The pancreas is a large glandular organ with mixed exocrine and endocrine functions, located in the abdominal cavity behind the stomach. The endocrine portion, 1-2 % of its total volume, is represented by islets of Langerhans and plays a significant role in the pathophysiology of diabetes. The islets mainly contain β cells, which produce and release the hormonal protein insulin into the bloodstream in order to reduce glucose concentrations in the blood . Dysfunction of this regulatory mechanism can lead to the development of type 2 diabetes mellitus (T2DM), a chronic metabolic disorder characterized by increased glucose levels in the blood and caused by either the resistance to insulin or the inability of β cells to produce (enough) insulin, or a combination of both [2,3]. The number of people affected by T2DM is growing world-wide, driven by the spread of obesity. The absence of characteristic symptoms complicates early diagnosis of the disease and can lead to premature death if left untreated . For all these reasons, in order to get a better understanding of the complex pathophysiology leading to the onset of T2DM and its progression, elucidation of the molecular mechanisms underlying the disorder is paramount. This PhD thesis aims to characterize, at the molecular level, the pancreas, focusing particularly on the islet of Langerhans and on their involvement in type two diabetes. cells failure, in type two diabetes, is caused by several factors: environmental factors, such as high-fat diets and sedentary lifestyle, and genetic predisposition . Individuals with high fasting levels of plasma free fatty acids (FFAs) have an elevated risk of developing T2DM . In fact, prolonged exposure to FFAs impairs insulin secretion in vivo and in vitro [7,8] inducing β cells death . Palmitate is the most common saturated FFA in human plasma and it has been used in vitro studies on isolated islets or β cells lines to investigate the mechanisms of lipotoxicity. Prolonged exposure to palmitate may promote the inhibition of insulin transcription , the induction of ER stress in β cells [11,12], the production of reactive oxygen species (ROS) , and ceramides  and finally to cells death. Some evidence suggest that palmitate could induce these effects through defects in mitochondrial function [13,15]. Nowadays, the relationship of lipotoxicity mechanisms to mitochondrial function is not well understood and remain under investigation. As far as mitochondria concerns, they play a central role in coupling glucose metabolism to insulin secretion. Mitochondrial dysfunction impairs glucose stimulated insulin secretion and may promote β cells death. Moreover, mitochondria are the major source of ROS but also the target of their damaging effects. An overproduction of free radicals in β cells by the mitochondrial respiratory chain produces peroxidation of mitochondrial membrane , impairment of ATP production  and damage of mitochondrial DNA  which regulates oxidative phosphorylation process involved in the insulin secretion from pancreatic β cells. The molecular mechanisms by which palmitate affects β cells function and survival, have been studied using different approaches such as RNA-based studies  and proteomic analysis . Very recently, Cnop et al. , mapped the transcriptome of human islets of Langherans, by using RNA-sequencinq (RNA-seq), following a 48h exposure to the saturated FFA palmitate and suggesting novel mechanisms of palmitate-induced β cells dysfunction and death. Little is known about mitochondrial responses to induced-palmitate stress and about the mechanisms through which glucagon-like peptide-1 (GLP-1) exerts its potential protective effect in β cells mitochondrial dysfunction. Brun et al. , using pharmacological and siRNA approaches, investigated the mitochondrial responses in isolated INS-1E cells mitochondria preparations exposed to different stressors: glucose, fatty acids and oxidative stress. They suggested a selective modification in expression levels of energy sensors and mitochondrial carriers after these different stress conditions. As far as the proteomic approach concerns, only one paper showed the changes of INS-1E mitochondrial proteome after stress induced by high glucose exposure . The purpose of the first part of this thesis was to investigate, for the first time, the lipotoxic effect of palmitate on mitochondria from rat INS-1E cells in the presence and in the absence of GLP-1 by using proteomics and metabolomics approaches. A different expression of mitochondrial proteins was evaluated by using two-dimensional electrophoresis (2-DE) coupled to tandem mass spectrometry (MS/MS) and quantitative shotgun analysis. The use of 2-DE allowed to validate shot-gun results and to overcome the limit of this technique by evaluating potential transformations which could occur in mitochondrial proteins such as post-translational modifications and protein degradation. Moreover, the metabolomic differences targeting aminoacids and carnitines, since they are related to the mitochondrial metabolism and activity, were measured. The study of mitochondrial alteration in rat INS-1E cells after treatment with palmitate and/or GLP- constitutes an important starting point before moving to the study of human cells and towards a better understanding of mitochondrial dysfunction in the context of type two diabetes. The second part of this thesis focused on the development of ultra-high resolution mass spectrometry imaging methods for the analysis of proteins in mouse and human pancreas tissues. The ability of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to simultaneously record the distributions of hundreds of molecules in tissue makes it a powerful discovery method for molecular pathology. MALDI-MSI combines the chemical specificity of modern mass spectrometry with the imaging capabilities of microscopy; it allows a highly multiplexed and untargeted analysis of biomolecular ions and enables their localization within the tissue section . In clinical applications and diagnostics MALDI-MSI has been used to analyse a large variety of analyte classes, such as xenobiotics , metabolites , lipids [25,26], N-linked glycans [27,28], peptides , and proteins [30,31]. Sample preparation is critical to the success of a MALDI-MSI experiment, and must be optimized prior to any clinical investigation. Several reports on method development for protein analysis from different tissues have been published, and indicate that the optimum sample preparation method may be tissue type and application specific [32–34]. To date only a few studies have been published for MALDI-MSI of pancreas tissues. The ability of the MALDI-MSI to measure the peptide hormones located in the endocrine and exocrine pancreas was shown [35–37]. Minerva et al. [38,39] reported two different methods for the analysis of endogenous peptides from the pancreases of obese and wild type mice. Another three studies focused on the analysis of proteolytic peptides from pancreas [40–42], one of which compared healthy and type 1 diabetes . Four studies focused on the analysis of intact proteins from pancreas: a 3D MALDI-MSI datasets from mouse pancreas in the mass m/z range 1600-15000 had been registered , and three focused on biomarker discovery on pancreas cancer tissue (ductal cancer, pre-neoplastic pancreatic lesions, pancreatic adenocarcinoma and insulinoma) [44–46]. When analysing intact proteins in clinical tissue samples the possibility of post-translational modifications (PTMs) and proteolytic processing must be considered, especially for pancreas tissue which is characterized by rapid post mortem degradation . The analysis of intact proteins allows the identification of any proteoforms by retaining any PTMs or proteolytic processing, and which can be clinically very relevant. Poté et al.  have demonstrated that a specific protein acetylation was indicative of microvascular invasion in hepatocellular carcinoma, and a specific truncation of thymosin beta 4 has been found to be associated with stromal activation in breast cancer and patient survival in malignant melanoma . MALDI-MSI of intact proteins has been performed predominantly using time-of-flight (TOF) based mass spectrometers, operated in linear mode [50,51]. Linear MALDI-TOF systems provide limited resolving power and mass accuracy (50-200 ppm) , which complicates protein identity assignments by mass matching MSI datasets with liquid chromatography (LC) MS/MS-based protein identifications. Recently Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been adapted for MALDI profiling [53–55] and MALDI-MSI . MALDI-FTICR-MSI provides the high mass accuracy and high resolving power required to analyse intact proteins (≤ 17.000 m/z) with isotopic resolution, and to assign protein identities with additional confidence . In the current work the workflows for the MSI analysis of intact proteins directly from pancreas tissue by MALDI-TOF-MS and MALDI-FTICR-MS had been developed. Method development, with special emphasis on sample preparation (e.g., tissue washing, matrix choice, MALDI-matrix deposition) was performed on mouse pancreas tissues. Afterward, the method optimization was extended to the analysis of endogenous peptides. The embedding of the tissue in a supporting material allows easy handling and precise microtoming of sections. In clinical laboratories, for histological applications, tissues cut on cryostat microtomes are usually embedded in the optimal cutting temperature (OCT) polymer. However, care should be taken to avoid contamination of the tissue sections with OCT, because its components can lead to ion suppression during mass spectrometry analysis by MALDI-TOF-MS. Recently, there is evidence  that it is feasible to analyse lipids from tissues embedded in OCT compound by MALDI-MSI after extensive tissue washing using water-based solutions. Also Green-Mitchell et al.  in the study on on-tissue reduction of insulin, used OCT embedded pancreas tissues. Seeley et al.  in a review of 2008 also reported that, after washing steps to remove OCT, “[…] spectra obtained from OCT-embedded samples are virtually identical to those obtained from fresh frozen tissue”. 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|Titolo:||Proteomics tools and mass spectrometry imaging techniques for the molecular characterization of pancreas|
|Appare nelle tipologie:||8.1 Tesi Dottorato|
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