Tesi sul tema "Molecular beam mass spectrometry"

With the aim to reduce the global pollution caused by the increasing levels of NOx, CO and CO2 emitted by internal combustion engines, the study of low-temperature chemistry (LTC) of the combustion process has been greatly enhanced. The goal of this thesis work is to evaluate the performances of the time-of-flight Molecular Beam Mass Spectrometry (TOF-MBMS) in the analysis of the oxidation of propane at low temperature with strong interest in the detection of fleeting species to verify the low-temperature oxidation mechanism. To do that we performed two different experiments: propane oxidation in jet stirred reactor and ozone activated cool diffusion flame in a counterflow setup. The first was carried out flowing a mixture of C3H8/O2/Ar with molar fraction 2/13/85%, respectively, corresponding to a equals to 0.77, in the temperature range 750-1100 K and residence time τ=1 s. To support the results obtained from MBMS, we analyzed the propane oxidation in the same conditions with the gas chromatograph (GC) technique. The results have been compared with the MBMS ones showing good agreement for most of the analysed species. Numerical simulations of the molar fraction of all species were performed and compared with experimental data. The results indicate that the model well predicts the trends of the molar fraction of the species, except for C2H2 and the allyl radical. The onset of the reaction is predicted at a lower temperature than in the experiment. In cool flame experiments the fuel/N2 and O2/O3 streams are facing each other and both propane and n-butane were used as fuel. In propane experiments a 50/50% C3H8/N2 mixture has been used with O2 flow rate of 5 dm3/min, which corresponds to an ozone concentration of 5.94%. For n-butane tests the mixture used was 45/55% for C4H10/N2, O2 flow rate 7 dm3/min and ozone concentration of 5.02%. Numerical analysis was performed showing a good prediction of flame position and species profiles through the reaction zone.

Doutoramento em Bioquímica
Os polissacarídeos são os componentes maioritários dos grãos de café verde e torrado e da bebida de café. Os mais abundantes são as galactomananas, seguindo-se as arabinogalactanas. Durante o processo de torra, as galactomananas e arabinogalactanas sofrem modificações estruturais, as quais estão longe de estar completamente elucidadas devido à sua diversidade e à complexidade estrutural dos compostos formados. Durante o processo de torra, as galactomananas e arabinogalactanas reagem com proteínas, ácidos clorogénicos e sacarose, originando compostos castanhos de alto peso molecular contendo nitrogénio, designados de melanoidinas. As melanoidinas do café apresentam diversas atividades biológicas e efeitos benéficos para a saúde. No entanto, a sua estrutura exata e os mecanismos envolvidos na sua formação permanecem desconhecidos, bem como a relação estrutura-atividade biológica. A utilização de sistemas modelo e a análise por espectrometria de massa permitem obter uma visão global e, simultaneamente, detalhada das modificações estruturais nos polissacarídeos do café promovidas pela torra, contribuindo para a elucidação das estruturas e mecanismos de formação das melanoidinas. Com base nesta tese, oligossacarídeos estruturalmente relacionados com a cadeia principal das galactomananas, (β1→4)-Dmanotriose (Man3), e as cadeias laterais das arabinogalactanas, (α1→5)-Larabinotriose (Ara3), isoladamente ou em misturas com ácido 5-Ocafeoilquínico (5-CQA), o ácido clorogénico mais abundante nos grãos de café verde, e péptidos compostos por tirosina e leucina, usados como modelos das proteínas, foram sujeitos a tratamento térmico a seco, mimetizando o processo de torra. A oxidação induzida por radicais hidroxilo (HO•) foi também estudada, uma vez que estes radicais parecem estar envolvidos na modificação dos polissacarídeos durante a torra. A identificação das modificações estruturais induzidas por tratamento térmico e oxidativo dos compostos modelo foi feita por estratégias analíticas baseadas principalmente em espectrometria de massa, mas também em cromatografia líquida. A cromatografia de gás foi usada na análise de açúcares neutros e ligações glicosídicas. Para validar as conclusões obtidas com os compostos modelo, foram também analisadas amostras de polissacarídeos do café obtidas a partir de resíduo de café e café instantâneo. Os resultados obtidos a partir dos oligossacarídeos modelo quando submetidos a tratamento térmico (seco), assim como à oxidação induzida por HO• (em solução), indicam a ocorrência de despolimerização, o que está de acordo com estudos anteriores que reportam a despolimerização das galactomananas e arabinogalactanas do café durante a torra. Foram ainda identificados outros compostos resultantes da quebra do anel de açúcares formados durante o tratamento térmico e oxidativo da Ara3. Por outro lado, o tratamento térmico a seco dos oligossacarídeos modelo (individualmente ou quando misturados) promoveu a formação de oligossacarídeos com um maior grau de polimerização, e também polissacarídeos com novos tipos de ligações glicosídicas, evidenciando a ocorrência de polimerização através reações de transglicosilação não enzimática induzidas por tratamento térmico a seco. As reações de transglicosilação induzidas por tratamento térmico a seco podem ocorrer entre resíduos de açúcares provenientes da mesma origem, mas também de origens diferentes com formação de estruturas híbridas, contendo arabinose e manose como observado nos casos dos compostos modelo usados. Os resultados obtidos a partir de amostras do resíduo de café e de café instantâneo sugerem a presença de polissacarídeos híbridos nestas amostras de café processado, corroborando a ocorrência de transglicosilação durante o processo de torra. Além disso, o estudo de misturas contendo diferentes proporções de cada oligossacarídeo modelo, mimetizando regiões do grão de café com composição distinta em polissacarídeos, sujeitos a diferentes períodos de tratamento térmico, permitiu inferir que diferentes estruturas híbridas e não híbridas podem ser formadas a partir das arabinogalactanas e galactomananas, dependendo da sua distribuição nas paredes celulares do grão e das condições de torra. Estes resultados podem explicar a heterogeneidade de estruturas de melanoidinas formadas durante a torra do café. Os resultados obtidos a partir de misturas modelo contendo um oligossacarídeo (Ara3 ou Man3) e 5-CQA sujeitas a tratamento térmico a seco, assim como de amostras provenientes do resíduo de café, mostraram a formação de compostos híbridos compostos por moléculas de CQA ligadas covalentemente a um número variável de resíduos de açúcar. Além disso, os resultados obtidos a partir da mistura contendo Man3 e 5-CQA mostraram que o CQA atua como catalisador das reações de transglicosilação. Por outro lado, nas misturas modelo contendo um péptido, mesmo contendo também 5-CQA e sujeitas ao mesmo tratamento, observou-se uma diminuição na extensão das reações transglicosilação. Este resultado pode explicar a baixa extensão das reações de transglicosilação não enzimáticas durante a torra nas regiões do grão de café mais ricas em proteínas, apesar dos polissacarídeos serem os componentes maioritários dos grãos de café. A diminuição das reações de transglicosilação na presença de péptidos/proteínas pode dever-se ao facto de os resíduos de açúcares redutores reagirem preferencialmente com os grupos amina de péptidos/proteínas por reação de Maillard, diminuindo o número de resíduos de açúcares redutores disponíveis para as reações de transglicosilação. Além dos compostos já descritos, uma diversidade de outros compostos foram formados a partir dos sistemas modelo, nomeadamente derivados de desidratação formados durante o tratamento térmico a seco. Em conclusão, a tipificação das modificações estruturais promovidas pela torra nos polissacarídeos do café abre o caminho para a compreensão dos mecanismos de formação das melanoidinas e da relação estrutura-atividade destes compostos.
Polysaccharides are the major components of green and roasted coffee beans, and coffee brew. The most abundant ones are galactomannans, followed by arabinogalactans. During the roasting process, galactomannans and arabinogalactans undergo structural modifications that are far to be completely elucidated due to their diversity and complexity of the compounds formed. During the roasting process, galactomannans and arabinogalactans react with proteins, chlorogenic acids, and sucrose, originating high molecular weight brown compounds containing nitrogen, known as melanoidins. Several biological activities and beneficial health effects have been attributed to coffee melanoidins. However, their exact structures and the mechanisms involved in their formation remain unknown, as well as the structure-biological activity relationship. The use of model systems and mass spectrometry analysis allow to obtain an overall view and, simultaneously, detailed, of the structural modifications in coffee polysaccharides promoted by roasting, contributing to the elucidation of the structures and formation mechanisms of melanoidins. Based on this thesis, oligosaccharides structurally related to the backbone of galactomannans, (β1→4)-D-mannotriose, and the side chains of arabinogalactans, (α1→5)-Larabinotriose, alone or in mixtures with 5-O-caffeoylquinic acid, the most abundant chlorogenic acid in green coffee beans, and dipeptides composed by tyrosine and leucine, used as models of proteins, were submitted to dry thermal treatments, mimicking the coffee roasting process. The oxidation induced by hydroxyl radicals (HO•) was also studied, since these radicals seem to be involved in the modification of the polysaccharides during roasting. The identification of the structural modifications induced by thermal and oxidative treatment of the model compounds was performed mostly by mass spectrometry-based analytical strategies, but also using liquid chromatography. Gas chromatography was used in the analysis of neutral sugars and glycosidic linkages. To validate the conclusions achieved with the model compounds, coffee polysaccharide samples obtained from spent coffee grounds and instant coffee were also analysed. The results obtained from the model oligosaccharides when submitted to thermal treatment (dry) or oxidation induced by HO• (in solution) indicate the occurrence of depolymerization, which is in line with previous studies reporting the depolymerization of coffee galactomannans and arabinogalactans during roasting. Compounds resulting from sugar ring cleavage were also formed during thermal treatment and oxidative treatment of Ara3. On the other hand, the dry thermal treatment of the model oligosaccharides (alone or when mixed) promoted the formation of oligosaccharides with a higher degree of polymerization, and also polysaccharides with new type of glycosidic linkages, evidencing the occurrence of polymerization via non-enzymatic transglycosylation reactions induced by dry thermal treatment. The transglycosylation reactions induced by dry thermal treatment can occur between sugar residues from the same origin, but also of different origins, with formation of hybrid structures, containing arabinose and mannose in the case of the model compounds used. The results obtained from spent coffee grounds and instant coffee samples suggest the presence of hybrid polysaccharides in these processed coffee samples, corroborating the occurrence of transglycosylation during the roasting process. Furthermore, the study of mixtures containing different proportions of each model oligosaccharide, mimicking coffee bean regions with distinct polysaccharide composition, subjected to different periods of thermal treatment, allowed to infer that different hybrid and non-hybrid structures may be formed from arabinogalactans and galactomannans, depending on their distribution in the bean cell walls and on roasting conditions. These results may explain the heterogeneity of melanoidins structures formed during coffee roasting. The results obtained from model mixtures containing an oligosaccharide (Ara3 or Man3) and 5-CQA and subjected to dry thermal treatment, as well as samples derived from spent coffee grounds, showed the formation of hybrid compounds composed by CQA molecules covalently linked to a variable number of sugar residues. Moreover, the results obtained from the mixture containing Man3 and 5-CQA showed that CQA acts as catalyst of transglycosylation reactions. On the other hand, in the model mixtures containing a peptide, even if containing 5-CQA and subjected to the same treatment, it was observed a decrease in the extent of transglycosylation reactions. This outcome can explain the low extent of non-enzymatic transglycosylation reactions during roasting in coffee bean regions enriched in proteins, although polysaccharides are the major components of the coffee beans. The decrease of transglycosylation reactions in the presence of peptides/proteins can be related with the preferential reactivity of reducing residues with the amino groups of peptides/proteins by Maillard reaction, decreasing the number of reducing residues available to be directly involved in the transglycosylation reactions. In addition to the compounds already described, a diversity of other compounds were formed from model systems, namely dehydrated derivatives formed during dry thermal treatment. In conclusion, the identification of the structural modifications in coffee polysaccharides promoted by roasting pave the way to the understanding of the mechanisms of formation of melanoidins and structure-activity relationship of these compounds.

Cells, whether bacterial, fungal or mammalian, are all equipped with metabolic pathways capable of producing an assortment of structurally and functionally distinct lipid species. Despite the structural diversity of lipids being recognized and correlated to specific cellular phenomena and disease states, the molecular mechanisms that underpin this structural diversity remain poorly understood. In part, this is due to the lack of adequate analytical techniques capable of measuring the structural details of lipid species in a direct, comprehensive and quantitative manner. The aim of my thesis study was to establish methodology for automated and quantitative analysis of molecular lipid species based on mass spectrometry. From this work a novel high-throughput methodology for lipidome analysis emerged. The main assets of the methodology were the structure-specific mass analysis by powerful hybrid mass spectrometers with high mass resolution, automated and sensitive infusion of total lipid extracts by a nanoelectrospray robot, and automated spectral deconvolution by dedicated Lipid Profiler software. The comprehensive characterization and quantification of molecular lipid species was achieved by spiking total lipid extracts with unique lipid standards, utilizing selective ionization conditions for sample infusion, and performing structure-specific mass analysis by hybrid quadrupole time-of-flight and ion trap mass spectrometry. The analytical routine allowed the comprehensive characterization and quantification of molecular glycerophospholipid species, molecular diacylglycerol species, molecular sphingolipid species including ceramides, glycosphingolipids and inositol-containing sphingolipids, and sterol lipids including cholesterol. The performance of the methodology was validated by comparing its dynamic quantification range to that of established methodology based on triple quandrupole mass spectrometry. Furthermore, its efficacy for lipidomics projects was demonstrated by the successful quantitative deciphering of the lipid composition of T cell receptor signaling domains, mammalian tissues including heart, brain and red blood cells, and the yeast Saccharomyces cerevisiae.

Cells, whether bacterial, fungal or mammalian, are all equipped with metabolic pathways capable of producing an assortment of structurally and functionally distinct lipid species. Despite the structural diversity of lipids being recognized and correlated to specific cellular phenomena and disease states, the molecular mechanisms that underpin this structural diversity remain poorly understood. In part, this is due to the lack of adequate analytical techniques capable of measuring the structural details of lipid species in a direct, comprehensive and quantitative manner. The aim of my thesis study was to establish methodology for automated and quantitative analysis of molecular lipid species based on mass spectrometry. From this work a novel high-throughput methodology for lipidome analysis emerged. The main assets of the methodology were the structure-specific mass analysis by powerful hybrid mass spectrometers with high mass resolution, automated and sensitive infusion of total lipid extracts by a nanoelectrospray robot, and automated spectral deconvolution by dedicated Lipid Profiler software. The comprehensive characterization and quantification of molecular lipid species was achieved by spiking total lipid extracts with unique lipid standards, utilizing selective ionization conditions for sample infusion, and performing structure-specific mass analysis by hybrid quadrupole time-of-flight and ion trap mass spectrometry. The analytical routine allowed the comprehensive characterization and quantification of molecular glycerophospholipid species, molecular diacylglycerol species, molecular sphingolipid species including ceramides, glycosphingolipids and inositol-containing sphingolipids, and sterol lipids including cholesterol. The performance of the methodology was validated by comparing its dynamic quantification range to that of established methodology based on triple quandrupole mass spectrometry. Furthermore, its efficacy for lipidomics projects was demonstrated by the successful quantitative deciphering of the lipid composition of T cell receptor signaling domains, mammalian tissues including heart, brain and red blood cells, and the yeast Saccharomyces cerevisiae.

Mass spectrometry imaging (MSI) is a MS-based technique. It provides a way of ascertaining both spatial distribution and relative abundance of a large variety of analytes from various biological sample surfaces. MSI is able to generate distribution maps of multiple analytes simultaneously without any labeling and does not require a prior knowledge of the target analytes, thus it has become an attractive molecular histology tool. MSI has been widely used in medicine and pharmaceutical fields, while its application in plants is recent although information regarding the spatial organization of metabolic processes in plants is of great value for understanding biological questions such as plant development, plant environment interactions, gene function and regulatory processes. The application of MSI to these studies, however, is not straightforward due to the inherent complexity of the technique. In this thesis, the issues of plant sample preparation, surface properties heterogeneity, fast MSI analysis for spatially resolved population studies and data analysis are addressed. More specifically, two MSI approaches, namely matrix assisted laser desorption ionization (MALDI) imaging and desorption electrospray ionization (DESI) imaging, have been evaluated and compared by mapping the localization of a range of secondary and primary metabolites in apple and grapes, respectively. The work based on MALDI has been focused on the optimization of sample preparation for apple tissues to preserve the true quantitative localization of metabolites and on the development of specific data analysis tool to enhance the chemical identification in untargeted MSI (chapter 3). MALDI imaging allows high-spatial localization analysis of metabolites, but it is not suitable for applications where rapid and high throughput analysis is required when the absolute quantitative information is not necessary as in the case of screening a large number of lines in genomic or plant breeding programs. DESI imaging, in contrast, is suitable for high throughput applications with the potential of obtaining statistically robust results. However, DESI is still in its infancy and there are several fundamental aspects which have to be investigated before using it as a reliable technique in extensive imaging applications. With this in mind, we investigated how DESI imaging can be used to map the distribution of the major organic acids in different grapevine tissue parts, aiming at statistically comparing their distribution differences among various grapevine tissues and gaining insights into their metabolic pathways in grapevine. Our study demonstrated that this class of molecules can be successfully detected in grapevine stem sections, but the surface property differences within the structurally heterogeneous grapevine tissues can strongly affect their semi-quantitative detection in DESI, thereby masking their true distribution. Then we decided to investigate this phenomenon in details, in a series of dedicated imaging studies, and the results have been presented in chapter 4. At the same time, during DESI experiments we have observed the production of the dianions of small dicarboxylates acids. We further studied the mechanism of formation of such species in the ion source proposing the use of doubly charged anions as a possible proxy to visualize the distributions of organic acid salts directly in plant tissues (chapter 5). The structural organization of the PhD thesis is as below: Chapter one and Chapter two describe the general MSI principle, compare the most widely used MSI ion sources, and discuss the current status in MSI data pre-processing and statistical methods. Due to the importance of sample preparation in MSI, sample handling for plant samples is independently reviewed in chapter two, with all the essential steps being fully discussed. The first two chapters describe the comprehensive picture regarding to MSI in plants. Chapter three presents high spatial and high mass resolution MALDI imaging of flavonols and dihydrochalcones in apple. Besides its importance in plant research, our results demonstrate that how data analysis as such Intensity Correlation Analysis could benefit untargeted MSI analysis. Chapter four discusses how sample surface property differences in a structurally/biologically heterogeneous sample affect the quantitative mapping of analytes in the DESI imaging of organic acids in grapevine tissue sections. Chapter five discusses the mechanism of formation of dicarboxylate dianions in DESI and ESI Chapter six summarizes the work in the thesis and discusses the future perspectives.

Mass spectrometry imaging (MSI) is a MS-based technique. It provides a way of ascertaining both spatial distribution and relative abundance of a large variety of analytes from various biological sample surfaces. MSI is able to generate distribution maps of multiple analytes simultaneously without any labeling and does not require a prior knowledge of the target analytes, thus it has become an attractive molecular histology tool. MSI has been widely used in medicine and pharmaceutical fields, while its application in plants is recent although information regarding the spatial organization of metabolic processes in plants is of great value for understanding biological questions such as plant development, plant environment interactions, gene function and regulatory processes. The application of MSI to these studies, however, is not straightforward due to the inherent complexity of the technique. In this thesis, the issues of plant sample preparation, surface properties heterogeneity, fast MSI analysis for spatially resolved population studies and data analysis are addressed. More specifically, two MSI approaches, namely matrix assisted laser desorption ionization (MALDI) imaging and desorption electrospray ionization (DESI) imaging, have been evaluated and compared by mapping the localization of a range of secondary and primary metabolites in apple and grapes, respectively. The work based on MALDI has been focused on the optimization of sample preparation for apple tissues to preserve the true quantitative localization of metabolites and on the development of specific data analysis tool to enhance the chemical identification in untargeted MSI (chapter 3). MALDI imaging allows high-spatial localization analysis of metabolites, but it is not suitable for applications where rapid and high throughput analysis is required when the absolute quantitative information is not necessary as in the case of screening a large number of lines in genomic or plant breeding programs. DESI imaging, in contrast, is suitable for high throughput applications with the potential of obtaining statistically robust results. However, DESI is still in its infancy and there are several fundamental aspects which have to be investigated before using it as a reliable technique in extensive imaging applications. With this in mind, we investigated how DESI imaging can be used to map the distribution of the major organic acids in different grapevine tissue parts, aiming at statistically comparing their distribution differences among various grapevine tissues and gaining insights into their metabolic pathways in grapevine. Our study demonstrated that this class of molecules can be successfully detected in grapevine stem sections, but the surface property differences within the structurally heterogeneous grapevine tissues can strongly affect their semi-quantitative detection in DESI, thereby masking their true distribution. Then we decided to investigate this phenomenon in details, in a series of dedicated imaging studies, and the results have been presented in chapter 4. At the same time, during DESI experiments we have observed the production of the dianions of small dicarboxylates acids. We further studied the mechanism of formation of such species in the ion source proposing the use of doubly charged anions as a possible proxy to visualize the distributions of organic acid salts directly in plant tissues (chapter 5). The structural organization of the PhD thesis is as below: Chapter one and Chapter two describe the general MSI principle, compare the most widely used MSI ion sources, and discuss the current status in MSI data pre-processing and statistical methods. Due to the importance of sample preparation in MSI, sample handling for plant samples is independently reviewed in chapter two, with all the essential steps being fully discussed. The first two chapters describe the comprehensive picture regarding to MSI in plants. Chapter three presents high spatial and high mass resolution MALDI imaging of flavonols and dihydrochalcones in apple. Besides its importance in plant research, our results demonstrate that how data analysis as such Intensity Correlation Analysis could benefit untargeted MSI analysis. Chapter four discusses how sample surface property differences in a structurally/biologically heterogeneous sample affect the quantitative mapping of analytes in the DESI imaging of organic acids in grapevine tissue sections. Chapter five discusses the mechanism of formation of dicarboxylate dianions in DESI and ESI Chapter six summarizes the work in the thesis and discusses the future perspectives.

Mass spectrometry imaging (MSI) is a MS-based technique. It provides a way of ascertaining both spatial distribution and relative abundance of a large variety of analytes from various biological sample surfaces. MSI is able to generate distribution maps of multiple analytes simultaneously without any labeling and does not require a prior knowledge of the target analytes, thus it has become an attractive molecular histology tool. MSI has been widely used in medicine and pharmaceutical fields, while its application in plants is recent although information regarding the spatial organization of metabolic processes in plants is of great value for understanding biological questions such as plant development, plant environment interactions, gene function and regulatory processes. The application of MSI to these studies, however, is not straightforward due to the inherent complexity of the technique. In this thesis, the issues of plant sample preparation, surface properties heterogeneity, fast MSI analysis for spatially resolved population studies and data analysis are addressed. More specifically, two MSI approaches, namely matrix assisted laser desorption ionization (MALDI) imaging and desorption electrospray ionization (DESI) imaging, have been evaluated and compared by mapping the localization of a range of secondary and primary metabolites in apple and grapes, respectively. The work based on MALDI has been focused on the optimization of sample preparation for apple tissues to preserve the true quantitative localization of metabolites and on the development of specific data analysis tool to enhance the chemical identification in untargeted MSI (chapter 3). MALDI imaging allows high-spatial localization analysis of metabolites, but it is not suitable for applications where rapid and high throughput analysis is required when the absolute quantitative information is not necessary as in the case of screening a large number of lines in genomic or plant breeding programs. DESI imaging, in contrast, is suitable for high throughput applications with the potential of obtaining statistically robust results. However, DESI is still in its infancy and there are several fundamental aspects which have to be investigated before using it as a reliable technique in extensive imaging applications. With this in mind, we investigated how DESI imaging can be used to map the distribution of the major organic acids in different grapevine tissue parts, aiming at statistically comparing their distribution differences among various grapevine tissues and gaining insights into their metabolic pathways in grapevine. Our study demonstrated that this class of molecules can be successfully detected in grapevine stem sections, but the surface property differences within the structurally heterogeneous grapevine tissues can strongly affect their semi-quantitative detection in DESI, thereby masking their true distribution. Then we decided to investigate this phenomenon in details, in a series of dedicated imaging studies, and the results have been presented in chapter 4. At the same time, during DESI experiments we have observed the production of the dianions of small dicarboxylates acids. We further studied the mechanism of formation of such species in the ion source proposing the use of doubly charged anions as a possible proxy to visualize the distributions of organic acid salts directly in plant tissues (chapter 5). The structural organization of the PhD thesis is as below: Chapter one and Chapter two describe the general MSI principle, compare the most widely used MSI ion sources, and discuss the current status in MSI data pre-processing and statistical methods. Due to the importance of sample preparation in MSI, sample handling for plant samples is independently reviewed in chapter two, with all the essential steps being fully discussed. The first two chapters describe the comprehensive picture regarding to MSI in plants. Chapter three presents high spatial and high mass resolution MALDI imaging of flavonols and dihydrochalcones in apple. Besides its importance in plant research, our results demonstrate that how data analysis as such Intensity Correlation Analysis could benefit untargeted MSI analysis. Chapter four discusses how sample surface property differences in a structurally/biologically heterogeneous sample affect the quantitative mapping of analytes in the DESI imaging of organic acids in grapevine tissue sections. Chapter five discusses the mechanism of formation of dicarboxylate dianions in DESI and ESI Chapter six summarizes the work in the thesis and discusses the future perspectives.

BACKGROUND Cardiovascular diseases are the world’s number one death cause, accounting for 17.1 million deaths a year. There is still much unknown about cardiovascular diseases and their physiological underlying mechanism. Understanding the nature of complex biological processes occurring in both healthy and diseased heart tissue requires identifying the compounds involved and determining where they are located. Summary METHODS We have investigated a complementary mass spectrometry imaging (MSI) approach using matrix-assisted laser desorption/ionization (MALDI) and secondary ion mass spectrometry (SIMS) on the major areas of rat heart: the pericardium, the myocardium, the endocardium, and the great vessels to study the native distribution and identity of atomics, lipids, peptides and proteins in rat heart sections. 40 layers of horizontal tissue slices were acquired and reconstructed into a 3 D dataset. RESULTS Surface rastering of heart tissue sections generated multiple secondary ions in a mass range up to 1500 m/z. In the negative spectra we identified cholesterol related ions that show high intensity in both atrias, the aorta, the pulmonary artery and the outline both ventricles. The m/z 105 (choline) signal localizes in both atrias, aorta, pulmonary artery, in the atrioventricular valves and semilunar valves but is not present in ventricles surface. DAG species with probable identifications as Oleic, Linoleic [OL]+ at m/z 602 and [OO]+ (Oleic, Oleic) at m/z 604, can be detected. The images of 3D reconstruction show a highly complementary localization between Na+, K+, ion at m/z 145 and ion at m/z 667. Na+ is localized to tissue regions corresponding to atrias, while K+ is strongly localized to tissue regions corresponding to ventricles surface.The ion at m/z 667 localized very precisely within the aortic wall and the ion at m/z 145 is primarily located to the atria regions. CONCLUSIONS To promote further research with cardiovascular disease, we report the identification of characteristic molecules that map the spatial organization in a rat heart’s structure. A series of images obtained from successive sections of animal heart could, in principle, be used to produce a molecular atlas. Such tissue atlases (based optical images) are widely used for anatomical and physiological reference. The specific aim of this project is to optimize the data obtained from Heart SIMS a analysis and the 3-D reconstructive techniques developed to aid in investigating and visualizing differential molecular localizations in heart rat structures. The results reported here represent the first 3D molecular reconstruction of rat heart by SIMS imaging.
Introduzione Le malattie cardiovascolari rappresentano nel mondo la prima causa di morte, contando 17.1 milioni di morti ogni anno. Attualmente i meccanismi fisiopatologici alla base delle patologie sono in larga parte ancora sconosciuti. Capire la natura dei complessi processi biologici in atto sia nel miocardio cardiaco sano che malato richiede l’identificazione e la localizzazione degli stessi elementi molecolari coinvolti. METODO Utilizzando tecniche complementari di spettrometria di massa d’immagine (SMI) quali la spettrometria di massa a ioni secondari (Secondary Ion Mass Spectrometry, SIMS) e la spettrometria di massa a desorbimento /ionizzazione laser assistita da matrice (Matrix-assisted laser desorption/ionization, MALDI) abbiamo analizzato le principali componenti del cuore del ratto: il pericardio, il miocardio, l’endocardio, le valvole e i grandi vasi al fine di studiare ed identificare l’originale distribuzione di atomi, lipidi, peptici e proteine nel tessuto cardiaco normale. Quaranta sezioni di tessuto cardiaco sono state acquisite e ricostruite ottenendo un database tridimensionale. RISULTATI L’analisi della superficie delle sezione di tessuto cardiaco ha generato molteplici ioni secondari con un intervallo di massa che raggiunge i 1500 m/z. Utilizzando la modalita’ negativa abbiamo identificato il colesterolo e gli ioni relativi ad esso che mostrato un alta intensita’ in entrambi gli atri, l’aorta, l’arteria polmonare e pericardio. La colina corrispondente a 105 m/z di massa molecolare risulta essere localizzata in entrambi gli atri, aorta, arteria polmonare, valvole atrioventricolari e valvole semilunari ma non risulta essere presente sulla superficie ventricolare. Sono state identificate molecole appartenenti al diacilglicerolo come acido Oleico, Linoleico [OL]+ corrispondenti alla massa molecolare di 602 m/z e [OO]+ (Oleico,Oleico) con massa molecolare di 604 m/z. Le immagini ottenute dalla ricostruzione tridimensionale mostrano una specifica localizzazione complementare tra il sodio, il potassio e gli ioni con massa molecolare di 145 m/z e 667 m/z. Il sodio e’maggiormente localizzato nelle regioni cardiache corrispondenti agli atri, mentre il potassio e’ maggiormente localizzato nelle regioni corrispondenti alla superficie ventricolari. Lo ione con massa molecolare di 667 m/z e’ localizzato con molta precisione all’interno della parete dell’aorta e lo ione con massa molecolare di 145 m/z e’ localizzato a livello delle regioni atriali. CONCLUSIONI Al fine di promuovere un’ulteriore ricerca in patologia cardiovascolare, riportiamo l’identificazione delle caratteristiche molecole che mappano l’organizzazione spaziale delle strutture cardiache del cuore del ratto. Una serie di immagini ottenute da sezioni successive del cuore potrebbero inizialmente essere utilizzate come un atlante molecolare e similmente, ad un atlante basato sulle immagini ottiche, essere ampiamente utilizzato come referente sia dal punto di vista fisiologico che anatomico. L’aiuto apportato da questo progetto e’ l’ottimizzazione dei dati ottenuti dall’analisi SIMS e lo sviluppo della tecnica per la ricostruzione tridimensionale al fine di investigare e visualizzare le differenti molecole localizzate nelle strutture del cuore di ratto. I risultati qui riportati rappresentano la prima ricostruzione tridimensionale ottenuta con immagini SIMS, del cuore di ratto.

Thesis: Ph. D., Joint Program in Oceanography (Massachusetts Institute of Technology, Department of Earth, Atmospheric, and Planetary Sciences; and the Woods Hole Oceanographic Institution), 2016.
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Includes bibliographical references.
Marine microbes produce a wide variety of metal binding organic ligands that regulate the solubility and availability of biologically important metals such as iron, copper, cobalt, and zinc. In marine environments where the availability of iron limits microbial growth and carbon fixation rates, the ability to access organically bound iron confers a competitive advantage. Thus, the compounds that microbes produced to acquire iron play an important role in biogeochemical carbon and metal cycling. However, the source, abundance, and identity of these compounds are poorly understood. To investigate these processes, sensitive methodologies were developed to gain a compound-specific window into marine iron speciation by combining trace metal clean sample collection and chromatography with inductively coupled plasma mass spectrometry (LCICPMS) and electrospray ionization mass spectrometry (LC-ESIMS). Coupled with isotope pattern assisted search algorithms, these tools provide a means to quantify and isolate specific iron binding ligands from seawater and marine cultures, identify them based on their mass and fragmentation spectra, and investigate their metal binding kinetics. Using these techniques, we investigated the distribution and diversity of marine iron binding ligands. In cultures, LC-ICPMS-ESIMS was used to identify new members of siderophore classes produced by marine cyanobacteria and heterotrophic bacteria, including synechobactins and marinobactins. Applications to natural seawater samples from the Pacific Ocean revealed a wide diversity of both known and novel metal compounds that are linked to specific nutrient regimes. Ferrioxamines B, E, and G were identified in productive coastal waters near California and Peru, in oligotrophic waters of the North and South Pacific Gyre, and in association with zooplankton grazers. Siderophore concentrations were up to five-fold higher in iron-deficient offshore waters (9pM) and were dominated by amphibactins, amphiphilic siderophores that partition into cell membranes. Furthermore, synechobactins were detected within nepheloid layers along the continental shelf. These siderophores reflect adaptations that impact dissolved iron bioavailability and thus have important consequences for marine ecosystem community structures and primary productivity. The ability to map and characterize these compounds has opened new opportunities to better understand mechanisms that link metals with the microbes that use them.
by Rene M. Boiteau.
Ph. D.

The existence of singly, doubly, and triply charged diatomic molecular ions was observed by using an Accelerator Mass Spectrometry (AMS) technique. The mean lifetimes of 3 MeV boron diatomic molecular ions were measured. No isotopic effects on the mean lifetimes of boron diatomic molecules were observed for charge state 3+. Also, the mean lifetime of SiF^3+ was measured.

Laser-Ionization Time-of-Flight Mass Spectrometry (LI-TOF-MS) is a sophisticated tool for the molecular-weight determination and structural characterization of a variety of molecules. Advances in instrumentation and ionization methods have recently expanded its role in the analysis of high-mass analytes. Large multimetallic complexes, which are efficient solar-energy converters, rely heavily on their chemical structure for optimum operation. Molecular mass determinations of these multimetallic complexes have been problematic due to their lability and high molecular weights.

This thesis describes the characterization of a LI-TOF-MS instrument and confirmation of theoretical time-of-flight mass-separation principles. Several test cases demonstrate the instrument's proper operation and calibration for a wide mass range of analytes. Mass spectral results of three organometallic compounds: i. [Ir(dpp)₂Cl₂](PF₆), ii. {[(bpy)₂Ru(dpp)]₂IrCl₂}(PF₆)₅, and iii. {[(bpy)₂Ru(dpp)]₂RuCl₂}(PF₆)₅ under a variety of laser ionization and sample preparation conditions are compared. A complete structural characterization of the monometallic complex, [Ir(dpp)₂Cl₂](PF₆), is presented. The two trimetallic analytes fragmented easily, but significant components of the molecules are successfully identified. After optimizing the ionization and analytical procedure, LI-TOF-MS proved useful in the analysis of high molecular mass metal complexes.
Master of Science

It has been presented the review and analysis of the main evolution stages of one of the most efficient methods for investigation of composition and properties of substances – mass spectrometry. The advantages and application area of the method were described, as well as physical and technical peculiarities of the devices for its realization were considered.

Molecular adsorption is a key feature in several disciplines of chemistry, covering as diverse fields as chromatographic separation to biomaterial development. This thesis aims at developing methods and techniques for the characterization of molecular adsorption at the liquid-solid interface. Two different experimental models were used, small molecular interaction characterization using liquid chromatography and complex protein adsorption on polymeric materials possible for biological sampling. Holistic approaches, where both detailed molecular interactions and identifications of trends, could improve the fundamental understanding of adsorption systems, were invariably part of the scientific process. The characterization of small molecular interactions on liquid chromatography stationary phases via adsorption isotherm determination used combined data from physical phase parameters i.e. carbon loading, linear-, and nonlinear-characterization methods. These experiments were conducted on high performance liquid chromatography systems, using both ordinary reversed phase stationary phases, and hybrid phases. The expansion of the improved elusion by characteristic point (ECP) for adsorption isotherm determination, led to that previous impossible isotherm types, having inflexion points, now could be determined by the method. It also reduced errors in isotherm parameters due to the elimination of inaccurate determined retention times where the mobile phase concentration was zero. The characterization of protein adsorption where performed in an unbiased way. Adsorbed proteins on different surfaces were identified using mass spectrometry (MS) and data dependent acquisition or a targeted method. Prior MS, an improved on surface enzymatic digestion (oSED) method was used to enable identification and quantitation of adsorbed protein originating from ventricular cerebrospinal fluid (vCSF). oSED was found to be able to experimentally determine large variations in protein adsorption characteristics between native and coated polycarbonate surfaces in contact with vCSF. The method was also confirmed being mechanistic in favor of enzymatic digestion of the proteins adsorbed on a surface, rather than a prior desorption into solution before digestion. An improvement of the overall understanding of adsorption systems was not only achieved with the oSED method as a promising tool for characterization of protein adsorption on arbitrary surfaces, but also the use of linear and nonlinear approaches in stationary phase characterization that strengthened drawn conclusions.

The work described in this thesis is concerned with the development of laser desorption laser photoionisation mass spectrometry (L2MS) towards spatially resolved analysis of real complex molecular systems. A broad overview of the main elements of the technique is presented. In addition, the experimental procedures and equipment used to carry out this work are described in some detail. Photoionisation mass spectra recorded for a series of azo dyes and porphyrin pigments revealed a marked wavelength dependence in their ionisation and fragmentation channels. The relationship between this behaviour and the known photochemical and photophysical properties of these molecules is discussed. The photochemistry of these molecules has been exploited to aid the differentiation between isomeric species. The selectivity inherent in the L2MS technique has been exploited for in situ studies of a number of real systems. Polymer additives, such as antioxidants and ultraviolet stabilisers, have been successfully detected directly from their host polymer matrices without recourse to extraction, separation or pre-concentration. The technique has been shown to be surface specific, suggesting that the long-term goal of spatially resolved analysis to monitor, for example, additive aggregation and migration to the surface are feasible. In further in situ studies, polycyclic aromatic hydrocarbons, an important class of priority pollutants, adsorbed onto aerosol particulates have been detected. Electrochemically polymerised indoles, known to form conducting films, have also been identified directly from the electrode surface. Finally, current limitations of the L2MS technique are discussed. It is suggested that many of the problems identified are inextricably linked to the laser desorption process. It is shown that energy imparted to neutral molecules during the desorption event can lead to fragmentation. This has implications for both the ionisation of high mass molecules and for quantitative studies. Possible ways of circumventing these problems are discussed. The future outlook for the technique, both for fundamental studies, and as an analytical tool, is also discussed.

The aim of thesis was to establish a methodology for 210Pb measurements by Accelerator Mass Spectrometry (AMS). The potential application is to measure 210Pb in people who have been exposed to radon. This will better our understanding of radon toxicity, which is not possible now with current radiometric and mass spectrometry techniques. The determination of 210Pb by AMS was done in two major studies 1) Studying Pb chemistry in a Cs+ sputter source used in AMS and 2) Evaluating 204,205 & 208Pb spikes for the quantification of 210Pb by isotope dilution. Pb chemistry was investigated using an 834 SIMS-type and a SO-110 Cs+ sputter source at the IsoTrace Laboratory and A.E Lalonde AMS facility, respectively. Different molecular anions of Pb were studied with the 834 SIMS-type Cs+ sputter source and the strongest molecular anion current of Pb and thus greatest ionization efficiency was achieved form the superhalogen PbF3-. The average 208PbF3- current was unaffected by varying the ratio of the fluorinating compounds (AgF2 and CsF) packed into a target. The average current of 208PbF3- was reproducibly increased by chemically mixing the targets of AgF2, CsF and PbF2 in concentrated HF rather than mechanically mixing them the powders with a stir rod. The count rate of 210Pb reproducibly increased by a factor of 20 when μg quantities of PbF2 were present in mg AMS targets compared to AMS targets that had pg quantities of PbF2. The average current of 208PbF3- for pure PbF2 targets in an SO-100 Cs+ sputter source was reproducibly increased when the Cs+ flux was decreased by a factor of 10. This phase of my work maximized the overall efficiency of PbF3-, to a value of 1.8x10-10 ±8x10-11s-, which was a key first step in the measurement by AMS. Then isotope dilution was tested to quantify 210Pb and the next stage of my work evaluated the use of 204,205 & 208Pb spikes. 210Pb was measured in the +3 charge state by isotope dilution assays using 204,205 & 208Pb spikes. 204Pb+3 reproducibly suffered from the molecular interference from 68Zn3+3, which could not be easily removed without negatively impacting the detection limit for 210Pb. 205Pb+3 continually suffered from 205Tl+3 interference which could be readily be removed but not without negatively affecting the II detection limit for 210Pb. 208Pb+3 suffers from no molecular interferences but if a large amount of 208Pb is needed to swamp the Pb in a sample, this could limit the detection limit for 210Pb as the abundance sensitivity is 210Pb/208Pb=1.3×10-12. A calibration curve is required when 208Pb is used as a spike due to a difference in collection efficiency of a Faraday cup, where 208Pb+3 is detected and the gas ionization chamber, where 210Pb+3 is detected. The quantification of 210Pb with 208Pb as a spike yielded a detection limit of 4.4mBq at the IsoTrace facility. A theoretical detection limit of ≤0.11mBq is expected at the A.E Lalonde AMS facility. The expected detection limit at the A.E Lalonde AMS facility is on par with α-spectroscopy but AMS samples can be counted in less than 1 hour whereas alpha spectrometry samples must be counted for about 1 day.

The physical properties of glycerophospholipids (GPLs) are not only determined by the head group (HG), but also by their fatty acid (FA) chains, which affect their distribution and function within membranes in the cell. Understanding the microheterogenity of lipid membranes on a molecular level requires qualitative and quantitative characterization of individual lipids and identification of their FA moieties. The aim of my study was to introduce the new technology of multiple precursor ion scanning (MPIS) on a QSTAR Pulsar time-of-flight mass spectrometer (QqTOF) to analyze lipids. Detailed information on fatty acid composition of individual GPL molecules could be obtained in parallel with conventional profiling of lipid classes, and this could be done by direct analysis of total lipid extracts. This method was termed Fatty Acid Scanning (FAS) and Head Group Scanning HGS, respectively. In this way the molecular GPL composition of total lipid extracts could be charted in a single analysis accurately and rapidly at a low picomole concentration level. Furthermore, combining FAS and HGS together with ion trap MS3 analysis allowed complete charting of the molecular composition of PCs, including quantification of their positional isomers, thus providing a detailed and comprehensive characterization of molecular composition of the pool of PCs. Development of the Lipid Profiler software allowed full automation and rapid processing of complex data, including identification and quantification of molecular GPLs. This approach was evaluated by preliminary applications. First, the molecular composition of PCs of total lipid extracts of MDCK cells and of human red blood cells (RBC) could accurately be charted. Significant presence of positional isomers was observed increasing the total number of individual PC species close to one hundred. Secondly, the molecular PC and SM species distribution in detergent resistant membranes (DRMs) prepared by Triton X-100 DRMs were analyzed and were found to be enriched in distinct GPLs. The distribution in PCs and SMs of Triton X-100 DRMs of RBC were compared with those of the DRMs of MDCK cells. Finally, combining the use of a 96 well plate and a robotic system demonstrated that these analyses can be automated and analyzed with high throughput. This system we termed Shotgun Lipidomics. Taken together, this mass spectrometric methodology provides rapid and detailed insight into the distribution of the molecular GPLs of membranes and membrane sub-fractions.

Le progrès partout dans le monde nécessite une variété de sources d'énergie propre. Les biocarburants liquides de type ester semblent être très efficaces dans ce contexte, car ils sont faciles à utiliser dans les véhicules modernes, ils peuvent être produits à partir de diverses ressources renouvelables et ils offrent des caractéristiques de combustion respectueuses de l'environnement. À cet égard, les esters éthyliques d'acides gras (EEAG) sont considérés comme une classe prometteuse de biocarburants. L'objectif principal de cette thèse était de développer un mécanisme cinétique chimique actualisé de la combustion des EEAG légers jusqu'au pentanoate d'éthyle et de le valider par rapport aux nouvelles données expérimentales sur la structure de flammes laminaires prémélangées à basse pression et pression atmosphérique. Les flammes alimentées par trois EEAG, l'acétate d'éthyle, le butanoate d'éthyle et le pentanoate d'éthyle, ont été étudiées au moyen de la spectrométrie de masse avec faisceau moléculaire et de la chromatographie en phase gazeuse. Plus de 40 espèces stables et intermédiaires comprenant des radicaux ont été détectées et quantifiées dans les flammes. Une analyse complète du mécanisme développé a été réalisée. La thèse se compose de 3 chapitres. Le premier chapitre présente une revue bibliographique. Les études expérimentales et théoriques les plus importantes sur la combustion des EEAG sont discutées. Le deuxième chapitre présente un aperçu des méthodes expérimentales et de simulation utilisées dans la thèse. Des détails sur le développement du mécanisme sont également fournis dans cette partie. Le dernier chapitre présente des résultats expérimentaux et de modélisation sur les esters étudiés en comparaison avec les mécanismes cinétiques de la littérature
Global progress all over the world requires a variety of clean energy sources. Liquid ester-based biofuels seem to be very effective in this context since they are easy to use in modern vehicles, they can be produced from a variety of renewable resources, and they provide environmentally friendly combustion characteristics. In this regard, fatty acid ethyl esters (FAEEs) are considered as a promising class of biofuels. The main goal of this thesis was to develop an updated chemical kinetic mechanism of combustion of light FAEEs up to ethyl pentanoate and validate it against the new experimental data on chemical speciation in low and atmospheric pressure premixed laminar flames. The flames fueled by three FAEEs, ethyl acetate, ethyl butanoate and ethyl pentanoate, were investigated by means of molecular-beam mass-spectrometry and gas-chromatography. More than 40 stable and intermediate species including radicals were detected and quantified in the flames. A comprehensive analysis of the developed mechanism was performed. The thesis consists of 3 chapters. In the first chapter a review of literature is presented. The most important experimental and theoretic studies on FAEEs are discussed. The second chapter presents an overview of experimental and simulation methods used in the work. Details on the mechanism development are also provided in this part. The last chapter present experimental and modeling results on the esters studied in comparison with the literature kinetic mechanisms

Liquid Chromatography/ Mass Spectrometry (LC/MS) is used to interface the separating power of LC with the sensitivity and specificity of MS for the determination of trace levels of organic compounds in a variety of matrices. The technique is finding increasing application in the field of environmental and pharmaceutical analysis. Particle Beam LC/MS (PB/LC/MS) uses a particle beam interface to connect the LC to the MS. This interface design has the advantage of being able to produce "classical" electron impact (El) spectra which can then be searched against commercial MS libraries. The aim of this work was to apply PB/LC/MS to a range of new problems in environmental analysis and evaluate the usefulness of this technique. PB/LC/MS was used to determine compounds that cannot easily be analysed by more conventional techniques such as gas chromatography with mass spectrometry (GC/MS) or liquid chromatography with UV/vis detection (LC/UV). For example, some polycyclic aromatic hydrocarbons (PAH) are too involatile to analyse by GC/MS, some commonly prepared isocyanate derivatives cannot be accurately identified by LC/UV and some classes of pesticides are thermally labile and so cannot be determined by GC/MS.The work presented in this thesis examines the factors affecting the sensitivity and performance of PB/LC/MS and comparisons are made with other analytical methods. Compound classes examined are polycyclic aromatic hydrocarbons (PAH), pesticides and isocyanate derivatives in a variety of environmental matrices. Methods for improving the sensitivity of PB/LC/MS are investigated and the results of these experiments used to compare the different models are used to explain PB/MS behaviour. Conclusions regarding the accuracy of these models are then made. The ability of PB/MS to provide useful El MS for identification purposes in complex environmental matrices is also investigated.

The performance of a Hewlett Packard Particle Beam LC/MS interface is evaluated using EPA appendix VIII and IX compounds. The behavior of these priority pollutants in the interface could determine its feasibility as a future EPA certified technique.

The evaluation process consists of studies to determine minimum detectable quantities (MDQ), linear response, and band broadening contributions. The MDQ's of the analytes in electron impact and chemical ionization modes are extrapolated from experimental signal to noise data. The linearity study involves ten compounds analyzed at five different concentrations. The response factors (RF) are calculated and discussed. The study concerning the band broadening contributions of the interface involves four independent variables (helium flow, desolvation chamber temperature, source temperature, and 96 methanol) and their effects on peak width. A Boxâ Behnken experimental design is used and described. Response surfaces are generated from the best fit equation describing the data.

Master of Science

Ge-Sb-Te basierte Phasenwechselmaterialen sind vielersprechende Kandidaten für die Anwendung in optischen und elektrischen nicht-flüchtigen Speicheranwendungen. Diese Materialien können mit Hilfe von elektrischen oder optischen Pulsen reversibel zwischen der kristallinen und amorphen Struktur geschaltet werden. Diese stukturellen Phasen zeigen einen großen Unterschied in ihren elektronischen Eigenschaften, der sich in einer starken Änderung der optischen Reflektivität und des elektrischen Widerstands zeigt.Diese Studie befasst sich mit epitaktischem Wachstum und Analyse der epitaktischen Schichten. Der erste Teil der Arbiet befasst sich mir dem epitaktischen Wachstum von GeTe. Dünne GeTe Schichten wurden auf Si(111) und Si(001) Substraten mit einer Gitterfehlanpassung von 10.8% präpariert. Auf beiden Substraten bildet sich in der GeTe Schicht die [111] Oberflächenfacette parallel zur Si(001) und Si(111) Oberfläche aus. Während des inertialen Wachstums findet eine Phasentransformation von amorph zu kristallin statt. Diese Phasentransformation wurde mittels azimuthaler in-situ Beugung hochenergetischer Elektronen sowie in-situ Röntgenbeugung unter streifendem Einfall untersucht. Der zweite Teil der Arbeit wird die Epitaxie sowie die strukturelle Charakterisierung dünner Sb2Te3 Schichten dargestellt. Der dritte Teil umfasst die Epitaxie terniärer Ge-Sb-Te Schichten . Zum Wachstum wurden sowohl die Substrattemperatur als auch die Ge, Sb und Te Flüsse variiert. Es wird gezeigt, dass die Komposition der Schicht stark von der Wachtumstemperatur abhängt und nur entlang der pseudibinären Verbindungslinie von GeTe-Sb2Te3 variiert. Zur Kontrolle des Wachstums wurde dabei die in-situ Quadrupol Massenspektroskopie verwendet. Es zeigen sich diverse inkommensurate Beugungsmaxima entlang der [111] Oberflächennormalen der Schichten, anhand derer die Ausbildung einer Lehrstellen Ordnung in Form einer Überstruktur diskutiert wird.
Ge-Sb-Te based phase change materials are considered as a prime candidate for optical and electrical data storage applications. With the application of an optical or electrical pulse, they can be reversibly switched between amorphous and crystalline state, thereby exhibiting large optical and electrical contrast between the two phases, which are then stored as information in the form of binary digits. Single crystalline growth is interesting from both the academic and industrial perspective, as ordered Ge-Sb-Te based metamaterials are known to exhibit switching at reduced energies. The present study deals with the epitaxial growth and analysis of Ge-Sb-Te based thin films. The first part of the thesis deals with the epitaxial growth of GeTe. Thin films of GeTe were grown on highly mismatched Si(111) and (001) substrates. On both the substrate orientations the film grows along [111] direction with an amorphous-to-crystalline transition observed during the initial stages of growth. The amorphous-to-crystalline transition was studied in-vivo using azimuthal reflection high-energy electron diffraction scans and grazing incidence x-ray diffraction. In the second part of the thesis epitaxy and characterization of Sb2Te3 thin films are presented. The third part of the thesis deals with the epitaxy of ternary Ge-Sb-Te alloys. The composition of the films are shown to be highly dependent on growth temperatures and vary along the pseudobinary line from Sb2Te3 to GeTe with increase in growth temperatures. A line-of-sight quadrupole mass spectrometer was used to reliably control the GeSbTe growth temperature. Growth was performed at different Ge, Sb, Te fluxes to study the compositional variation of the films. Incommensurate peaks are observed along the [111] direction by x-ray diffraction. The possibility of superstructural vacancy ordering along the [111] direction is discussed.

Phosphorus (P) regulates trophic status in most aquatic systems. However, only bioavailable P contributes to primary production. In most lakes and shallow seas, mineralisation of sediment P into its bioavailable form and its release to the water column is important for maintaining primary production. Sediment organic P forms a substantial proportion of this P to be mineralised and can originate from different sources on land (farmland, forests, etc.) or from primary production in the lake. These organic P forms can thus be expected to have differing composition, degradability and recyclable P content. Knowledge of the chemical structure of sediment organic P compounds is scarce, mainly due to lack of appropriate analytical techniques. The commonly used 31P-nuclear magnetic resonance (31P-NMR) technique, only identifies P binding groups, so a mass spectrometric (MS) analysis method was developed that allows individual sediment organic P compounds to be identified. EDTA as pre-extractant resulted in the highest P yield in subsequent NaOH extraction. Extracted organic P compound groups were identified using 31P-NMR. For identification of specific P compounds with MS, a sample preparation method prior to electrospray tandem mass spectrometry (ESI-MS/MS) analysis was developed. Liquid chromatography (LC) with porous graphitic carbon prior to ESI-MS/MS enhanced sensitivity and selectivity, enabling several of the ions detected to be identified as nucleotides. 31P-NMR analysis showed P monoesters to be the most stabile P compounds throughout a lake sediment profile. The developed LC-ESI-MS/MS analysis method revealed that some monoester-P (nucleotides) were labile, while other P compounds increased in concentration with Baltic Sea sediment depth and were therefore considered stabile. Differences in patterns of P compounds detected were also shown depending on catchment characteristics in relation to Baltic Sea sediment age. For cost-effective management of eutrophication, knowledge of the sources of degradable organic P forms, contributing to internal loading, is needed. This thesis showed the developed LC-ESI-MS/MS analysis method to be a powerful analytical tool for this purpose.

The present study employs a range of soft-ionisation mass spectrometry techniques to study the degradation of model compounds of poly(methyl methacrylate), poly(n-butyl acrylate) and poly(2-hydroxyethyl methacrylate) under conditions designed to simulate the worst-case scenario that would be experienced by a polymer used in a surface coating on a steel roof. Vinyl-terminated and saturated polymers were degraded for periods of up to 2 years under simulated solar radiation at a temperature of 95??C, temperature of 95 ??C in the dark, and simulated solar radiation at 35??C. Similar degradation mechanisms were observed under heat and UV radiation. The presence of UV radiation accelerated the degradation occurring at high temperature, and vice versa. The combination of heat and UV radiation is far more detrimental to the polymers than either of these conditions alone. Both vinyl-terminated and saturated pMMA degraded under UV radiation at 95??C, whereas under conditions of UV radiation alone or high temperature alone, the saturated polymer was found to be stable. The vinyl-terminated pMMA degrades in all cases via the formation of ethylene oXide-type end groups, which subsequently rearrange under the expulsion of formaldehyde and 2-oxo-propionic acid methyl ester. This is in contrast to all previous literature, in which pMMA degrades via depolymerisation and is stable at 95??C. Degradation of pBA included a degradation mechanism similar to that of pMMA in addition to other polymer fragments, some of which cannot be assigned. pBA (both saturated and vinyl-terminated) showed a tendency to crosslink under all degradation conditions in this study. Only saturated pHEMA was stable under thermal degradation. In all other cases, pHEMA showed some degradation, but displayed a much greater tendency to crosslink rapidly. Terminal vinyl bonds were shown in all cases to be a weak point in the polymer with respect to degradation. pMMA was found to be the least reactive of these polymers. pHEMA showed some small degradation but had a greater tendency to crosslink via the hydroxyethyl side groups. pBA does not have any such reactive groups, and its crosslinking reaction may be explained via the acrylate backbone, or the longer alkyl ester group.

Alzheimer’s disease (AD) is the most prevalent form of dementia affecting the elderly, and as the aging population increases the social and economic burden of AD grows substantially. Pathological hallmarks of AD include the accumulation of extracellular amyloid plaques and intracellular neurofibrillary tangles (NFTs), as well as significant neuron loss. Amyloid plaques consist of aggregated amyloid beta (Aβ) peptide, which is generated from the proteolytic processing of amyloid precursor protein (APP) in addition to several other peptides. While the processing of APP has been characterized, its primary physiological function and its involvement in AD pathology are poorly understood. Developing a greater understanding of the function of APP, and the molecular and cellular functions it is involved in or other proteins it is associated with, could provide insight into its role in AD pathology. To investigate the function of APP695, the neuronal isoform of APP, we used mass spectrometry to compare changes in protein expression and phosphorylation between APP-null B103 and APP695-expressing B103-695 rat neuroblastoma cells. Mass spectrometry-based proteomics has become a powerful technique for the unbiased identification of proteins from complex mixtures. Quantitative proteomics using labeling techniques, such as stable isotope labeling by amino acids in cell culture (SILAC), allow relative quantitation of multiple samples at once. More recently, with advances in mass spectrometer technology, label-free quantitation has become a reliable quantitative proteomics approach. Additionally, mass spectrometry can be used for the analysis of post-translational modifications, such as phosphorylation, a dynamic modification involved in the regulation of many cellular processes. Phosphoproteomics identifies site-specific phosphorylation and surrounding sequence information, which can be used for consensus motif analysis to provide further information about potential changes in kinase activity. Identifying changes in phosphorylation and kinase activity also provides information about signaling pathways and functions that may be affected by APP695 expression. Comprehensive proteomic and phosphoproteomic datasets can be used to gain insight into the molecular mechanisms that may be regulated by APP695 expression, or involved in AD progression and pathology, leading to the development of novel therapeutic and preventative strategies for AD. Proteomic and phosphoproteomic analysis of B103 and B103-695 cells identified several significant protein expression and phosphorylation changes that may be mediated by APP695-expression. Global-scale proteomic analysis identified increased expression of Ras and ƴ-synuclein in B103-695 cells, which was further validated in human AD brain tissue. Phosphoproteomic analysis showed increased phosphorylation of Histone H4 at Ser47, and led to the investigation of PCTAIRE-2 (Cdk17), and PCTAIRE-3 (Cdk18) expression, which were all shown to be increased in AD transgenic mouse tissue, culture primary rat neurons treated with Aβ, as well as mild cognitive impairment (MCI) and AD human brain tissue. Label-free quantitative proteomics was used for the analysis of human brain tissue from the cortex of individuals affected by AD, MCI, Parkinson’s disease (PD), and progressive supranuclear palsy (PSP) compared to cognitively normal, control samples. A number of differentially expressed proteins were identified in AD, MCI, PD, and PSP tissue. Bioinformatic analysis of the comprehensive proteomic datasets from AD, MCI, PD, and PSP human brain tissue identified several proteins consistent with corresponding disease pathology and neurodegeneration, such as inflammatory proteins. While some of the molecular and cellular functions were unique among neurodegenerative diseases, there also appears to be overlap of affected functions, suggesting there may be a more common mechanism of neurodegeneration.

In this thesis, the detection of global proteomics alteration and changes in neuropeptide distribution caused by neuropathic pain in rat spinal cord tissue was the main focus. Neuropathic pain (NP) is a major clinical syndrome caused by disease or dysfunction of the nervous system and often mediated by neuronal networks in the spinal cord. The estimated prevalence of NP is 6-8% in general population. Only in the United States, the indirect cost associated with chronic pain has been estimated to 100 billion dollars each year and NP substantially contributes to this cost. So far, the underlying mechanisms of NP are not well understood. Proteomics techniques are commonly used in biology system studies, due to its high throughput, capability of unbiased analysis and sensitivity. It builds up a bridge to link genes, peptides, proteins, and the disease. Two proteomic/peptidomic approaches were developed, evaluated and discussed in this thesis. Both of them were further applied in the studies of neuropathic pain. First approach is a quantitative proteomic approach using liquid chromatography combined with Fourier transform mass spectrometry (LC-FTMS), which is developed for quantitative analysis of proteins originated from small central nervous system (CNS) samples. This approach was successfully applied in the study of the rat spinal cord tissue proteome in a neuropathic pain model. Another approach is using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) for the visualization of the distribution of neuropeptides in rat spinal cord, which in the future will be applied in investigating the ongoing signal transmission under neuropathic pain conditions. Results provided by these two methods are of high importance for the general understanding of the underlying pathophysiological mechanisms and potential identification of new targets for novel treatment of neuropathic pain.

The last century has seen a steady increase in the extension of the average lifespan. This has concomitantly produced higher incidences of age-related chronic degenerative diseases like Alzheimer’s and Parkinson’s diseases. Age is the single greatest risk factor for the development of not just these degenerative conditions but cancer as well. The aged niche undergoes a number of maladaptive changes that allow underlying conditions to present and progress. Exactly which changes, contribute to the progression of which disease is currently an area of intense study. However, these answers often present therapeutic targets for disease prevention. Age is characterized by a progressive loss of tissue function that eventually leads to the death of the organism. At the cellular level, aged tissues are characterized by a loss of resident stem cell populations, senescence, and low-grade inflammation. While aging is heterogeneous in terms of its ultimate effect on tissue function the underlying changes have a degree of overlap. Cells often experience increased oxidative stress and a diminished activity in pathways like NRF2 whose role it is to provide resistance to such stress. Aged cells also have some change in their overall chromatin and nucleosome structure contributing to observable changes in gene expression and regulation. When these disruptions occur in tissues that can affect the larger organism such as the hypothalamus they affect the organism as a whole and contribute to syndromes seen in age such as insulin resistance. The immune system, in particular, is sensitive to both the cell-autonomous and systematic changes that occur with age. Immune and endocrine signaling pathways have a considerable amount of overlap, and mounting evidence points to the role of inflammation in the metabolic syndromes that occur with age. Immune dysfunction in the CNS has a special significance because of the dual roles of microglia. These cells exist not just to protect against foreign invasion but play vital roles in the maintenance of brain architecture and in processes central to cognition like long-term potentiation and the differentiation of stem cells in the hippocampus. The aged microglial phenotype contributes to the decline that occurs normally with age but can also be central to the progression of underlying pathologies including several degenerative diseases. Therapies targeting the maintenance of microglial function with age hold the potential to delay disease onset and possibly preserve cognitive function further into life. Top-down research approaches are well suited for the study of interactions between complex systems. The rapid improvement of mass spectrometry over the past decade has allowed researchers to examine more complex samples with fewer preparation steps and improved accuracy. This approach has thus far worked very well in the study of aging with the number of “Omics” techniques in aging models increasing rapidly. We use both label-free mass spectrometry and the more traditional real-time PCR to analyze signaling pathways and systems in both tissue homogenates and isolated cells from aged animals. By analyzing inflammatory and neurogenic pathways in animals treated with polyphenolic compounds we were able to postulate that the improved behavioral effect of these compounds is likely related to the decrease of pro-inflammatory cytokines and a restoration of WNT signaling. Proteomic analysis of aged microglia revealed widespread changes in chromatin structure and cellular machinery responsible for the regulation of transcription. In addition, we uncovered a shift in the underlying metabolic state of aged microglia and identified several pathways upstream of these changes. These upstream pathways included mTOR, a well-studied nutrient sensing pathway that plays a role in regulating microglial phenotype. Modulation of identified pathways through the use of both genetic (siRNA) and pharmacological (allosteric inhibitor) was able to recapitulate the aged phenotype in normal cells, confirming the role of these pathways in pathological changes.

A molecular hydrogen laser, with an output of 7.8 eV photons in the vacuum ultraviolet, is evaluated as a selective source for photoionization mass spectrometry. Types of compounds ionized by the laser include a variety of amines, nitrogen heterocycles, drugs of abuse, pharmaceuticals, and polynuclear aromatic hydrocarbons (PAHS). The laser is coupled to a time-of-flight mass analyzer, which allows a spectrum to be recorded with each laser pulse. The laser is a "soft" ionization source and mass spectra of nearly all of the compounds studied yield single ion peaks due to the parent molecule with no fragments. This results in simplified mass spectra with a one-to-one correspondence of photoactive molecules with molecular ion peaks. Since the photoionization threshold of the laser is relatively low, selectivity of the photoactive species is high in the presence of a complex sample matrix. The performance of the laser source is improved with a few changes in the original design. In addition, the previous method of recording mass spectra with a photographic emulsion is replaced with a digital oscilloscope, which averages spectra over many laser pulses. As a result, a true assessment of the technique's sensitivity is finally achieved. The time-of-flight mass spectrometer is modified with a new microchannel plate ion detector and preamplifier. As a result, detection limits for PAH's improve by nearly three orders of magnitude, from the 100 ng range to the 100 pg range. Selectivity of the laser photoionization source in complex mixture analysis is demonstrated with the ability to detect PAH's in a drinking water sample at concentrations below 100 parts-per-trillion, using a simple solid-phase extraction technique. Application of the technique for rapid screening of drugs of abuse in urine is demonstrated where solid-phase extraction columns are utilized for sample pretreatment. Urine samples spiked with drugs such as cocaine, codeine, morphine, phencyclidine, and methadone, yield photoionization mass spectra consisting of parent molecular ions for the drugs with a few noninterfering ion signals from the matrix. The technique is evaluated and compared to other drug screening techniques such as enzyme-multiplied immunoassay.

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 [1]. 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 [4]. 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 [5]. Individuals with high fasting levels of plasma free fatty acids (FFAs) have an elevated risk of developing T2DM [6]. In fact, prolonged exposure to FFAs impairs insulin secretion in vivo and in vitro [7,8] inducing β cells death [9]. 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 [10], the induction of ER stress in β cells [11,12], the production of reactive oxygen species (ROS) [13], and ceramides [14] 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 [16], impairment of ATP production [16] and damage of mitochondrial DNA [17] 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 [18] and proteomic analysis [19]. Very recently, Cnop et al. [18], 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. [20], 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 [21]. 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 [22]. In clinical applications and diagnostics MALDI-MSI has been used to analyse a large variety of analyte classes, such as xenobiotics [23], metabolites [24], lipids [25,26], N-linked glycans [27,28], peptides [29], 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 [42]. 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 [43], 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 [47]. 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. [48] 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 [49]. 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) [52], 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 [56]. 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 [56]. 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 [57] 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. [42] in the study on on-tissue reduction of insulin, used OCT embedded pancreas tissues. Seeley et al. [58] 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”. However, most of the studies principally showed how the PEG contamination in the spectra is reduced after removing OCT compound with suitable washing steps [34,59], but any of them showed the comparison between data from OCT-embedded and non-embedded tissues after the application of the same sample preparation procedure. On this basis, here an in-depth comparison between mass spectrometry imaging data obtained from OCT-embedded and non-embedded tissues was performed. The optimized methods were applied to a small set of human pancreas samples (3x T2DM and 3x control), so that small endocrine compartments (islets of Langerhans) may be analysed in control and pathological tissues. In particular, human pancreas samples were collected from the same individual both OCT-embedded and non-embedded. References [1] Gittes, G., Developmental biology of the pancreas: a comprehensive review. Developmental biology 2009, 326, 4–35. 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[11] Laybutt, Preston, Åkerfeldt, Kench, et al., Endoplasmic reticulum stress contributes to beta cell apoptosis in type 2 diabetes. Diabetologia 2007, 50, 752–763. [12] Cunha, D., Hekerman, P., Ladrière, L., Bazarra-Castro, A., et al., Initiation and execution of lipotoxic ER stress in pancreatic β-cells. Journal of Cell Science 2008, 121, 2308–2318. [13] Carlsson, Borg, H., Welsh, Sodium Palmitate Induces Partial Mitochondrial Uncoupling and Reactive Oxygen Species in Rat Pancreatic Islets in Vitro 1 1999. [14] Shimabukuro, M., Higa, M., Zhou, Y.-T., Wang, M.-Y., et al., Lipoapoptosis in Beta-cells of Obese Prediabeticfa/fa Rats ROLE OF SERINE PALMITOYLTRANSFERASE OVEREXPRESSION. Journal of Biological Chemistry 1998, 273, 32487–32490. [15] Lowell, B., Shulman, G., Mitochondrial Dysfunction and Type 2 Diabetes. Science 2005, 307, 384–387. [16] Li, N., Frigerio, F., Maechler, P., The sensitivity of pancreatic β-cells to mitochondrial injuries triggered by lipotoxicity and oxidative stress. Biochemical Society Transactions 2008, 36, 930–934. [17] Chen, X., Wang, X., Kaufman, B.A., Butow, R.A., Aconitase Couples Metabolic Regulation to Mitochondrial DNA Maintenance. Science 2005, 307, 714–717. [18] Cnop, M., Abdulkarim, B., Bottu, G., Cunha, D., et al., RNA Sequencing Identifies Dysregulation of the Human Pancreatic Islet Transcriptome by the Saturated Fatty Acid Palmitate. Nestle Nutr Works Se 2014, 63, 1978–1993. [19] Maris, M., Robert, S., Waelkens, E., Derua, R., et al., Role of the saturated nonesterified fatty acid palmitate in beta cell dysfunction. Journal of proteome research 2012, 12, 347–62. [20] Brun, He, K., Lupi, Boehm, The diabetes-linked transcription factor Pax4 is expressed in human pancreatic islets and is activated by mitogens and GLP-1 2008. 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