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Beta-cel dood en overleving in modellen voor type-1 diabetes: proteomische analyse
No full textT1D is an autoimmune disease characterized by a selective destruction of the insulin-producing ß-cells in the pancreas. We used 2D-DIG E as proteomic platform to elucidate the effects at the protein level of ß-cell death inducing agents. One of the biases of every proteomic research is the inability to study the complete proteome at the same time, due to its inherent bewilderingl y complex nature. By creating a reference map (chapter 4) of the INS-1E cells, we sought to gain insight in the proportion of the proteome readi ly accessible to us by 2D-GE. We identified 592 spots on the gel, and cl assified the proteins according to function and subcellular localization . This reference map will serve as a starting point and guide for future proteomic analyses in diabetes-related research. Future quantitative ap proaches under different experimental conditions will hopefully provide new insights in the mechanisms involved in altered ß-cell func tion and viability associated with diabetes development. ß-cell destruction is partially mediated through cytokines secreted by immune cells causing activation of ER stress and pro-apoptotic pathw ays. We sought for the mechanisms involved in ß-cell destructi on and the changes evoked at the protein level. We analyzed the effect o f the cytokines IL-1ß and IFN-γ on ß-cell apoptosis and proteome. We firstly (chapter 5) studied the effect on the insulin-p roducing INS-1E cells, a rat model cell line for ß-cells. The individual cytokines did not generate profound effects on apoptosis indu ction in this cell line. We mainly identified proteins with a protective or anti-apoptotic role in INS-1E cells treated with IFN-γ alone. W hen we considered the effects of IL-1ß alone, the proteome changes in the INS-1E cells were indicative of imbalanced cell functionality. Th e combination of IL-1ß and IFN-γ elicited profound cellular ch anges, translated in a high rate of apoptosis induction and numerous pro teins affected. Different cellular mechanisms were affected by the cytok ine treatment. Metabolism, as observed through differential expressed en zymes involved in the Krebs cycle and glycolysis, was impaired, as well as cytoskeleton formation. Chaperones, necessary for proper protein fold ing, were inactivated or downregulated. The proteins responsible for sta bilization of insulin mRNA and proper processing of the insulin protein displayed a decreased expression, as was the case for proteins with prot ective roles in ROS quenching. Bioinformatical network analysis demonstr ated a close interconnectivity of these diverse effects. In a follow-up study (chapter 6), we used mouse islets deficient in two important members of the IFN-γ signaling pathway. Disruption of the signaling pathway at the level of STAT-1 completely prevented cytokine- induced apoptosis, while disruption more downstream, at the level of IRF -1, was responsible for a partial protection. Wild-type mouse islets sho wed a similar response at the protein level as compared to INS-1E cells. Mouse islets deficient in STAT-1 or IRF-1 displayed an alteration in th eir proteome in concordance with their reduced sensitivity for cytokine- induced apoptosis. Proteins with a protective effect which were downregu lated in wild-type mouse islets did not display such a regulation in the disrupted mouse islets. Moreover, the PTM and inactivation of GRP78, an important chaperone in ß-cells, as observed in INS-1E cells a nd wild-type mouse islets, did not occur in the disrupted mouse islets. We identified a protein, namely GRP75, whose function in the ß -cell and expression profile in the different mouse strains could explai n the differences observed in sensitivity to cytokine-induced cell death in STAT-1-/- and IRF-1-/- mouse islets. Taken together, we identified t he proteins which were STAT-1 or IRF-1 dependent and had a role in the p rotection of the islets against cytokine-induced apoptosis. With these s tudies, we were able to validate and complement previous research on cyt okine-induced apoptosis in ß-cells, one of the mechanisms invo lved in T1D. We identified the proteomic profile of a ß-cell g oing into apoptosis by cytokines, and proposed some proteins with possib le key roles in the road to ß-cell apoptosis or survival. We c hose not only to further focus on the important JAK/STAT pathway for&nbs p;ß-cell death upon cytokine challenge, but also a more mechanistic level was investigated. IL-1ß and IFN-γ cause ß-cel l apoptosis partially through ER stress. We used the reversible ER stres sor CPA in order to elucidate the changes at the protein level this ER s tressor generates. Low levels of ER stress were insufficient to trigger apoptosis, but we detected at the proteome level the picture of an imbal anced cell, with the inherent profile of a cell destined to go into apop tosis. At later time points however, this proteomic profile reversed and apoptosis was averted. Key proteins for this reversal were identified ( 14-3-3 proteins and the modification status of GRP78). At elevated level s of ER stress, still not toxic to other cell types, INS-1E cells demons trated a high rate of apoptosis. We identified the changes imposed on th e proteome using a high dose of CPA. Proteins responsible for cell metab olism, cytoskeleton, protein synthesis and RNA synthesis are all downreg ulated with no recovery at a later time point.status: Publishe
Moleculaire mechanismen van beta cel dood en beta cel dysfunctie in modellen voor type 2 diabetes
No full textType 2 diabetes (T2D) is a dual disease that develops when the pancreatic beta cellscan no longer compensate for peripheral insulin resistance in the skeletalmuscle, liver and fat tissue by increasing their insulin secretion. Underphysiological conditions, there is an interplay between glucose and free fattyacids (FFA) that are oriented towards oxidation or storage depending onnutrient availability and cellular needs. Many in vitro and in vivo studiesdemonstrated that chronic elevation of glucose and FFA, a hallmark of T2D,leads to dysfunction of beta cells (a process referred to asglucolipotoxicity). To date, the exact molecular mechanisms and effectors thatexert these harmful effects are still not understood. When unfolded proteinsoverwhelm the folding capacity of the endoplasmic reticulum (ER), the ER stressresponse is activated. ER stress has been implicated in the development of T2D,via an effect on obesity and insulin resistance and via an impact on pancreaticbeta cell health. As the C/EBP homologous protein (CHOP) is induced by ERstress, it can play a central role in insulin resistance, as well as in betacell dysfunction as observed in T2D.We applied a proteomic 2D-DIGE approach on glucose and FFA-exposed INS-1Ecells, a reliable pancreatic beta cell model. The analysis revealed thatessential molecular pathways, involved in either production and secretion ofinsulin, or in the protection of beta cells against harmful stimuli, werehampered by high glucose. Exposure of INS-1E cells to the saturated FFApalmitate induced ER stress and subsequent cell death. As only a limited numberof proteins could be detected, a subcellular fractionation protocol wasoptimized in order to study ER-associated proteins. This technique led us todetect low abundance proteins and pathways that have never been associated withpalmitate-induced beta cell dysfunction and death before. In addition, wedemonstrated that the mono-unsaturated FFA oleate also interferes with insulinbiosynthesis and secretion, as well as with protective pathways. Importantly,the combination of high levels of FFA and glucose together aggravated beta celldysfunction and had a detrimental outcome on survival. In conclusion, thepresent findings provide new insights in glucolipotoxicity and beta celldysfunction and provide new targets for drug discovery.In the second part of this thesis, we studied the role of CHOP in thepathogenesis of insulin resistance and T2D. It has been reported that deletionof this protein delayed the onset of T2D in a spontaneous mouse model. Whereasno protection could be observed in terms of beta cell death, systemic deletionof CHOP in mice induced abdominal obesity and hepatic steatosis.Despite the obesity, CHOP-/- mice preserved normal fasting plasma insulin andglucose levels and normal peripheral and hepatic insulin sensitivity. Thisdiscrepancy was accompanied by lower expression of pro-inflammatory cytokinesand less infiltration of immune cells in fat and liver tissue of CHOP-/- mice.These observations suggest that insulin resistance is not induced by fataccumulation per se, but rather by the inflammatory status induced by theectopic fat. CHOP may play a key role in the crosstalk between excessive fatdeposition, inflammation and insulin resistance. CHOP represents a potentialkey target for the prevention and treatment of T2D.status: Publishe
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
Effects of Cytokine Exposure on the Proteome of Human Islets as Evaluated by Shotgun and 2DE Analysis
No full textPro-inflammatory cytokines (CYT) lead to several changes in human islet (HI) protein expression, but few data are available on CYT-induced alterations at the whole proteome level, including post-translational modifications (PTM). We used label-free shotgun analysis (SG) and bi-dimensional gel electrophoresis (2DE) to explore how CYT modify the proteome of isolated HI. For this purpose, HI were isolated from the pancreas of 5 nondiabetic organ donors (age: 74±6 years; gender: 3M/2F; BMI: 27.9±3.6 kg/m2) and then cultured for 48h in the presence of 50 U/ml interleukin (IL)-1β + 1000 U/ml interferon (IFN)-γ. Then, 40 or 250 μg protein extracts were used respectively for SG or 2DE (to assess lysine-acetylation) analysis, with the SG mass spectrometry data acquired by nano-liquid chromatography tandem mass spectrometry. SG identified around 3000 proteins, of which 286 were differentially expressed after CYT exposure. Of these, 176 were upregulated and 110 downregulated. Among the former, we found mediators of inflammation, proteins of the ER and the immunoproteasome system and oxidative stress modulators (e.g., CXCL2, CXCL10, PSMB8, PSMB9, PSMB10, SOD2). Among the downregulated proteins, there were cathepsins, antioxidant molecules and enzymes of glycolysis and Krebs’ cycle (e.g., CTSH (a candidate gene for T1D), PRDX2, MDH). Ingenuity Pathway Analysis indicated that several upstream regulators were predicted to be differentially affected. Of them, 118 were presumably activated, including STAT1 and STAT2, NF-κB, JAK1, IRF1, HMGB1 and 36 inhibited, such as ACKR2, TRIM24, MAPK1, SUMO3. Bi-dimensional gel electrophoresis showed 151 spots that were lysine-acetylated, of which 2 were upregulated (PRDX3 and SOD1) and 4 downregulated (GDH, CTSD, ACAD9 and EIF4A1) by CYT. The present study shows novel proteomic changes, including specific PTM, induced in human islets by cytokine treatment that could play a role in beta cell dysfunction and demise and be the target for specific treatments
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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