No description available.

No description available.

The endoplasmic reticulum (ER) is the organelle responsible for synthesis and folding of secreted and membranous protein and lipid biosynthesis. It also functions as one of the main cellular calcium stores. Pancreatic beta-cells evolved to produce and secrete insulin upon demand in order to regulate blood glucose homeostasis. In response to increases in serum glucose, insulin synthesis represents nearly 50% of the total protein biosynthesis by beta-cells. This poses an enormous burden on the ER, rendering beta-cells vulnerable to agents that perturb ER function. Alterations of ER homeostasis lead to accumulation of misfolded proteins and activation of an adaptive response named the unfolded protein response (UPR). The UPR is transduced via 3 ER transmembrane proteins, namely PERK, IRE-1 and ATF6. The signaling cascades activated downstream of these proteins: a) induce expression of ER resident chaperones and protein foldases. Increasing the protein folding capacity of the ER; b) attenuate general protein translations which avoids overloading the stressed ER with new proteins; c) upregulate ER-associated degradation (ERAD) genes, which decreases the unfolded protein load of the ER. In severe cases, failure by the UPR to solve the ER stress leads to apoptosis. The mechanisms linking ER stress to apoptosis are still poorly understood, but potential mediators include the transcription factors Chop and ATF3, pro-apoptotic members of the Bcl-2 familly, the caspase 12 and the kinase JNK. Accumulating evidence suggest that ER stress contributes to beta-cell apoptosis in both type 1 and type 2 diabetes. Type 1 diabetes is characterized by a severe insulin deficiency resulting from chronic and progressive destruction of pancreatic beta-cells by the immune system. During this autoimmune assault, beta-cells are exposed to cytokines secreted by the immune cells infiltrating the pancreatic islets. Our group has previously shown that the pro-inflamatory cytokines interleukin-1beta (IL1-beta and interferon-gamma (IFN-gamma), via nitric oxide (NO) formation, downregulate expression and function of the ER Ca2+ pump SERCA2. This depletes beta-cell ER Ca2+ stores, leading to ER stress and apoptosis. Of note, IL1-beta alone triggers ER stress but does not induce beta-cell death, while IFN-gamma neither causes ER stress nor induces beta-cell death. Together, these cytokines cause beta-cell apoptosis but the mechanisms behind this synergistic effect were unknown. Type 2 diabetes is characterized by both peripheral resistance to insulin, usually as a result of obesity, and deficient insulin secretion secondary to beta cell failure. Obese patients have high levels of circulating free fatty acids (FFA) and several studies have shown that the FFA palmitate induces ER stress and beta-cell apoptosis. In the present work we initially established an experimental model to specifically activate the ER stress response in pancreatic beta-cells. For this purpose, insulinoma cells (INS-1E) or primary rat beta-cells were exposed to the reversible chemical SERCA pump blocker cyclopiazonic acid (CPA). Dose-response and time course experiments determined the best conditions to induce a marked ER stress without excessive cell death (<25%). The first goal of the work was to understand the synergistic effects of IL1-beta and IFN-gamma leading to pancreatic beta-cell apoptosis. Our group previously observed, by microarray analysis of primary beta-cells, that IFN-gamma down-regulates mRNAs encoding for some ER chaperones. Against this background, our hypothesis was that IFN-gamma aggravates beta-cell ER stress by decreasing the ability of these cells to mount an adequate UPR. To test this hypothesis, we investigated whether IFN-gamma pre-treatment augments CPA-induced ER stress and beta cell death. The results obtained indicated that IFN-gamma pre-treatment potentiates CPA-induced apoptosis in INS-1E and primary beta-cells. This effect was specific for IFN-gamma since neither IL1-beta nor a low dose CPA pre-treatment potentiated CPA-induced apoptosis in INS-1E cells. These effects of IFN-gamma were mediated via the down regulation of genes involved in beta cell defense against ER stress, including the ER chaperones BiP, Orp150 and Grp94 as well as Sec61, a component of the ERAD pathway. This had functional consequences as evidenced by a decreased basal and CPA-induced activity of a reporter construct for the unfolded protein response element (UPRE) and augmented expression of the pro-apoptotic transcription factor Chop. We next investigated the molecular regulation of the Chop gene in INS-1E cells in response to several pro-apoptotic and ER stress inducing agents, namely cytokines (IL1-beta and IFN-gamma), palmitate, or CPA. Detailed mutagenesis studies of the Chop promoter showed differential regulation of Chop transcription by these compounds. While cytokines (via NO production)- and palmitate-induced Chop expression was mediated via a C/EBP-ATF composite and AP-1 binding sites, CPA induction required the C/EBP-ATF site and the ER stress response element (ERSE). Cytokines, palmitate and CPA induced ATF4 protein expression and further binding to the C/EBP-ATF composite site, as shown by Western blot and EMSA experiments. There was also formation of distinct AP-1 dimers and binding to the AP-1 site after exposure to cytokines or palmitate. The third objective of this work was to obtain a broad picture of the pancreatic beta-cell molecular responses during and after (recovery period) a severe ER stress. For this purpose, we utilized an “in home” spotted microarray, the APOCHIP, containing nearly 600 probes selected for the study of beta-cell apoptosis. Time-dependent gene expression profiles were measured in INS-1E cells exposed to CPA. CPA-induced ER-stress modified expression of 183 genes in at least one of the time points studied. Most of theses genes returned to control levels 3h after CPA removal from the culture medium. We observed full beta-cell recovery and survival, indicating that these cells trigger efficient defenses against ER stress. Beta-cell recovery is associated with a sustained increase in the expression of ER chaperones and a rapid decrease of pro-apoptotic mRNAs following CPA removal. Two groups of genes were particularly affected by CPA, namely those related to the cellular responses to ER stress, which were mostly up-regulated, and those related to differentiated beta-cell functions, which were down-regulated. Among this last group, we observed a 40-90% decrease of the mRNAs for insulin-1 and -2. These findings were confirmed in INS-1E cells exposed to cytokines or thapsigargin (another SERCA blocker), and in primary beta-cells exposed to the same treatments. This decrease in insulin mRNA expression is due to transcript degradation, most probably caused by IRE-1 activation and triggering of its endoribonuclease activity, as recently described in Drosophila cells. In conclusion, our work enabled a better understanding of the pancreatic beta-cell responses to ER stress: 1.)We identified a sensitizing effect of IFN-gamma to ER stress in beta-cells via downregulation of key ER chaperones. 2.)We observed a differential regulation of Chop transcription by different treatments suggesting distinct responses of pancreatic beta-cells to diverse ER stress inducers. 3.)We provided the first global analysis of gene expression modifications in pancreatic beta-cells following ER stress. 4.)We demonstrated a high capacity of beta-cells to cope and recover from a severe ER stress. 5.)We identified a new protective mechanism against ER stress, namely the degradation of insulin mRNA which limits the load posed on the ER by insulin synthesis. This, coupled to a marked increase in ER chaperones and a fast degradation of pro-apoptotic mRNAs, enables beta cells to recover from ER stress after the causes of this stress are removed.

Parents of children with a chronic disease are usually highly involved in their child’s treatment and may be affected by the heavy demands and constant stress. This can increase the risk of developing burnout, which is an individual reaction to long-term stress consisting of symptoms associated with emotional exhaustion, as well as physical and cognitive fatigue. The overall aim was to estimate the prevalence of burnout in parents of children with Type 1 Diabetes Mellitus (T1DM) and inflammatory bowel disease (IBD) (paper I), identify the risk factors associated with parenting a child with T1DM (paper II), explore how mothers suffering from burnout describe their mothering of a child with diabetes, with special focus on their need for control and Performance-based self-esteem (PBSE) (paper IV), and to evaluate the effect of a group intervention aimed at reducing stress-related symptoms (paper III). A total of 251 parents of children with T1DM, 38 parents of children with IBD and 124 parents of healthy children participated in a population-based study (I, II). The validated Shirom-Melamed Burnout Questionnaire (SMBQ) was used to assess burnout. 16 parents (SMBQ ≥3.75) participated in a group intervention and were evaluated for changes in SMBQ and PBSE (III). A total of 21 mothers of children with T1DM who scored for clinical burnout (SMBQ) participated in a qualitative study. Semi-structured interviews were conducted and Inductive content analysis was used (IV). In the study group 36.0% parents of children with a chronic disease scored for clinical burnout (SMBQ ≥3.75) compared to 20.2% of the reference parents (p=0.001) with a preponderance of mothers compared to fathers, 42% vs. 20.5% (p=0.001), respectively (I). Less support from the social network, sleep disturbances and lack of personal leisure time and recovery seem to be important risk factors for clinical burnout in parents of children with T1DM, especially mothers (II). Mothers’ experiences of mothering a child with T1DM were interpreted as one theme; Mission impossible, illustrating the extremely difficult circumstances under which they bring up the child with diabetes to adulthood (IV). Parents’ subjective evaluation of the intervention group was mainly positive and SMBQ (p=0.01) and PBSE scale (p= 0.04) measurements were significantly reduced 6 months after completion of the intervention (III). It is important to pay attention to how parents and especially mothers experience their daily life in order to support those who are at risk of developing burnout.

Bakgrund: Typ 1 diabetes mellitus (T1DM) bland ungdomar ökar i Europa och också i Sverige. Sjukdomen är kronisk och kräver livsstilsförändringar samt egenvård. Ungdomsåren kännetecknas av frigörelse från föräldrar och att hitta sin sociala tillhörighet. Att drabbas av kronisk sjukdom under denna del av livet kan antas vara särskilt omvälvande. Syfte: Syftet var att beskriva ungdomars upplevelser av att leva med T1DM. Metod: Studien var baserad på en litteraturöversikt med tio vetenskapliga artiklar som systematiskt söktes fram från databaserna Cinahl Complete och PubMed. Analysen genomfördes enligt fem steg där sista steget bestod av kategorisering och tematisering av artiklarnas resultat. Resultat: Genom analysen identifierades tre teman; Ett liv som ungdom med T1DM i isolering och utanförskap, mötet med sjukvårdspersonal, upplevelser av diagnostiseringen. Slutsats: Litteraturöversikten visar att ungdomarna hade svårigheter att hantera den förändrade livssituationen efter diagnosen. De beskriver känslor av utanförskap och isolering. Ungdomarnas kontakter med vården är överlag negativ då de inte blir respekterade som individer. Resultatet diskuteras med utgångspunkt från Dorotea Orems egenvårdsteori. / Background: Type 1 diabetes mellitus (T1DM) among young people is increasing in Europe and also in Sweden. The disease is chronic and requires lifestyle changes and selfcare. Adolescence is characterized by liberation from parents and finding one’s social affiliation. Suffering from a chronic illness during this part of life can assumed to be particularly transformative. Aim: The aim was to describe young people’s experiences of living with type 1 diabetes mellitus. Method: The study was based on a literature review with ten scientific articles systematically retrieved from the databases Cinahl Complete and PubMed. The analysis was carried out according to five steps, the last step consisted of categorizing and thematizationthe results from the included articles. Results: The analysis resulted in three themes: A life as a youth in isolation and exclusiondue to T1DM, encounters with healthcare professionals and Experiences of getting thediagnosis. Conclusion: This literature review highlights that young people had difficulty coping with the changed life situation after the diagnosis. They described feelings of exclusion and isolation. Their contacts with healthcare are generally perceived as negative as they experienced not being respected as individuals. The results are discussed in relation to Dorotea Orem’s self-care theory.

Bakgrund Varje dag insjuknar cirka två barn i diabetes mellitus typ 1 (T1DM) i Sverige. När ett barn blir sjukt är det föräldrarna till barnet som ansvarar för omvårdnaden vardagen. Omvårdnadsansvaret läggs på föräldrarna och för en del kan detta leda till stress och i värsta fall till utbrändhet. Syfte Syftet var att beskriva hur föräldrar upplever och hanterar att deras barn insjuknar i diabetes mellitus typ 1. Metod En kvalitativ semistrukturerad intervjustudie genomfördes. Sju föräldrar till barn med diabetes mellitus typ 1 från Stockholm och Uppsala intervjuades. Samtlig insamlade data analyserades utifrån en kvalitativ innehållsanalys. Resultat De kategorier som framkom under resultatanalysen var; att hantera sitt barns sjukdom, det stora tunga ansvaret, sjukdomens påverkan i det dagliga livet och behov av stöd. Föräldrar upplevde deras barns insjuknande i T1DM som traumatiskt och något som påverkar hela familjens liv. Samtliga föräldrar som deltog i studien uttryckte ett behov av stöd i olika former. Att känna sig ensamma utan stöd från varken anhöriga eller hälso- och sjukvården är en vardag för dessa föräldrar. Stödet som hälso- och sjukvården erbjöd upplevdes bristfälligt och med flera förbättringsområden. Flertalet föräldrar uppgav att de lider av sömnbrist, vilket försvårar det dagliga livet på många sätt. Slutsats Föräldrar till barn med T1DM lär sig, över tid hantera omvårdnaden som sjukdomen medför, men det finns svårigheter kring hanteringen av ansvaret, sorgen och oron. Föräldrarna upplever brister i det långsiktiga stödet som hälso- och sjukvården erbjuder och önskar mer avlastning för att minska riskerna att drabbas av psykisk och fysisk ohälsa. Vi anser att hälso och sjukvården måste erbjuda ett utökat stöd till dessa föräldrar för att tillgodose deras behov innan de riskerar att drabbas av utbrändhet.

Selective molecular recognition between proteins is a fundamental event in biological processes that governs cellular growth, function, survival, and differentiation. The immune system, for example, is a complex network of cellular processes regulated by protein-protein interactions (PPIs) between cells, receptors, and secreted molecules. Generating and maintaining an appropriate immune response and regulation requires coordination across many cell types and components, while dysregulation of these interactions can lead to disease. A major obstacle in small molecule therapy development towards these PPIs is their restriction to small protein-protein interfaces and a well-defined hydrophobic pocket. Most PPIs have large contact surface areas and lack traditional binding pockets making them historically challenging for the development of potent small-molecule modulators. To address this limitation, we utilized two binding-based approaches, a unique peptidomimetic fragment library and high-throughput small-molecule microarrays to design and discover molecules that target three important immunoregulatory PPIs: the DQ8-insulin complex, the KEAP1-Nrf2 complex, and the IL-4/IL-4R receptor complex. Many autoimmune diseases involve the ternary PPI complex between immunogenic peptides presented to T cell receptors through the major histocompatibility complex (MHC). Inhibiting this interaction may provide a therapeutic approach for delaying or preventing disease. To target type 1 diabetes, we developed a unique library consisting of 125 fragment-sized molecules that mimic glutamic acid and tyrosine residues from the immunogenic insulin B:9-23 peptide responsible for CD4+ T cell activation. Screening of our library after generation of the MHC class II protein responsible for insulin B:9-23 presentation, DQ8, has resulted in identification of 15 lead fragment compounds to date. Application of our fragment library towards pharmaceutically validated target for inflammation and neurogenerative diseases, the Kelch like ECH-associated protein 1 (KEAP1) and nuclear factor erythroid 2 like 2 (Nrf2), resulted in a 30% hit rate. These are promising results for the further development of selective compounds to inhibit these interactions. For treating inflammatory diseases, such as asthma or cancer, we report the identification of a first-in-class small molecule inhibitor to the cytokine Interleukin-4 (IL-4). The PPI between IL-4 and its receptor complex (IL-4Rα) contains no conventional binding pockets and binding is driven through clusters of complementary residues. Through the combination of small-molecule microarrays and cell-based assays we identified the lead compound, Nico-52, with micromolar inhibitory potency and micromolar affinity. A library of 60 analogs of Nico-52 was synthesized and preliminary structure activity relationships suggest amenability of the p-fluorophenyl substituent and importance of the diol substituent to retain binding potency. These studies resulted in development of a more potent inhibitor to IL-4 with a p-aniline substituent, which could be developed into a targeting ligand to deliver additional therapeutic payloads to an IL-4 enriched microenvironment. In summary, we have developed a peptidomimetic small molecule fragment library as a toolkit for screening against challenging PPI targets with applications towards type 1 diabetes and developed a first-in-class small molecule inhibitor towards IL-4 with applications towards inflammatory diseases. / 2025-09-21T00:00:00Z

The endoplasmic reticulum (ER) is the organelle responsible for synthesis and folding of secreted and membranous protein and lipid biosynthesis. It also functions as one of the main cellular calcium stores. Pancreatic beta-cells evolved to produce and secrete insulin upon demand in order to regulate blood glucose homeostasis. In response to increases in serum glucose, insulin synthesis represents nearly 50% of the total protein biosynthesis by beta-cells. This poses an enormous burden on the ER, rendering beta-cells vulnerable to agents that perturb ER function. Alterations of ER homeostasis lead to accumulation of misfolded proteins and activation of an adaptive response named the unfolded protein response (UPR). The UPR is transduced via 3 ER transmembrane proteins, namely PERK, IRE-1 and ATF6. The signaling cascades activated downstream of these proteins: a) induce expression of ER resident chaperones and protein foldases. Increasing the protein folding capacity of the ER; b) attenuate general protein translations which avoids overloading the stressed ER with new proteins; c) upregulate ER-associated degradation (ERAD) genes, which decreases the unfolded protein load of the ER. In severe cases, failure by the UPR to solve the ER stress leads to apoptosis. The mechanisms linking ER stress to apoptosis are still poorly understood, but potential mediators include the transcription factors Chop and ATF3, pro-apoptotic members of the Bcl-2 familly, the caspase 12 and the kinase JNK. <p>Accumulating evidence suggest that ER stress contributes to beta-cell apoptosis in both type 1 and type 2 diabetes. Type 1 diabetes is characterized by a severe insulin deficiency resulting from chronic and progressive destruction of pancreatic beta-cells by the immune system. During this autoimmune assault, beta-cells are exposed to cytokines secreted by the immune cells infiltrating the pancreatic islets. Our group has previously shown that the pro-inflamatory cytokines interleukin-1beta (IL1-beta and interferon-gamma (IFN-gamma), via nitric oxide (NO) formation, downregulate expression and function of the ER Ca2+ pump SERCA2. This depletes beta-cell ER Ca2+ stores, leading to ER stress and apoptosis. Of note, IL1-beta alone triggers ER stress but does not induce beta-cell death, while IFN-gamma neither causes ER stress nor induces beta-cell death. Together, these cytokines cause beta-cell apoptosis but the mechanisms behind this synergistic effect were unknown.<p>Type 2 diabetes is characterized by both peripheral resistance to insulin, usually as a result of obesity, and deficient insulin secretion secondary to beta cell failure. Obese patients have high levels of circulating free fatty acids (FFA) and several studies have shown that the FFA palmitate induces ER stress and beta-cell apoptosis.<p>In the present work we initially established an experimental model to specifically activate the ER stress response in pancreatic beta-cells. For this purpose, insulinoma cells (INS-1E) or primary rat beta-cells were exposed to the reversible chemical SERCA pump blocker cyclopiazonic acid (CPA). Dose-response and time course experiments determined the best conditions to induce a marked ER stress without excessive cell death (<25%).<p>The first goal of the work was to understand the synergistic effects of IL1-beta and IFN-gamma leading to pancreatic beta-cell apoptosis. Our group previously observed, by microarray analysis of primary beta-cells, that IFN-gamma down-regulates mRNAs encoding for some ER chaperones. Against this background, our hypothesis was that IFN-gamma aggravates beta-cell ER stress by decreasing the ability of these cells to mount an adequate UPR. To test this hypothesis, we investigated whether IFN-gamma pre-treatment augments CPA-induced ER stress and beta cell death. The results obtained indicated that IFN-gamma pre-treatment potentiates CPA-induced apoptosis in INS-1E and primary beta-cells. This effect was specific for IFN-gamma since neither IL1-beta nor a low dose CPA pre-treatment potentiated CPA-induced apoptosis in INS-1E cells. These effects of IFN-gamma were mediated via the down regulation of genes involved in beta cell defense against ER stress, including the ER chaperones BiP, Orp150 and Grp94 as well as Sec61, a component of the ERAD pathway. This had functional consequences as evidenced by a decreased basal and CPA-induced activity of a reporter construct for the unfolded protein response element (UPRE) and augmented expression of the pro-apoptotic transcription factor Chop. <p>We next investigated the molecular regulation of the Chop gene in INS-1E cells in response to several pro-apoptotic and ER stress inducing agents, namely cytokines (IL1-beta and IFN-gamma), palmitate, or CPA. Detailed mutagenesis studies of the Chop promoter showed differential regulation of Chop transcription by these compounds. While cytokines (via NO production)- and palmitate-induced Chop expression was mediated via a C/EBP-ATF composite and AP-1 binding sites, CPA induction required the C/EBP-ATF site and the ER stress response element (ERSE). Cytokines, palmitate and CPA induced ATF4 protein expression and further binding to the C/EBP-ATF composite site, as shown by Western blot and EMSA experiments. There was also formation of distinct AP-1 dimers and binding to the AP-1 site after exposure to cytokines or palmitate. <p>\ / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

No description available.

The purpose of this study was to explore the feasibility of using human patient simulation (HPS) to teach Type 1 diabetes (T1DM) management to grandparents of grandchildren with T1DM. Thirty grandparents (11 male, 19 female) of young grandchildren (aged 12 and under) with T1DM were recruited from an urban medical center. Experimental group (n = 14) grandparents received hands-on visual T1DM management education using an HPS intervention, and control group (n = 16) grandparents received similar education using a non-HPS intervention. Post-intervention, researchers interviewed twelve grandparents (50% HPS, 50% non-HPS) who scored highest and lowest on the Hypoglycemia Fear Survey. Using a mixed-method design, researchers integrated study instrument data and post-intervention interview data to describe grandparent’s experience learning T1DM management. Post-intervention, grandparent scores for knowledge, confidence, and fear showed no significant difference by group assignment, however, all grandparent scores showed improvement from Time 1 to Time 2. Grandparents described how taking part in T1DM education heightened their awareness of T1DM risks. GP T1DM knowledge gains aided GPs to make sense of T1DM risks. Newfound T1DM knowledge enhanced GP T1DM management confidence. Improved T1DM knowledge and confidence helped to defuse T1DM management fear. Although study instruments did not measure significant difference between grandparents who received the HPS intervention and those who did not, the consistency of larger HPS-taught grandparent score improvement is suggestive of a benefit for HPS.