Lychnophora (Vernonieae: Asteraceae) é um gênero micro-endêmico dos \"campus rupestris\" do bioma Cerrado. Os extratos foliares de Lychnophora ericoides (\"Arnica-da-serra\") são usados na terapêutica popular principalmente como analgésico. Seus óleos essenciais são quimicamente ricos em sesquiterpenos. Tais compostos, principalmente os de esqueletos bisabolano e cadinano são produtos biossintéticos do precursor cátion nerolidila. Óleos essenciais de folhas de L. ericoides são bioativos frente a invertebrados do Táxon Acari. Um de seus componentes majoritários, o orto-acetoxi bisabolol, é antinociceptivo em ensaios in vitro. Terpenos são valorizados pela indústria de química fina, haja vista sucessos como Taxol® e Acheflan®. Nossos objetivos em Fitoquímica e Ciências Farmacêuticas se alinham a esse interesse. As terpeno-sintases (TerpS) envolvidas na biossínteses dos compostos são ainda alvo de pesquisas com em Bioquímica. Sua identificação é frequentemente conclusiva em diversos aspectos. Como exemplo, citamos a compreensão dos mecanismos da variabilidade metabólica temporal em espécies nativas e selvagens e os avanços na bioengenharia de Produtos Naturais (PN). Frente a este cenário de fronteira este manuscrito traz um resumo da investigação que objetivou determinar se há diversas isoformas de TerpS operando na produção temporalmente variável de sesquiterpenos bioativos em tecidos foliares de Lychnophora ericoides ou se não as há. Acessamos diferentes amostras selvagens de L. ericoides in situ. Nossos esforços em responder a questão supracitada foram divididos em tarefas, a seguir: (1) Determinação da fração rica em sesquiterpenos inspirada na \"Química Analítica Verde\"; (2) MALDI imaging das proteínas das folhas; (3) Estratégia \"omics\" combinada na identificação e clonagem das TerpS. Visitamos campos de ocorrência (CampO) georreferenciados bem como desconhecidos. O desenvolvimento de método de quantificação por Headspace-SPME permitiu uma comparação rápida e livre de solventes de amostras vegetais mínimas (10 mg). A separação e identificação foi conduzida por Gas Chromatography-Mass Spectrometry (GC-MS). As amostras de Diamantina, CampO mais Boreal de todos, apresentam a maior quantidade de derivados bisabolano. A hidrodestilação do material vegetal excedente ao HS-SPME forneceu óleos essenciais para o isolamento de moléculas. Dados espectrais diversos fundamentam a descrição de um derivado cadinano, o 11-dehidro cadinol, entre outros. O MALDI imaging determinou como exequível a geração de imagens moleculares em folhas de L. ericoides. Foram geradas imagens subepidermais dos íons com m/z que se assemelham a cadinano e bisabolano synthases (CadS and BS). A limitação do emprego da técnica é a determinação previa da massa nominal da proteína nativa. Polyacrylamide Gel Electrophoresis (1D-PAGE) gerou mapas de proteínas separadas por tamanho (A, B, C, e D) (30KDa> m/z <80KDa). A digestão com tripsina das bandas geraram peptídeos para análise em MS. Íons resultantes alimentaram o algorítimo MASCOT. Uma isoforma de germacrano-sintase foi identificada. Prospecção de genes codificadores com o cDNA (Ubiquitina +) determinou uma BS, de comprimento 1600pb, amplificada com primers de design para genes de Helianthus spp. A BS de L. ericoides foi clonada. Concluindo, destacamos dois pontos. (1) O controle enzimático na produção dos sesquiterpenos poderá enfim ser averiguada a nível do transcriptoma. (2) A busca pela produção biotecnológica de PN sofreu um pequeno incremento. Esperamos ter contribuído humildemente na produção sustentável de PN e por conseguinte na preservação da biodiversidade. / Lychnophora (Vernonieae: Asteraceae) is micro endemic to \"campus rupestris\" from Brazilian \"Cerrado\". Leaves extracts of Lychnophora ericoides (\"Arnica-da-serra\") are used as folk medicine and mainly as wound healer. Its essential oils were chemically profiled as sesquiterpene-rich. Such sesquiterpenes, both bisabolene-like and cadinane-like carbon skeletons, are derivatives of the nerolidyl cation. L. ericoides leaves essential oils are bioactive against invertebrate Acari. An anti-hypernociceptive ability of its component orto-acetoxy bisabolol was also displayed in vitro. Terpenes are valuable in fine chemistry industry, e.g. Taxol® and Acheflan®. Our phytochemical and pharmaceutical goals are aligned to such an interest. Terpene synthases (TerpS) behind their biosynthesis are target of researches in plant sciences and biochemistry. Identification of TerpS often led to conclusions with diverse impacts. Fundamental concepts on time-dependent shifts of terpene productions in wild type species and advances towards plant metabolites bioengineering are examples. Facing such a frontier field we share here an abstract for the investigation aimed to determine whether there were many isoforms of sesquiterpene synthases operating at leaves tissues in Lychnophora ericoides shift-able production of bioactive sesquiterpenes or whether there were not. We have accessed different in situ samples of L. ericoides. Efforts to answer the above question were sectioned as follows: (1) \"Green-Analytical-Chemistry\" oriented profiling of sesquiterpene-rich fraction; (2) MALDI imaging of proteins in leaves; (3) Combined \"omics\" approaches towards identifying TerpS and gene cloning. We visited both known and novel L. ericoides sites of occurrence (SoO). The development of a quantitative Headspace-Solid Phase Micro Extraction (HS-SPME) method enabled a rapid and solvent-free comparison of minimized samples (10mg). Separation and identification were carried out using Gas Chromatography-Mass Spectrometry (GC-MS). Samples harvested in Diamantina, Northern Most SoO, accumulate the highest amount of bisabolene-like derivatives. Hydrodistillation of leftovers material from HS-SPME yield essential oils used to purify unknown compounds. Based on a diverse spectra collection we report a novel cadinane-like derivative, one 11dehydro Cadinol. MALDI imaging has been determined as suitable for imaging proteins in L. ericoides. In a prospective fashion we generate sub-epidermal images of ions within the m/z frame comprising reported isoforms of both cadinane and bisabolane synthases (CadS and BS). The limitation to its use is the awareness of molecular weight of targeted native proteins. Polyacrylamide Gel Electrophoresis (1D-PAGE) protein mapping determined broad bands of protein distribution in different mass ranges (A, B, C, and D) (30KDa> m/z <80KDa). Bands tryptic digestion, followed by sample clean up, generated peptide pools feasible for MS. The output ions data feed MASCOT algorithm. A germacrane synthase could have been identified. When prospecting encoding genes, viable cDNA (Ubiquitin +) was used. A BS, lengthened 1600bp, was amplified with BS primers designed for Helianthus spp. genes. BS presented in L. ericoides was successfully cloned. In conclusion we headline two topics. (1) Hypothetical enzymatic control of the sesquiterpenes production can now be further investigated at transcriptome level. (2) The seek of a platform that guarantee natural products production in a controlled system has been moved forward. Future production of valued compounds can slightly rely in our humble contribution to support biodiversity conservation.

Lychnophora (Vernonieae: Asteraceae) é um gênero micro-endêmico dos \"campus rupestris\" do bioma Cerrado. Os extratos foliares de Lychnophora ericoides (\"Arnica-da-serra\") são usados na terapêutica popular principalmente como analgésico. Seus óleos essenciais são quimicamente ricos em sesquiterpenos. Tais compostos, principalmente os de esqueletos bisabolano e cadinano são produtos biossintéticos do precursor cátion nerolidila. Óleos essenciais de folhas de L. ericoides são bioativos frente a invertebrados do Táxon Acari. Um de seus componentes majoritários, o orto-acetoxi bisabolol, é antinociceptivo em ensaios in vitro. Terpenos são valorizados pela indústria de química fina, haja vista sucessos como Taxol® e Acheflan®. Nossos objetivos em Fitoquímica e Ciências Farmacêuticas se alinham a esse interesse. As terpeno-sintases (TerpS) envolvidas na biossínteses dos compostos são ainda alvo de pesquisas com em Bioquímica. Sua identificação é frequentemente conclusiva em diversos aspectos. Como exemplo, citamos a compreensão dos mecanismos da variabilidade metabólica temporal em espécies nativas e selvagens e os avanços na bioengenharia de Produtos Naturais (PN). Frente a este cenário de fronteira este manuscrito traz um resumo da investigação que objetivou determinar se há diversas isoformas de TerpS operando na produção temporalmente variável de sesquiterpenos bioativos em tecidos foliares de Lychnophora ericoides ou se não as há. Acessamos diferentes amostras selvagens de L. ericoides in situ. Nossos esforços em responder a questão supracitada foram divididos em tarefas, a seguir: (1) Determinação da fração rica em sesquiterpenos inspirada na \"Química Analítica Verde\"; (2) MALDI imaging das proteínas das folhas; (3) Estratégia \"omics\" combinada na identificação e clonagem das TerpS. Visitamos campos de ocorrência (CampO) georreferenciados bem como desconhecidos. O desenvolvimento de método de quantificação por Headspace-SPME permitiu uma comparação rápida e livre de solventes de amostras vegetais mínimas (10 mg). A separação e identificação foi conduzida por Gas Chromatography-Mass Spectrometry (GC-MS). As amostras de Diamantina, CampO mais Boreal de todos, apresentam a maior quantidade de derivados bisabolano. A hidrodestilação do material vegetal excedente ao HS-SPME forneceu óleos essenciais para o isolamento de moléculas. Dados espectrais diversos fundamentam a descrição de um derivado cadinano, o 11-dehidro cadinol, entre outros. O MALDI imaging determinou como exequível a geração de imagens moleculares em folhas de L. ericoides. Foram geradas imagens subepidermais dos íons com m/z que se assemelham a cadinano e bisabolano synthases (CadS and BS). A limitação do emprego da técnica é a determinação previa da massa nominal da proteína nativa. Polyacrylamide Gel Electrophoresis (1D-PAGE) gerou mapas de proteínas separadas por tamanho (A, B, C, e D) (30KDa> m/z <80KDa). A digestão com tripsina das bandas geraram peptídeos para análise em MS. Íons resultantes alimentaram o algorítimo MASCOT. Uma isoforma de germacrano-sintase foi identificada. Prospecção de genes codificadores com o cDNA (Ubiquitina +) determinou uma BS, de comprimento 1600pb, amplificada com primers de design para genes de Helianthus spp. A BS de L. ericoides foi clonada. Concluindo, destacamos dois pontos. (1) O controle enzimático na produção dos sesquiterpenos poderá enfim ser averiguada a nível do transcriptoma. (2) A busca pela produção biotecnológica de PN sofreu um pequeno incremento. Esperamos ter contribuído humildemente na produção sustentável de PN e por conseguinte na preservação da biodiversidade. / Lychnophora (Vernonieae: Asteraceae) is micro endemic to \"campus rupestris\" from Brazilian \"Cerrado\". Leaves extracts of Lychnophora ericoides (\"Arnica-da-serra\") are used as folk medicine and mainly as wound healer. Its essential oils were chemically profiled as sesquiterpene-rich. Such sesquiterpenes, both bisabolene-like and cadinane-like carbon skeletons, are derivatives of the nerolidyl cation. L. ericoides leaves essential oils are bioactive against invertebrate Acari. An anti-hypernociceptive ability of its component orto-acetoxy bisabolol was also displayed in vitro. Terpenes are valuable in fine chemistry industry, e.g. Taxol® and Acheflan®. Our phytochemical and pharmaceutical goals are aligned to such an interest. Terpene synthases (TerpS) behind their biosynthesis are target of researches in plant sciences and biochemistry. Identification of TerpS often led to conclusions with diverse impacts. Fundamental concepts on time-dependent shifts of terpene productions in wild type species and advances towards plant metabolites bioengineering are examples. Facing such a frontier field we share here an abstract for the investigation aimed to determine whether there were many isoforms of sesquiterpene synthases operating at leaves tissues in Lychnophora ericoides shift-able production of bioactive sesquiterpenes or whether there were not. We have accessed different in situ samples of L. ericoides. Efforts to answer the above question were sectioned as follows: (1) \"Green-Analytical-Chemistry\" oriented profiling of sesquiterpene-rich fraction; (2) MALDI imaging of proteins in leaves; (3) Combined \"omics\" approaches towards identifying TerpS and gene cloning. We visited both known and novel L. ericoides sites of occurrence (SoO). The development of a quantitative Headspace-Solid Phase Micro Extraction (HS-SPME) method enabled a rapid and solvent-free comparison of minimized samples (10mg). Separation and identification were carried out using Gas Chromatography-Mass Spectrometry (GC-MS). Samples harvested in Diamantina, Northern Most SoO, accumulate the highest amount of bisabolene-like derivatives. Hydrodistillation of leftovers material from HS-SPME yield essential oils used to purify unknown compounds. Based on a diverse spectra collection we report a novel cadinane-like derivative, one 11dehydro Cadinol. MALDI imaging has been determined as suitable for imaging proteins in L. ericoides. In a prospective fashion we generate sub-epidermal images of ions within the m/z frame comprising reported isoforms of both cadinane and bisabolane synthases (CadS and BS). The limitation to its use is the awareness of molecular weight of targeted native proteins. Polyacrylamide Gel Electrophoresis (1D-PAGE) protein mapping determined broad bands of protein distribution in different mass ranges (A, B, C, and D) (30KDa> m/z <80KDa). Bands tryptic digestion, followed by sample clean up, generated peptide pools feasible for MS. The output ions data feed MASCOT algorithm. A germacrane synthase could have been identified. When prospecting encoding genes, viable cDNA (Ubiquitin +) was used. A BS, lengthened 1600bp, was amplified with BS primers designed for Helianthus spp. genes. BS presented in L. ericoides was successfully cloned. In conclusion we headline two topics. (1) Hypothetical enzymatic control of the sesquiterpenes production can now be further investigated at transcriptome level. (2) The seek of a platform that guarantee natural products production in a controlled system has been moved forward. Future production of valued compounds can slightly rely in our humble contribution to support biodiversity conservation.

MALDI imaging allows the creation of a molecular image of a tissue slice. This image is reconstructed from the ion abundances in spectra that are obtained while rastering the laser over the tissue. These images can then be correlated with tissue histology to detect potential biomarkers of, for example, aberrant cell types. MALDI is known to have problems with ion suppression, making it difficult to correlate measured ion abundance with concentration. It would be advantageous to have a method that can provide more accurate protein concentration measurements, particularly for screening applications or for precise comparisons between samples. My hypothesis was that a method based on multiple reaction monitoring (MRM) with isotopically-labelled internal standards can be developed which would allow the accurate quantitation of proteins in MALDI Imaging. This study reports on the development of this novel MALDI Imaging method for the localization and accurate quantitation of proteins in tissues. This method involves optimization of in-situ tryptic digestion, followed by reproducible and uniform deposition of an isotopically-labelled standard peptide from a target protein onto the tissue, using an aerosol-generating device. Data is acquired by MALDI-MRM-MS and accurate peptide quantitation is determined from the ratio of MRM transitions for the endogenous unlabelled proteolytic peptides to the corresponding transitions from the applied isotopically-labelled standard peptides. In a parallel experiment, the quantity of the labelled peptide applied to the tissue was determined using a standard curve generated from MALDI-TOF-MS data. This external calibration curve was then used to extrapolate the quantity of endogenous peptide in a given area. All standard curves generated by this method had coefficients of determination greater than 0.97. These proof-of-concept experiments using MALDI MRM-based imaging show the feasibility of obtaining precise and accurate quantitation of tissue protein concentrations over two orders of magnitude, while maintaining the spatial localization information for the proteins. / Graduate

Regulatorische Foxp3+CD4+ T-Zellen sind essentiell für das Gleichgewicht des intestinalen Immunsystems. Eine Einschränkung ihrer Suppressionsfunktion wird bei Patienten mit Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX)-Syndrom beobachtet und führt im Tiermodell zu lymphoproliferativen Erkrankungen und intestinalen Entzündungen. Von entscheidender Bedeutung für Homöostase und Suppressionsfunktion regulatorischer T-Zellen ist das Signalmolekül Interleukin-2 (IL-2). Im Gegensatz zu Effektor-T-Zellen exprimieren Foxp3+CD4+ T-Zellen den hochaffinen IL-2-Rezeptor αβγ konstitutiv. IL-2 wird von regulatorischen T-Zellen nicht in relevanten Mengen exprimiert. Sie sind somit auf von anderen Zellen sezerniertes IL-2 angewiesen. In der vorliegenden Arbeit wird gezeigt, dass im Tiermodell regulatorische Foxp3+CD4+ T-Zellen durch Applikation eines IL-2/anti-IL-2-Antikörperkomplex nicht nur in mesenterialen Lymphknoten und Milz, sondern auch lokal in der Lamina propria mucosae des Kolons der Versuchstiere expandiert werden. Als relevante Quelle von IL-2 in situ könnten aktivierte proliferierende T-Zellen dienen. Um dies näher zu untersuchen, wurde die Proteinexpression proliferierender Einzelzellen mittels Matrix assisted laser desorption/ionisation-Time of flight-Massenspektrometrie-Imaging (MALDI-Imaging) analysiert. Es gelang die Identifikation präferentiell in lymphoiden Geweben exprimierter Peptidmassen. Obwohl die Einzelzellanalyse mittels MALDI-Imaging prinzipiell möglich erscheint, ist ein Nachweis von Zytokinen wie IL-2 derzeit aufgrund fehlender Sensitivität im Proteinmassebereich zwischen 10kDa und 20kDa nicht möglich. Die therapeutischen Möglichkeiten der Expansion regulatorischer Foxp3+ T-Zellen durch stabile IL-2-Rezeptor-Agonisten und die Rolle von IL-2 für die intestinale Immunregulation sollten weiter untersucht werden.

Biological functions within cells and organisms are mainly carried out by the translational products; proteins and peptides. The analysis and characterization of these biomolecules are of great importance for the progress in disease research and biomarker and drug discovery. The term peptidomics was introduced to describe the comprehensive analysis of peptides (e.g. neuropeptides) in biological tissues. In this thesis, a peptidomics approach using nanoflow liquid chromatography coupled to electrospray mass spectrometry (MS) has been developed for detection, identification, and quantification of neuropeptides in different disease models. A thoroughly controlled sample preparation technique and targeted neuropeptide sequence collections have been used to improve sample quality and to increase the number of identified neuropeptides. In particular, neuropeptide changes in experimental models of Parkinson’s disease (PD), with or without L-DOPA treatment, and the effect of antidepressant treatment on neuropeptide expression have been investigated. Several novel, potentially bioactive, neuropeptides have been identified and a number of peptides derived from precursors such as secretogranin-1, preproenkephalin-B, and somatostatin have been found differentially expressed. Some of them represent novel findings, not previously associated with PD or treatment with antidepressants. In addition, MALDI imaging MS (IMS), a technology that permits detection and spatial distribution determination of endogenous compounds and/or administered drugs directly on tissue sections, has been used in both small protein and drug applications. MALDI IMS on tissue samples from experimental models of PD revealed differential expression patterns of two small proteins involved in calcium regulation, PEP-19 and FKBP-12. Biomolecular interaction analysis was performed on FKBP-12 using surface plasmon resonance together with MS and several potential binding partners were identified. In a second approach, MALDI IMS was used to study the distribution of the anticholinergic bronchodilator tiotropium in rat lung following inhalation of the drug. The distribution of the drug was monitored in both MS and MS/MS mode and the levels where linearly quantifiable in the range of 80 fmol – 5 pmol. Conclusively, in this thesis mass spectrometry based technologies have successfully been developed to detect, identify, and characterize small proteins, peptides, and drugs in various tissue samples.

L'imagerie par spectrométrie de masse MALDI est une technique jeune dotée d'un très<br />fort potentiel ; elle est capable de nous donner la localisation de près d'une centaine de<br />composés sur un tissu en une seule analyse.Appliquée dans le domaine de la protéomique ou du biomédical, cette dernière a déjà permis de repérer de nombreux biomarqueurs régulés dans des cas de cancers ou dans de nombreuses autres pathologies. D'autres applications comme l'étude de médicaments ou de lipides ont également été décrites avec succès en utilisant l'imagerie ou le « profiling » c'est à-dire l'acquisition directement sur le tissu par le spectromètre de masse MALDI. Cependant comme toute technologie récente, il est nécessaire de repousser ses limites pour tenter d'améliorer certains points et de valoriser cette stratégie.<br />Le but de mon travail a été de développer de nouveaux systèmes pour l'imagerie<br />MALDI, notamment en ce qui concerne le choix de la matrice à utiliser, ou le traitement de<br />l'echantillon en vue d'améliorer la qualité de l'acquisition. La possibilité d'analyser des tissus<br />fixés et paraffinés conservés dans les bibliothèques des anatomopathologistes a également été<br />étudiée, permettant d'augmenter considérablement le nombre d'échantillons analysables.<br />De plus, afin d'étendre le type de molécule pouvant être localisé en imagerie MALDI,<br />nous avons développé un nouveau concept appelé Tagmass, nous permettant de réaliser des<br />images d'acides nucléiques ou de protéines de très hautes masses moléculaires. Ce type de<br />localisation restait inaccessible par cette technique jusqu'à présent.<br />Enfin, dans le cadre de l'étude du cancer de l'ovaire, nous avons utilisé l'imagerie et le<br />profiling sur tissu afin de rechercher des biomarqueurs potentiels de cette maladie. Ce type<br />d'information peut s'avérer crucial pour un éventuel dépistage précoce ou pour une meilleure<br />compréhension des processus de la maladie.

A l'heure de la protéomique, la spectrométrie de masse s'est révélée un outil puissant<br />pour la recherche et l'identification des biomolécules à partir d'échantillons purifiés. Une<br />nouvelle ère s'ouvre, avec l'imagerie MALDI, permettant en plus la localisation de<br />biomolécules telles que les peptides, les protéines ou les lipides au sein des tissus. Des<br />développements cruciaux restent encore à réaliser pour améliorer les performances de cette<br />technologie. Dans ce contexte, nous nous sommes tout d'abord intéressés à la mise au point<br />de nouveaux protocoles adaptés à l'analyse directe et l'imagerie par spectromètrie de masse<br />de petits organismes en particulier la sangsue Hirudo medicinalis. Ce modèle est<br />particulièrement intéressant du point de vue des phénomènes de régénération nerveuse et nous<br />avons débuté des études sur les lipides pouvant y être impliqués. Le deuxième point abordé<br />est l'étude des apports de la métallisation pour la spectromètrie de masse. Tout d'abord un<br />dépôt métallique sur des lames histologiques permet à la fois une corrélation des informations<br />morphologiques obtenues en microscopie optique avec les images moléculaires d'IMS. La<br />métallisation de l'échantillon quand à elle, a permis de supprimer les décalages de pics vers<br />les plus hauts rapports m/z, d'obtenir des spectres MALDI de meilleures qualités et grâce à<br />une reproductibilité plus importante entre 2 spectres, de produire des images MALDI de plus<br />grandes qualités. Enfin, une partie des développements a été dédiée à la possibilité<br />d'améliorer la résolution de l'image grâce à l'utilisation d'un système permettant de diminuer<br />la zone accessible au laser

The human brain (lat. cerebrum) is the most complex and heterogeneous organ in the human body. It is involved in a great number of body functions like movement, touch sensing, vision, hearing, smelling, hormone regulation and many more. In no other organ, the molecular communication mechanisms between different cells are so poorly understood. Due to the extensive diversity of processes that are controlled by the brain, diseases and injuries of the nervous system affect the human body significantly. Because of the immense complexity of the brain, the molecular mechanisms underlying the pathology of the diseases remain largely unknown. Hence, there is an urgent need for the development of new analytical strategies in order to investigate these conditions on a molecular level. Here, a central focus lies in the study of protein and peptide expression profiles, which can provide an insight in ongoing molecular mechanisms underlying the pathophysiology of the diseases. A powerful approach for studying proteins and peptide dynamics is mass spectrometry based proteomics, which is defined as the comprehensive study of all proteins expressed in a biological matrix at a certain point of time. The central objective of this thesis was to develop and employ different mass spectrometric techniques to study protein and peptide dynamics in the central nervous system in different neurological diseases. The individual studies comprise different aspects of proteome research. The first two studies included clinical proteomic applications for investigating protein dynamics in traumatic brain injury and amyotrophic lateral sclerosis. A further study was focused on method development for MS analysis of intact neural cells. The final three projects described in this thesis comprised MS based protein and peptide imaging in brain and spinal cord tissue samples. Here, the aim was to elucidate topological changes in protein expression in ALS as well as neuropeptide alterations in distinct brain structures in L-DOPA induced dyskinesia (LID) in Parkinson’s disease. / Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 713

Matrix assisted laser desorption/ionization mass spectrometry (MALDI MS) and the corresponding visualization technique MALDI MS Imaging (MSI) have emerged as important analytical tools in biochemical sciences, e.g., for drug development or to trace the metabolomic changes in cancerous tissues. Initially developed for the detection of high molecular weight compounds (HMWC; M > 1000 Da), in recent years the reliable and reproducible detection of low molecular weight compounds (LMWC; M < 1000 Da) has gained high attention, e.g., in the research fields of metabolomics and lipidomics. By using a protective matrix, the MALDI technique is capable of soft ionization of analytes to prevent their fragmentation or degradation. This matrix is responsible for the spatial separation of the analyte molecules, their protection from the strong laser shots, and their ionization. Commonly used matrices are small organic matrices (SOMs; M < 500 Da), which are utilized in HMWC analytics and recently also in LMWC analytics since they show sufficient absorption of the laser radiation, high crystallinity, and good ionization efficiency. However, their utilization can cause several drawbacks: (i) High background interferences below m/z = 1000 (not MALDI silent), which is disadvantageous specifically for LMWC analytics; (ii) low vacuum stability, which is especially problematic for standard instruments operated under high vacuum (HV); (iii) challenging homogeneous thin-layer coating, potentially causing inconsistent measurement conditions; and (iv) usually no suitability for dual polarity mode experiments, i.e., carrying out positive and negative mode measurements with the same matrix. Polymeric materials are promising candidates for MALDI silent matrices, as the large variety of possible molecular layouts potentially allows to meet all prerequisites of a MALDI matrix: (a) Sufficient ultra-violet (UV) laser radiation absorption, implemented by introducing conjugated π-electron systems in the polymer backbone or side chains; (b) high ionization efficiency, enhanced by adding acidic and/or basic functional groups to the polymer’s molecular structure, potentially also allowing dual polarity mode measurements; (c) MALDI silence, enabled by the high molar mass of the polymer chains; (d) high vacuum stability, also granted by the polymer’s molar mass; and (e) homogeneous thin-films, achieved by multiple available coating methods. Yet, despite their high potential only a handful of polymeric matrices were reported in literature and so far, investigations to develop conscious design strategies are missing. The target of this thesis is to contribute to the field of MALDI silent matrices by developing and investigating different polymers as macromolecular MALDI MS and MSI matrices for LMWC analytics. Therefore, two different strategies were explored: (i) Investigating conjugated polymers, and (ii) polymerizing SOMs. For the first strategy, five conjugated polymers were tested as MALDI matrices for the detection of various LMWCs. Among these, four were found to be excellent matrices, with sufficient ionization efficiencies and rare dual polarity mode suitability and allowed LMWC detection with low background interferences (MALDI silent). A high crystallinity of the matrix (SOM) is reported to be crucial to ensure successful measurements, yet conjugated polymer matrices (CPMs) are semi-crystalline, i.e., they contain crystalline and amorphous domains. Hence, the analytes are expected to be incorporated in the crystalline domains of the CPMs, depending on their degree of crystallization. Therefore, two amorphous CPMs were synthesized and tested, showing similar matrix performances (e.g., ionization efficiencies, dual polarity mode, MALDI silence) as a structurally related semi-crystalline CPM. This indicates that the analytes are incorporated in the amorphous parts of the CPM. The second strategy towards polymeric matrices (PMs) is the polymerization of standard SOMs. As the matrix performance of the corresponding SOMs is known, the performance of the respective polymerized SOMs (P(SOMs)) can be validated against this benchmark. At the same time, polymerization can induce the properties needed to enable efficient LMWC analytics. Therefore, two standard SOMs were modified and polymerized, resulting in P(SOMs), which were vacuum stable and MALDI silent, and showed similar optical properties, analyte scopes and ionization efficiencies in benchmark tests with their respective SOMs. For the fast and facile comparison of the matrix performances of PMs and standard matrices, the graphing software OriginPro was used to visualize, process, and evaluate the acquired mass spectra. To automatize these tasks, a script was programmed using the OriginPro-native programming languages LabTalk and OriginC: X Functions.

Regulatorische Foxp3+CD4+ T-Zellen sind essentiell für das Gleichgewicht des intestinalen Immunsystems. Eine Einschränkung ihrer Suppressionsfunktion wird bei Patienten mit Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX)-Syndrom beobachtet und führt im Tiermodell zu lymphoproliferativen Erkrankungen und intestinalen Entzündungen. Von entscheidender Bedeutung für Homöostase und Suppressionsfunktion regulatorischer T-Zellen ist das Signalmolekül Interleukin-2 (IL-2). Im Gegensatz zu Effektor-T-Zellen exprimieren Foxp3+CD4+ T-Zellen den hochaffinen IL-2-Rezeptor αβγ konstitutiv. IL-2 wird von regulatorischen T-Zellen nicht in relevanten Mengen exprimiert. Sie sind somit auf von anderen Zellen sezerniertes IL-2 angewiesen. In der vorliegenden Arbeit wird gezeigt, dass im Tiermodell regulatorische Foxp3+CD4+ T-Zellen durch Applikation eines IL-2/anti-IL-2-Antikörperkomplex nicht nur in mesenterialen Lymphknoten und Milz, sondern auch lokal in der Lamina propria mucosae des Kolons der Versuchstiere expandiert werden. Als relevante Quelle von IL-2 in situ könnten aktivierte proliferierende T-Zellen dienen. Um dies näher zu untersuchen, wurde die Proteinexpression proliferierender Einzelzellen mittels Matrix assisted laser desorption/ionisation-Time of flight-Massenspektrometrie-Imaging (MALDI-Imaging) analysiert. Es gelang die Identifikation präferentiell in lymphoiden Geweben exprimierter Peptidmassen. Obwohl die Einzelzellanalyse mittels MALDI-Imaging prinzipiell möglich erscheint, ist ein Nachweis von Zytokinen wie IL-2 derzeit aufgrund fehlender Sensitivität im Proteinmassebereich zwischen 10kDa und 20kDa nicht möglich. Die therapeutischen Möglichkeiten der Expansion regulatorischer Foxp3+ T-Zellen durch stabile IL-2-Rezeptor-Agonisten und die Rolle von IL-2 für die intestinale Immunregulation sollten weiter untersucht werden.:Bibliographische Beschreibung 3 Inhaltsverzeichnis 4 Abkürzungsverzeichnis 7 1. Einleitung 9 1.1. Störung der Barrierefunktion des intestinalen Immunsystems als Ursache chronisch entzündlicher Darmerkrankungen 9 1.2. Foxp3+ regulatorische T-Zellen 10 1.3. Die Rolle von Interleukin-2 für regulatorische T-Zellen 11 1.4. Signaltransduktion in regulatorischen T-Zellen als Grundlage ihrer selektiven Expansion und Induktion 12 1.5. Möglichkeiten der präferentiellen Expansion regulatorischer T-Zellen 15 1.5.1. Expansion regulatorischer T-Zellen durch Agonisten des hochaffinen IL-2-Rezeptors 15 1.5.2. Induktion regulatorischer T-Zellen durch TGF-β 16 1.5.3. Expansion regulatorischer T-Zellen durch Rapamycin (Sirolimus) 17 1.5.4. Expansion regulatorischer T-Zellen durch UVB-Bestrahlung bzw. Vitamin D-Rezeptor-Agonisten 18 1.5.5. Expansion regulatorischer T-Zellen durch Histon-Deacetylaseinhibitoren 19 1.6. Suppression von Effektor-T-Zellen durch regulatorische T-Zellen 20 1.6.1. Zellkontaktabhängige Mechanismen 20 1.6.2. Zellkontaktunabhängige Mechanismen 22 1.7. Matrix assisted laser desorption ionisation-Time of flight-Massenspektromie (MALDI-TOF-MS): Bedeutung und Funktion 23 1.8. Zielstellung 25 2. Materialien und Methoden 26 2.1. Versuchstiere 26 2.2. IL-2/IgG2b-Fusionsprotein-Vorexperiment 26 2.2.1. Induktion von 2,4,6-Trinitrobenzensulfonsäure (TNBS)-Kolitis 26 2.2.2. Durchführung des IL-2/IgG2b-Fusionsprotein-Vorexperimentes 26 2.3. Durchführung des IL-2/anti-IL-2-Antikörperkomplex-Experiments 27 2.4. Durchflusszytometrie 27 2.5. Histologische Färbungen 28 2.5.1. Probenvorbereitung 28 2.5.2. Hämatoxylin/Eosin (HE) Färbung 29 2.5.3. Immunfluoreszenz-Färbungen 29 2.5.4. Ki67-Schnellfärbung 30 2.5.5. Mikroskopie und Photographie 30 2.6. Histologische Auswertungen 31 2.6.1. Kolitis-Score 31 2.6.2. Bildanalyse 31 2.6.3. Validierung der automatischen Bildanalyse mittels CellProfiler 33 2.7. MALDI-Imaging 35 2.7.1. Probenvorbereitung für MALDI-Imaging 35 2.7.2. Analyse der Peptidexpression in Gewebeschnitten mittels MALDI-Imaging 36 2.8. Statistische Auswertungen 36 2.8.1. Statistische Tests 36 2.8.2. Berechnung der zu erwartenden Zahl von Kontakten zwischen Ki67+ und Foxp3+ Zellen 36 3. Ergebnisse 38 3.1. Design des IL-2/anti-IL-2-Antikörperkomplex Experimentes 38 3.2. Mit IL-2/anti-IL-2-Antikörperkomplex behandelte Tiere zeigen Splenomegalie und Lymphadenomegalie 40 3.3. Behandlung mit einem IL-2/anti-IL-2-Antikörperkomplex führt zur präferentiellen Expansion regulatorischer T-Zellen in mesenterialen Lymphknoten und Milz 41 3.4. Behandlung mit IL-2/anti-IL-2-Antikörperkomplex führt nicht zu Kolitis 43 3.5. Behandlung mit IL-2/anti-IL-2-Antikörperkomplex führt zur präferentiellen Expansion regulatorischer T-Zellen in der Lamina propria mucosae 45 3.6. IL-2/anti-IL-2-Antikörperkomplex steigert die Proliferation regulatorischer T-Zellen in der Lamina propria mucosae und lymphoiden Follikeln des Kolons 47 3.7. Regulatorische T-Zellen sind nach Behandlung mit IL-2/anti-IL-2-Antikörperkomplex weiter mit proliferierenden Zellen assoziiert. 50 3.8. MALDI-Imaging als Möglichkeit der Proteinexpressionsanalyse in situ 52 3.8.1. Vergleich der Proteinexpression in verschiedenen Geweben von Ileum und Zäkum mit der Expression in Thymus und mesenterialem Lymphknoten 55 3.8.2. MALDI-Imaging nach Schnellfärbung Ki67+ Zellen mit Streptavidin 63 3.8.3. Analyse der Massenspektren von Einzelzellen mittels MALDI-Imaging 66 4. Diskussion 68 4.1. Applikation von IL-2/anti-IL-2-Antikörperkomplex hat keine fatalen Nebenwirkungen 68 4.2. IL-2/anti-IL-2-Antikörperkomplex führt zur präferentiellen Expansion regulatorischer T-Zellen in mesenterialen Lymphknoten, Milz und Kolon 70 4.3. IL-10 als wichtiger Vermittler der Suppressionsaktivität durch IL-2/anti-IL-2-Antikörperkomplex expandierter regulatorischer T-Zellen 71 4.4. Expansion regulatorischer T-Zellen beim Menschen: Voraussetzungen und Chancen 72 4.5. Regulatorische T-Zellen akkumulieren an proliferierenden Zellen 73 4.6. Nachweis spezifischer Massen in Gewebe und Einzelzellen mittels MALDI-Imaging 74 5. Zusammenfassung 80 Literaturverzeichnis 83 Publikationen 90 Erklärung über die eigenständige Abfassung der Arbeit 97 Lebenslauf 98 Danksagungen 99