MBNL1 situés de part et autre de l’exon 5 et entraine la formation d’une structure à longue distance via des interactions protéiques MBNL1-MBNL1. Cette structure isolerait l’exon 5 dans une boucle et limiterait la fixation du spliceosome. Par ailleurs, j’ai mis en œuvre une approche de purification de RNP formées en extrait nucléaire pour identifier d’autres facteurs régulant l’inclusion de l’exon 5. La protéine hnRNP H a ainsi pu être identifiée. Sa capacité à activer l’inclusion de l’exon 5 in cellulo et à entrer en compétition avec MBNL1 pour la régulation de l’inclusion de l’exon 5 via sa fixation sur des sites localisés dans l’exon 5 et en aval de cet exon a pu être confirmée. La seconde partie de ma thèse a porté sur l’étude de l’effet d’un stress oxydatif généré par 500 µM d’H2O2 sur le profil global d’épissage alternatif des pré-ARNm de cellules HeLa. Lors de ce travail, j’ai pu établir que la réponse des cellules HeLa au stress oxydatif implique deux phases de réponse : une phase précoce (1h-8h) caractérisée par un fort taux de mortalité associé à une forte augmentation du taux de d’entités oxygénées réactives (ROS) intracellulaire et une phase tardive (16h-24h) corrélée à une diminution du taux de ROS intracellulaires et une surexpression des ARN satellite III. Sur la base de ces données, une analyse globale du transcriptome par emploi de puces à exons (Affymetrix) a été réalisée à partir d’ARN totaux isolés 1h, 2h, 4h et 24h après le début du stress. Nous avons ainsi identifié des modulations d’expression et d’épissage spécifiques de chacune des deux phases. L’analyse des données par des outils bio-informatiques a permis de mettre en évidence des fonctions cellulaires bien définies qui sont plus particulièrement affectées lors d’un stress oxydant. Enfin, pour comprendre l’origine des variations d’épissage observées lors d’un stress oxydant, j’ai entrepris d’analyser les effets de ce stress sur le niveau d’expression et la localisation cellulaire des composants du spliceosome ou des facteurs qui s’associent pour réguler son activité / Myotonic distrophy of type 1 (DM1) is a genetic disease characterized by skeletal muscle degeneration associated to myotonia. DM1 results from the instable expansion of CTG repeats within the 3’ untranslated region of the DMPK gene. The accumulation of mutated DMPK mRNAs within nuclear foci leads to the sequestration of the MBNL1 splicing factor and causes splicing misregulation of numerous pre-mRNAs. Among altered events the increase of the inclusion of exon 5 in the human cardiac troponin T (hcTNT) mRNA is of particular importance, since it contributes to the cardiac symptoms presented by the patients. Through collaborative work with N. Sergeant’s team from Lille, the team has studied the molecular bases of hcTNT exon 5 inclusion regulation and mapped 8 MBNL1 binding sites, including 6 new ones, within intronic regions surrounding exon 5. They also identified positive and negative splicing regulatory elements of which protein partners remain unidentified. The first objective of my PhD thesis was to test the functional importance of each individual MBNL1 binding site. The obtained results established that the 6 newly identified MBNL1 binding sites are involved in splicing regulation by MBNL1 and lead us to propose a new regulation model in which MBNL1 binds on triplets of MBNL1 sites present on each side of exon 5 and form a long distance structure via MBNL1-MBNL1 protein interaction. The formation of this looping-structure is expected to isolate exon 5 and limit its recognition by the spliceosome. In addition I searched for protein partners of the identified regulatory elements by affinity chromatography. By this way, I identified hnRNP H as a positive regulator of exon 5 inclusion. Its capacity to compete with MBNL1 to regulate splicing in cellulo by binding on exonic and intronic binding sites was further confirmed. The second part of my PhD work corresponds to the study of the global impact of oxidative stress, generated by exposition of HeLa cells to 500 µM of H2O2, on alternative splicing. This allows us to establish that the response of HeLa cells to oxidative stress involve two distincts phases: an early one (1h-8h) characterized by poor survival rate and high intracellular ROS content and a late phase (16-24h), associated with a decrease of the intracellular ROS level and the overexpression of the long non coding sat III RNAs. Based on this observation, we performed a transcriptome global analysis by using exon arrays from Affymetrix on RNA samples isolated 1, 2, 4 or 24 hours after the induction of the oxidative stress. We identified changes of the gene expression level or mRNA splicing pattern specific of each of the response phases. Data computing by bio-informatic tools identified the most affected cellular processes and functions during the cell response to oxidative stress. In order to better understand the mechanisms underlying alternative splicing modulation during oxidative stress, I started to study the impact of oxidative stress on the expression level and the cellular localization of spliceosome components and most common splicing regulation factors

Introduction: Diabetes mellitus is an incurable, widespread disease increasing globally. 2017 there were 425 million people with the diagnosis, and circa 10% of these have type 1 diabetes mellitus. Diabetes mellitus is a disease which requires a lot of self-management. Living with a chronic disease may impact the patient’s mental and psychosocial health. Aim: The aim of this literature review was to describe how living with type 1 diabetes mellitus may affect the patient’s psychosocial health.  Method: The study was conducted as a literature review using Polit and Beck’s (2017) nine steps. Data was collected using two databases, PubMed and CINAHL. The articles chosen were critiqued according to Polit and Beck’s (2017, p. 102-109) “Guide to an Overall Critique of a Qualitative Research Report” and “Guide to an Overall Critique of a Quantitative Research Report”. Twelve articles in total were chosen for the result, six quantitative and six qualitative.  Results: Two main themes and seven subthemes were developed based on the results. Conclusion: Healthcare professionals did not take these patients’ psychosocial health into consideration. Many patients also felt that they could not self-manage their disease in public due to others’ perception of them. This could result in complications.

O objetivo geral deste trabalho foi avaliar a presença de sintomas depressivos e as possíveis influências do Diabetes Mellitus tipo I (DM1) na qualidade de vida de adolescentes com esta doença. Para alcançar tal objetivo foram avaliados adolescentes com DM1 (58) e sem DM1 (61) completando um total de 119 adolescentes avaliados; de ambos os sexos. Os locais de coleta de dados foram o Ambulatório de Endocrinologia da Criança e do Adolescente (ECA) do Hospital das Clínicas da Faculdade de Medicina da USP (HCRP-USP) para os adolescentes diabéticos e para os não diabéticos o colégio público regular Escola Municipal de Ensino Fundamental Prof. Raul Machado, no município de Ribeirão Preto-SP. Foram utilizados dois instrumentos para coleta de dados, o The World Health Organization Quality of Life-Bref (Whoqol-bref); instrumento de Qualidade de Vida; e o Inventário de Depressão de Beck; instrumento que avalia sintomas depressivos. Os dados coletados foram organizadas em banco de dados no programa estatístico Stata, versão 12.0, e analisadas a partir de técnicas de estatística descritiva e inferencial. Os resultados desta pesquisa mostraram que, nas avaliações de qualidade de vida, quando não há presença de sintomas depressivos em comparação com o grupo sem diabetes, o grupo de diabéticos apresenta pior qualidade de vida nos domínios físico, social, ambiente e satisfação, todavia, quando há presença destes sintomas, a qualidade de vida é pior nos dois grupos. Conclui-se que o DM1 e a presença de sintomas depressivos, ambos interferem na qualidade de vida dos indivíduos em que acomete, nem sempre de forma igual. Desta maneira, foi possível compreender a importância do olhar ao adolescente com e sem a doença, afim de observar as peculiaridades dos indivíduos nesta fase da vida / The aim of this study was to assess the presence of depressive symptoms and the possible influences of type I diabetes mellitus (DM1) the quality of life of adolescents with this disease. To achieve this goal were evaluated adolescents with type 1 diabetes (58) and without DM1 (61) for a total of 119 adolescents evaluated; of both sexes. Data collection sites were the Endocrinology Clinic of Child and Adolescent (ECA) of the Hospital of the Faculty of Medicine, USP (HCRP-USP) for diabetic patients and nondiabetic patients regular public school Municipal School Elementary Education Prof. Raul Machado, in Ribeirão Preto-SP. Two instruments for data collection were used, The World Health Organization Quality of Life-Bref (WHOQOL-BREF); Quality of Life instrument; and the Beck Depression Inventory; instrument that assesses depressive symptoms. The collected data were organized in a database using Stata, version 12.0, and analyzed from descriptive and inferential statistical techniques. Our results show that in life quality assessments where there is presence of depressive symptoms compared with the group without diabates, the group of diabetics has worse quality of life in the physical, social, environment and satisfaction, however, when there is presence of these symptoms, quality of life is worse in both groups. It is concluded that the presence of DM1 and depressive symptoms, both interfere with the quality of life of individuals in that affects not always equal. In this way it was possible to understand the importance of looking adolescents with and without the disease in order to observe the peculiarities of individuals at this stage of life.

Background: There are significant psychological challenges faced by people throughout their lives and many of these challenges can be readily understood from a contextual behavioural science perspective, and Acceptance and Commitment Therapy (ACT) shows promise as a theoretically and practically relevant intervention. Some problems faced in delivering such an intervention are volume and access to healthcare. A potential solution to this is to design theoretically driven interventions which can be delivered through technology. These need to be interactive, individually shaped and will combine mindfulness, acceptance and values. Such interventions need to be evaluated scientifically according to acceptability, quality, safety and effectiveness. Aims: This thesis has two sections. Firstly, a systematic review aiming to assess the acceptability and effectiveness of using technology to deliver ACT. Secondly, an empirical research study aiming to analyse the experiences of using an ACT based app for young people with type 1 diabetes (TD1). Methods: The review searched 11 databases, and a related website. Included studies were required to use a form of technology to deliver ACT, with no real-time therapist. Two independent researchers determined inclusion of articles into the review and rated the studies according to the quality criteria. Where there was uncertainty a third reviewer was used. For the empirical study, individual interviews of 9 young people aged 13-22 years with TD1 were asked about their experiences of using the ACT based app. Framework analysis was used to determine themes. Results: The review search yielded 18 studies which met inclusion criteria. Findings highlighted that generally these interventions were seen as acceptable and satisfactory. All of these interventions were conducted in an adult population, and qualitative data was not robustly accounted for. The empirical research found two main themes: ‘Desire for apps to represent my needs’ and ‘How diabetes impacts me and how this could potentially be addressed in an app’. Discussion: Both the review and empirical study found that participants were positive about the use of technology to deliver ACT. Developmental progress needs to be made in the app to truly represent the needs of young people with TD1. These interventions could enhance the availability of psychological therapies. This has been highlighted as a government objective in several countries. Methodological weaknesses limit conclusions, such as underpowered studies. As this is a fast growing body of research it is hoped that future studies could be more similar methodologically. It would still be interesting to determine whether asynchronous contact enhances the cost-effectiveness of this form treatment. This thesis has provided me with the opportunity to design an ACT protocol for young people with type 1 diabetes (TD1). It has helped me to fully understand the undertaking which goes into designing apps and the scope of how responsive apps can be. It has given me the chance to communicate with people from different professional backgrounds to create a shared language, an opportunity to lead and manage a project and much more. With the help of my supervisor in my first year of training, we established links with the informatics department to see if students would be able to help with the programming of such an app. We had to create a synopsis of the proposed study to entice students to undertake the project as part of their degree. An interested student was assigned the project and meetings were held to determine our expectations and to establish the scope of what could be created. A second student took on the project during my second year or training. During this time my supervisor and I created a protocol of the content for the app. This was based on previous ACT protocols and tools we were aware of, which we thought might be helpful. A lot of thought had to go into trying to keep the content concise, including different modes of delivery (MP3s, video, animation etc), making the content applicable to young people with TD1 based on previous literature, and thinking about how interactive the app could be. Friends were also relied on to create graphics for the app. I went to different health boards across Scotland to meet with Diabetes teams to inform them about the project and to gather advice on the appropriateness of the diabetes information within the content of the app, and to determine whether they were interested in taking part in the study. I tested the initial prototype and glitches were ironed out. The next stage was to test the app on professionals working in the field, and to gather their feedback through focus groups. Adaptations to the app were made based on this. The app was initially made for Android phone devices based on general market research indicating that there was little evidence that one platform was more popular in adolescents. The diabetes teams and I tried to recruit young people with TD1 from their usual diabetes clinics. Initial barriers to recruitment were that at least 50% of young people had iPhones so could not download the app, and others did not seem interested in downloading the app to take part in the study. Funding of 10 Android tablets was agreed by the University. I attended the usual diabetes clinics in NHS Lothian and young people with TD1 started to volunteer to take part in the study. Originally it was hoped a trial of the effectiveness of the app would be carried out, but the difficulties in recruitment meant that instead I decided to use a qualitative methodology to explore young people’s experiences of using the app.

O diabetes mellitus tipo 1 é uma doença metabólica, caracterizada pela desregulação glicêmica, que ocorre devido a um ataque autoimune. A insulinoterapia é o tratamento clássico para o DM1. Contudo, alguns pacientes que apresentam essa doença não respondem de forma eficiente a este tratamento e apresentam episódios frequentes de hipoglicemia severa e despercebida (pacientes hiperlábeis). Essas complicações comprometem de forma significativa a qualidade de vida dessas pessoas. O transplante de ilhotas é uma importante alternativa para o tratamento de pacientes hiperlábeis com DM1. No entanto, essa terapia apresenta restrições como a necessidade de mais de um doador por transplante e significativa morte das ilhotas devido ao estresse provocado pelo procedimento de isolamento, além da morte promovida pelo sistema imune do paciente nos primeiros momentos pós-transplante. A autofagia é um mecanismo de reciclagem de componentes citoplasmáticos que é fundamental para a homeostase celular. Em condições de estresse, este mecanismo é ativado acima do seu nível basal, promovendo a degradação de agregados proteicos e organelas defeituosas, evitando assim, danos celulares que comprometam a viabilidade da célula. Trabalhos realizados por nosso grupo têm mostrado a citoproteção que PRL promove em células-beta, reduzindo a apoptose induzida por citocinas pró-inflamatórias. Também demonstramos o papel essencial de HSPB1 na inibição de apoptose induzida por PRL após o tratamento com citocinas. Além disso, resultados recentes de nosso laboratório mostraram um aumento nos níveis de autofagia em células-beta após sua exposição a citocinas, bem como uma restauração a níveis normais na presença de PRL. Visando um melhor entendimento do papel da PRL na modulação da autofagia em células-beta, o objetivo desse projeto foi estudar se HSPB1 também é essencial no mecanismo de regulação da autofagia induzido por PRL.Para tal, fizemos experimentos em modelos de células-beta MIN6, MIN6 silenciadas para HSPB1 (MIN6-shHSPB1) e MIN6 com sequencia short hairpin aleatória (MIN6- SsC), medindo a morte celular através de ensaios de viabilidade, e ensaios de western blot para avaliar os níveis de marcadores de autofagia e fluxo autofágico (degradação de autofagossomos), tratando as células com citocinas, prolactina e indutores ou inibidores de autofagia. Os resultados mostraram que a modulação da autofagia ocasionada pela prolactina em células-beta se dá, em parte, através de HSPB1. O tratamento com prolactina foi capaz de inibir a morte celular induzida por citocinas, mesmo na presença de cloroquina, um bloqueador de autofagia, o que nos levou a concluir que a autofagia não é uma via envolvida na citoproteção de células beta induzida por PRL. Os resultados gerados nesse estudo contribuíram para uma melhor compreensão dos eventos moleculares induzidos por PRL em células-beta, e poderão permitir a inferência de novas abordagens que melhorem a citoproteção, cultura e transplante dessas células em pacientes com diabetes tipo 1. / Type 1 diabetes mellitus is a metabolic disease characterized by glycemic dysregulation, which occurs due to an autoimmune destruction of beta-cells. Insulin therapy is the gold standard treatment for DM1. However, some DM1 patients do not respond efficiently to this treatment and suffer frequent episodes of severe hypoglycemia unawareness. Since this complication jeopardizes the quality of life of these people, Islet transplantation is a therapeutic alternative indicated to treat these patients. However, besides the lack of enough organ donors, the loss of beta cells during both the isolation as well as the infusion of islets into the recipient induce a great estresse and thus a significant cell death is one of the drawbacks of this procedure. Autophagy is a mechanism of recycling cytoplasmic components and is essential for cellular homeostasis. Under estresse conditions, this mechanism is activated above basal levels, promoting the degradation of protein aggregates and defective organelles, thus avoiding cell damage that could compromise cell viability. Studies carried out by our group have shown not only that PRL promotes cytoprotection in beta-cells, reducing pro-inflammatory cytokines-induced apoptosis, but also that HSPB1 plays an essential role in this inhibition of apoptosis mediated by PRL after treatment with cytokines. Moreover, recent results from our laboratory showed an increase in autophagy levels in beta-cells after exposure to cytokines, as well as a restauration to normal levels in the presence of PRL. In order to better understand the role of PRL in the modulation of autophagy in these cells, the aim of this project is to study whether HSPB1 is also essential in the mechanism of autophagy regulation induced by PRL. Using MIN6 beta cell models where HSPB1 was silenced (MIN6-shHSPB1) or not (MIN6-SsC), we studied cell death by viability assays. Moreover, western blot assays were performed in order to assess levels of autophagy and autophagic flux markers in the cells.Our results showed that HSPB1 in one of the mediators of PRL-induced modulation of autophagy. Nevertheless, since hormonal treatment was still able to inhibit cytokinesinduced cell death even in the presence of chloroquin, an autophagy blocker, we conclude that autophagy is not a signaling pathway involved in PRl-induced beta-cell cytoprotection. Altogether, the results shown in this study may help to increase the knowledge of the molecular events induced by PRL in beta-cells, and may allow to infer new approaches to improve cytoprotection, culture and transplantation of these cells into type 1 diabetic patients.