Cocoa beans (Theobroma cacao) are a highly concentrated source of dietary flavanols- bioactive compounds associated with the health protective properties of cocoa. Cocoa beans undergo processing steps, such as fermentation, roasting, winnowing, grinding, pressing, etc., to produce a final product with specific desirable sensory attributes. It is well established that these processing steps, specifically fermentation and roasting, result in dramatic degradation of cocoa's native flavanols, but it is possible that these processing steps may generate compounds with novel activities, potentially preserving or enhancing bioactivity. Raw unfermented cocoa beans were processed by way of a partial factorial approach to produce cocoa powders from the same batch of raw beans using various combinations of fermentation [unfermented, cool fermented (maximum 46°C), hot fermented (maximum 60°C))] and roasting [unroasted, cool roasted (120°C), hot roasted (170°C)]. To simulate cocoa fermentation in a highly controlled environment, a pilot-scale fermentation model system was employed to eliminate many external unknowns and ensure that the differences between our cocoa powders were due to our various treatments, rather than unknown factors occurring during fermentation and roasting. Low and high molecular weight fractions (8-10 kDa cutoff) were produced from cocoa powder extracts (CPE) of each treatment to quantify Maillard reaction products (MRP). A HILIC-UPLC MS/MS method was developed to more efficiently and sensitively quantify cocoa flavanols with high degrees of polymerization (DP) produced during processing. Overall, cocoa processing significantly (p<0.05) decreased the total phenolic and total flavanol concentrations of CPEs. Hot roasting had the greatest impact on native flavanol degradation yet produced CPEs with the highest mean degree of polymerization (mDP). All CPEs dose-dependently inhibited α-glucosidase enzyme activity, with cool fermented/cool roasted cocoa powder exhibiting the best inhibition (IC50 of 62.2 µg/mL). Increasing flavanol mDP was correlated with decreasing IC50 values, suggesting that the complex flavanols produced during processing enhance cocoa's bioactivity (or their production is associated with other products that enhance bioactivity). Alternatively, high molecular weight CPE fractions were correlated with increasing IC50 values, suggesting that MRPs interfere with enzyme inhibition or are associated with other products (polyphenols, macronutrients, etc.) that interfere with enzyme inhibition. Overall, the data presented within this work indicate that the components of processed cocoa powders are promising inhibitors of α-glucosidase, despite a significant reduction in native flavanol composition induced by processing, and moreover that fermentation and roasting conditions can positively influence the bioactivity of cocoa despite losses of native flavanols. / Master of Science in Life Sciences / According to the Centers for Disease Control and Prevention, obesity-related chronic conditions such as cardiovascular disease and type 2 diabetes mellitus (T2D) are the leading cause of preventable and/or premature death, with 51% of the American population predicted to be obese by 2030. Cocoa (Theobroma cacao) is a highly concentrated source of polyphenols, and these compounds have been shown to interact with and inhibit digestive enzymes responsible for carbohydrate breakdown. By inhibiting the activity of these digestive enzymes, it is possible to slow down carbohydrate absorption after a meal and ultimately reduce large spikes in blood glucose levels, being a promising strategy in the prevention and maintenance of T2D. Cocoa beans undergo processing steps to produce a final product, such as cocoa powder, and it is known that these processing steps reduce the levels of beneficial polyphenols. Yet, how this processing-induced degradation effects the health protective activities of cocoa is still widely unknown and is the focus of this work. Through highly controlled cocoa bean processing, cocoa powders of different processing conditions were produced and used to assess how various processing parameters impacted digestive enzyme activity. Overall, processing steps did reduce levels of native polyphenols. However, these losses did not demonstrate a reduction in enzyme inhibition and certain processing conditions actually enhanced digestive enzyme inhibition. This research shows promise for the potential use of processed cocoa powder as an effective strategy in the prevention and maintenance of T2D and further work must be done to understand the mechanisms behind this relationship.

Die Rückstandsanalytik tierischer Lebensmittel ist eine anspruchsvolle Aufgabe aufgrund des hohen Lipidanteils der Proben sowie des sich stetig vergrößernden Wirkstoffspektrums. Heutzutage werden für die Probenaufarbeitung die DFG S 19 Methode, mit der vorrangig unpolare Analyten nachgewiesen werden und zunehmend die QuEChERS Methode eingesetzt, die insbesondere auf die Erfassung polarer Pestizide abzielt. In dieser Arbeit wurde eine moderne Multirückstandsmethode für Hühnereier entwickelt, um sowohl gas- als auch flüssigkeitschromatographisch (GC, LC) erfassbare Wirkstoffe zu analysieren. Dazu gehören unpolare PCBs, Pyrethroide und Organochlorpestizide, aber auch polarere Organophosphate, Triazole und Carbamate. Das Verfahren basiert auf der Extraktion mittels Matrix Solid Phase Dispersion, der Reinigung auf Grundlage einer modifizierten Gelpermeationschromatographie (GPC) und zwei verschiedenen Festphasenextraktionen (SPEs) für GC- und LC-erfassbare Pestizide sowie der Quantifizierung mittels GC- und LC-MS/MS. Dünnschichtchromatographisch wurde die effektive Entfernung hochmolekularer Lipide durch die modifizierte GPC und niedrigmolekularer Fette durch die SPEs belegt. Laut der für Ei durchgeführten Validierung erfüllten 164 der 172 untersuchten Pestizide und alle sechs PCBs die Leistungskriterien für die amtliche Rückstandskontrolle - zumeist am niedrigsten validierten Level (5 µg/kg bzw. 0,5 µg/kg). Ausnahmen bildeten sehr polare LC-Pestizide (z.B. Aminopyralid, Clopyralid, MCPA, Quinmerac) und pH-Wert-abhängige GC-Analyten (Nicotin, Tolylfluanid, Dichlofluanid), die auch mit den etablierten Verfahren schwierig zu analysieren sind. Weiterhin verdeutlichte die erfolgreiche Untersuchung von verschiedenen Ringversuchsmaterialien, dass die ursprünglich für Eier entwickelte Methode auch für mageres Geflügelfleisch und Sahne genutzt werden kann. Gegenüber den etablierten Verfahren wies die neue Methode deutliche Vorzüge auf. So belegte die Dünnschichtchromatographie, dass mit der neuen Methode Cholesterin, aber auch freie Fettsäuren besser abgetrennt werden als mit den etablierten Verfahren. Die neue Methode verbrauchte im Vergleich zur DFG S 19 Methode 46 % weniger Lösungsmittel und ermöglichte eine Verdopplung des Probendurchsatzes innerhalb von 8 h. Zudem eignete sich das entwickelte Verfahren laut den Validierungsdaten für GC-Analyten deutlich besser als die QuEChERS Methode und etwas besser als die DFG S 19 Methode (v.a. für Pyrethroide). Hinsichtlich der LC-Analyten unterschieden sich die neue und die QuEChERS Methode nur bei wenigen Analyten. Mit dem neuen Verfahren konnten folglich im Gegensatz zu den etablierten Methoden sowohl unpolare GC- als auch polare LC-Analyten sicher erfasst werden.:1 EINLEITUNG UND ZIELSTELLUNG 1 2 THEORETISCHE GRUNDLAGEN 3 2.1 Grundlagen der Pestizidanalytik 3 2.1.1 Definitionen 3 2.1.2 Rechtliche Grundlagen in Bezug auf tierische Lebensmittel 3 2.2 Herausforderung der Analytik tierischer Lebensmittel 6 2.2.1 Erfassen eines breiten Analytspektrums 6 2.2.2 Komplexität der tierischen Matrix 8 2.3 Probenaufarbeitung tierischer Lebensmittel 11 2.3.1 Extraktion 11 2.3.2 Möglichkeiten der Lipid-Reinigung 14 2.3.3 Messmethoden in der modernen Pestizidanalytik 18 2.4 Multimethoden in der Pestizidanalytik tierischer Lebensmittel 21 2.4.1 Vorstellung etablierter Multimethoden 21 2.4.2 Vergleich etablierter Multimethoden 24 2.4.3 Forschungsergebnisse bezüglich fetthaltiger Matrizes bis 2010 26 3 ERGEBNISSE 28 3.1 Auswahl relevanter Analyten in tierischen Lebensmitteln 28 3.2 Auswahl der Matrix Hühnerfrischei 30 3.3 Messung mittels GC-MS/MS und LC-MS/MS 32 3.3.1 Ansatz 32 3.3.2 Erstellung einer GC-MS/MS Datenbank 33 3.3.3 Besonderheiten der SRM-Messmethoden 36 3.3.4 Vorsäulen-Backflush in der Gaschromatographie 38 3.3.5 Leistungsfähigkeit der SRM-Methoden 44 3.4 Beurteilung des Reinigungseffektes 45 3.5 Entwicklung der Probenaufarbeitungsmethode 47 3.5.1 Ziele und allgemeines Vorgehen 47 3.5.2 Extraktion mittels Matrix Solid Phase Dispersion 48 3.5.3 Konzentrierung von Lösungsmittelextrakten 55 3.5.4 Lipidentfernung durch Ausfrieren 56 3.5.5 Modifizierung der GPC zur Verbesserung der Wirtschaftlichkeit 57 3.5.6 Festphasenextraktion 63 3.6 Analysemethode für Eier 78 3.7 Validierung der neuen Analysemethode 82 3.7.1 Hintergrund und Durchführung 82 3.7.2 Linearität 83 3.7.3 Spezifität 84 3.7.4 Wiederfindungsraten und Wiederholbarkeit 84 3.7.5 Bestimmungsgrenze 88 3.7.6 Matrix-spezifische Auswirkung auf die Analyten 88 3.8 Vergleich mit etablierten Probenaufarbeitungsmethoden 89 3.8.1 Ansatz 89 3.8.2 Reinigungseffekt 90 3.8.3 Validierungsdaten 93 3.8.4 Identifizierung und Quantifizierung anhand gewachsener Rückstände 95 3.8.5 Zusammenfassende Bewertung 97 3.9 Untersuchung verschiedener Ringversuchsmaterialien 99 4 DISKUSSION 103 4.1 Entwickelte Analysemethode 103 4.2 Ausblick 111 5 ZUSAMMENFASSUNG 113 6 LITERATUR 117 7 ANHANG A-1

No / A novel ion trap time-of-flight hybrid mass spectrometer (qIT-TOF MS) has been applied for peptide sequencing in proteolytic digests generated from spore mixtures of Bacilli. The method of on-probe solubilization and in situ proteolytic digestion of small, acid-soluble spore proteins has been recently developed in our laboratory, and microorganism identification in less than 20 min was accomplished.1 In this study, tryptic peptides were generated in situ from complex spore mixtures of B. subtilis 168, B. globigii, B. thuringiensis subs. Kurstaki, and B. cereus T, respectively. MALDI analysis of bacterial peptides generated was performed with an average mass resolving power of 6200 and a mass accuracy of up to 10 ppm using a trap-TOF tandem configuration. Precursor ions of interest were usually selected and stored in the quadrupole ion trap with their complete isotope distribution by choosing a window of ±2 Da. Sequence-specific information on isolated protonated peptides was gained via tandem MS experiments with an average mass resolving power of 4450 for product ion analysis, and protein and bacterial sources were identified by database searching.

No / Lipidomics aim to generate qualitative and quantitative information on different classes of lipids and their species, and when applied in conjunction with proteomic and genomic assays, facilitate the comprehensive study of lipid metabolism in cellular, organ or body systems. Advances in mass spectrometry have underpinned the expansion of lipidomic methodologies. Prostanoids are potent autacoids present in a plethora of cellular systems, known best for their intimate role in inflammation. Electrospray ionisation (ESI) allows the efficient ionisation of prostanoids in aqueous systems. ESI can be readily coupled to liquid chromatography (LC) followed by tandem mass spectrometry (MS/MS)-based detection, thus allowing the development of a potent and selective LC/ESI-MS/MS quantitative assays. The protocol we describe in this chapter outlines the steps we follow to a) extract prostanoids from solid or liquid samples, b) semi-purify the metabolites using solid phase extraction c) set-up the HPLC separation using reverse phase chromatography and d) set up the MS/MS assay using a triple quadrupole mass spectrometer. The experimental details and notes presented here are based on the detailed protocols followed in our group

No / Piperacillin, a potent ß-lactam antibiotic, is effective in a large variety of Gram+ and Gram¿ bacterial infections but its administration is limited to the parenteral route as it is not absorbed when given orally. In an attempt to overcome this problem, we have synthesized a novel series of charged liposaccharide complexes of piperacillin comprising a sugar moiety derived from d-glucose conjugated to a lipoamino acid residue with varying side-chain length (cationic entity) and the piperacillin anion. A complete multiple reaction monitoring LC¿MS/MS method was developed to detect and characterize the synthesized complexes. The same method was then successfully applied to assess the in vitro apparent permeability values of the charged liposaccharide complexes in Caco-2 monolayers. / BBSRC

Voxelotor (GBT440) is a haemoglobin S polymerization inhibitor used to treat anaemia in sickle cell disease. Due to an increase of arterial oxygen saturation as well as serum erythropoietin and haemoglobin, the World Anti-Doping Agency included voxelotor in the list of prohibited substances and methods in 2023. The objective of the present study was to identify and characterize metabolites of voxelotor to detect a potential misuse by athletes. The biotransformation was studied in vitro using the human hepatocellular cell line HepG2 and pooled human liver microsomes. The metabolites were analysed using high-performance liquid chromatography (high-resolution) mass spectrometry. In total, three phase I metabolites and six phase II metabolites (resulting from glucuro-conjugation and O-methylation) were formed by the HepG2 cells in a time-dependent manner, and two phase I metabolites were generated by the liver microsomes, among them one also found in the HepG2 incubations. A reduced metabolite and the glucuro-conjugate of a reduced metabolite were the most abundant formed by HepG2 cells. In addition, metabolites resulting from mono-hydroxylation, reduction and O-methylation in different combinations were identified. Voxelotor was also found as glucuro-conjugate with a low abundance. With the spectrometric behaviour of voxelotor and its in vitro metabolites described herein, an implementation in doping control screening and, consequently, a detection of an abuse in an athlete urine sample might be possible.

Le long bio-polymère d'ADN est condensé à l’intérieur du noyau des cellules eukaryotes à l'aide de petites protéines appelées histones. En plus de leurs fonctions condensatrices,ces histones sont également la cible de nombreuses modifications post-traductionnelles(MPT), particulièrement au niveau de leur section N-terminale. Ces modifications réversibles font partie d’un code d’histones épi-génétique transmissible qui orchestre et module dynamiquement certains événements impliquant la chromatine, tels l’activation et la désactivation de gènes ainsi que la duplication et la réparation d’ADN. Ces modifications sont impliquées subséquemment dans la signalisation et la progression de cancers, tels que la leucémie. En conséquence, l'élucidation des modifications d’histones est importante pour comprendre leurs fonctions biologiques. Une méthodologie analytique a été mise au point en laboratoire pour isoler, détecter, et quantifier les MPT d’histones en utilisant une approche rapide à deux volets à l’aide d’outils bioinformatiques spécialisés. La méthodologie développée en laboratoire a été validée en utilisant des histones de souche sauvage ainsi que deux types d’histones mutants déficients en enzymes acétyltransferase. Des trois sources d’histones utilisées, la seule MPT qui a démontré un changement significatif est l’acétylation de l’histone H3 à lysine 56 (H3K56ac). L’expression et la stoechiométrie de cette MPT, issue de cellules de souche sauvage et de cellules mutantes, ont été déterminées avec précision et comparées. Les fonctions de balayage polyvalentes d'un instrument à trappe ionique quadrupôle linéaire hybride ont été utilisées pour améliorer la détection de protéines intactes. Le mode de balayage « enhanced multiply charged » (EMC) a été modifié pour contenir et détecter les ions de protéines intactes situées dans la trappe ionique linéaire. Ce mode de balayage nommé « targeted EMC » (tEMC) a permis de quadrupler le niveau de sensibilité (signal/interférence), et quintupler la résolution du mode de balayage conventionnel. De plus, la capacité de séparation des charges du tEMC a réduit de façon significative les effets de « space charge » dans la trappe ionique linéaire. La résolution supérieure du mode tEMC a permis de différencier plusieurs isoformes modifiées, particulièrement pour l’histone H3. L’analyse des peptides d’histones trypsiques à l’aide du mode de balayage « MRM » a permis le séquençage et la quantification de MPT avec un haut degré de précision. La seule MPT qui était sous-exprimée entre l’histone de souche sauvage et le mutant DOT1L fut la méthylation de l’histone H3 lysine 79(H3K79me1). Les effets de deux inhibiteurs d’enzymes HDAC (HDACi) sur l’expression de MPT d’histone ont été évalués en utilisant la méthodologie analytique mentionnée. Les histones extraites de cellules normales et cancéreuses ont été exposées à du Vorinostat(SAHA) ou du Entinostat (MS-275) pour une période de 24 à 72 heures. Deux histones furent principalement affectées, soit H3 et H4. Étonnamment, les mêmes effets n'ont pas été détectés lorsque les cellules normales ont été traitées avec le HDACi pour une période de 48 à 72 heures. Une méthode absolue de quantification avec une courbe d’étalonnage a été développée pour le peptide H3K56ac. Contrairement à certaines publications, nos résultats démontrent que cette MPT est présente dans les cellules mammifères avec une stoechiométrie très basse (< 0,1%) et n'est pas surexprimée de façon significative après le traitement au HDACi. / In eukaryotic cells, the lengthy DNA biopolymer is condensed into the cell nucleus with the aid of small packaging proteins called histones. In addition to their packing functions,histones are also targets of numerous post translational modifications (PTMs), especially on their N-terminus. These reversible modifications are believed to be constituents of a heritable epigenetic “histone code” that dynamically orchestrate and modulate chromatin based events such as gene activation and silencing, DNA replication and repair, and are also involved in the downstream signaling and progression of cancers, such as leukemia. Thus, the elucidation of histone PTMs is important in understanding their biological function. An analytical workflow was designed and set-up in the laboratory to isolate, detect, and quantitate histone PTM, using a two-pronged, unbiased, and rapid approach with specialized bioinformatic tools. The workflow was validated using histones from wildtype, and 2 mutants deficient in acetyltransferase activity. Between the three histone sources, the only PTM that demonstrated any change was acetylation at histone H3 lysine 56 (H3K56ac). The down-regulation and stoichiometry of this PTM was accurately assessed between wild-type and mutant cells. The versatile scan functions of a hybrid quadrupole-linear ion trap instrument were exploited to enhance the detection of intact histone proteins. The enhanced multiply charged (EMC) scan was modified in order to contain and detect intact protein ions within the linear ion trap. This targeted EMC (or tEMC) resulted in not only a 4-fold increase in signal-to-noise, but also a 5-fold increase in resolution. Furthermore, the charge separation capability of the tEMC dramatically reduced space charge effects within the linear ion trap. The superior resolution of the tEMC mode allowed for the discimination of many modified histone isoforms, especially for histone H3. Using the bottom-up strategy with multiple reaction monitoring (MRM), histone peptides were quantified and sequenced with a high degree of precision. The only PTM that was down-regulated between wild-type and DOT1L mutant histones was methylation at histone H3 lysine 79 (H3K79me1). The effects of two clinically relevant small molecule HDAC inhibitors (HDACi) on histone PTMs patterns were assessed using the analytical workflow developed. Histones derived from both normal and cancer cells were exposed to either Vorinostat (SAHA) or Entinostat (MS-275) over a 24- to 72 hour period. The two core histones primarily affected were H3 and H4. Surprisingly, the same effects were not observed when normal cells were treated with three doses of SAHA at 24-hour intervals over a 72-hour period. An absolute quantitation method using a calibration curve was developed for H3K56ac. In opposition to other published literature, our findings demonstrate that this PTM is present in very low stoichiometry (< 0.1%) in mammalian cells, and exhibits no significant up-regulation in different cell lines treated with several types of HDACi.

Neospora caninum (filo Apicomplexa) é um parasita obrigatório intracelular como todos os membros deste filo, alguns reconhecidos por causarem doenças com impacto relevante na saúde humana (Plasmodium e Toxoplasma) e veterinária (Babesia, Eimeria e Cryptosporidium). Causador da neosporose, N. caninum vem emergindo como um dos maiores causadores de abortos infecciosos em bovinos, levando a consideráveis perdas econômicas na bovinocultura mundial. Devido à sua recente descoberta, o conhecimento sobre diversos processos bioquímicos de N.caninum ainda é limitado, demandando novas pesquisas para a compreensão de seus mecanismos de sobrevivência e consequente identificação de alvos para intervenção terapêutica. O processo de invasão celular é bastante investigado em pesquisas envolvendo apicomplexas, uma vez que a sobrevivência desses parasitas depende do sucesso de sua entrada na célula hospedeira. Proteínas secretadas de organelas filo-específicas (micronemas, roptrias e grânulos densos) estão intimamente envolvidas com a invasão celular. Elas são responsáveis pela interação inicial com a célula hospedeira, participam da junção de movimento formada no momento da invasão, e contribuem para a estabilização do vacúolo parasitóforo. Neste trabalho as proteínas secretadas por taquizoítas de N. caninum foram investigadas de duas formas: (1) por caracterização molecular de proteínas com domínio Apple; e (2) por estudo do secretoma do parasita. Os domínios proteicos do tipo Apple são caracterizados pela capacidade de interação proteína-proteína e proteína-carboidrato, e estão presentes em algumas proteínas micronêmicas com propriedades adesivas. Neste trabalho três proteínas de N. caninum contendo domínios Apple foram caracterizadas: MIC17A, MIC17B e MIC17C. A análise das sequências proteicas e das estruturas dos domínios Apple, obtidas por modelagem molecular, mostraram alta identidade sequencial e estrutural entre MIC17A e MIC17C. Apesar de ser paráloga às outras duas, MIC17B apresenta diferenças importantes em sua sequência e estrutura. Para MIC17B e MIC17C foram realizados experimentos de detecção das proteínas nativas nos extratos total e secretado do taquizoíta que sugerem diferentes formas de processamento entre essas proteínas no parasita. Para MIC17B foi confirmada a localização em micronemas, num padrão diferente do observado para MIC17C. Os ensaios de invasão combinados aos de localização indicam que estas proteínas estejam relacionadas ao processo de invasão celular, porém, suas funções permanecem desconhecidas. O secretoma é o conjunto de proteínas secretadas pelo parasita e, para explorar a composição deste extrato (ESA) no taquizoíta de N. caninum, duas abordagens complementares foram utilizadas. Na primeira abordagem foram identificadas as proteínas presentes no ESA por espectrometria de massas. Na segunda abordagem realizou-se uma ii quantificação relativa das proteínas, marcadas por dois isótopos, nos extratos totais de taquizoítas submetidos ou não ao estímulo secretório. O resultado esperado seria com as proteínas secretadas diminuídas no parasita estimulado. Em ambas as abordagens foram utilizadas técnicas de espectrometria de massas de alta resolução (nanoLC-MS/MS), o que resultou num alto número de identificações; 615 proteínas no ESA e 2011 proteínas quantificadas. A comparação das duas abordagens permitiu o reconhecimento de proteínas com maior probabilidade de secreção. Uma rede de interação entre as proteínas diferencialmente expressas foi predita, gerando resultados que, associados às informações sobre as proteínas aumentadas, permitiram uma investigação sobre proteínas potencialmente envolvidas com a regulação do metabolismo relacionado à secreção. Os resultados obtidos por ambos os estudos aqui demonstrados somam conhecimento acerca do parasita N. caninum e demonstram ser úteis para guiar a busca e seleção de alvos a serem investigados para o desenvolvimento de terapêutica contra a neosporose. / Neospora caninum (Apicomplexa phylum) is an obligatory intracellular parasite like all members from this phylum, some causing diseases with relevant impact on human (Plasmodium and Toxoplasma) and veterinary (Babesia, Eimeria and Cryptosporidium) health. Causative agent of neosporosis, N. caninum has emerged as one of the leading causes of infectious abortion in cattle, generating huge economical losses in worldwide livestock. Due to its recent discovery, knowledge of N. caninum biochemical processes remains scarce, demanding new research for comprehending its survival mechanisms and, consequently, identifying new targets for therapeutic intervention. The invasion process has often been investigated in apicomplexans since their survival depends on the success of their entry into the host cell. Proteins secreted from phylum-specific organelles (micronemes, rhoptries and dense granules) are deeply involved with invasion. They are responsible for the initial interaction with the host cell; participate of the moving junction formed in the moment of invasion; and contribute for the stabilization of the parasitophorus vacuole. In this study, the proteins secreted by N. caninum tachyzoites were investigated in two ways: (1) the molecular characterization of Apple domaincontaining proteins; and (2) exploring the parasite secretome. The Apple protein domains are characterized by the ability to interact as protein-protein and proteincarbohydrate, and are present in some microneme proteins with adhesive properties. Here three N. caninum proteins containing Apple domains were characterized: MIC17A, MIC17B and MIC17C. Analyses of the Apple domains sequences and structures, obtained by molecular modeling, revealed high sequential and structural identities between MIC17A and MIC17C. Although being a paralog of the other two proteins, MIC17B presents significant differences in its sequence and structure. Experiments were performed for native MIC17B and MIC17C detection in the total and secreted tachyzoite extracts, suggesting different processing forms for these proteins in the parasite. For MIC17B, the microneme localization was confirmed, differently from the pattern observed for MIC17C. Invasion and localization assays indicated that these proteins are related to the cell invasion process; nevertheless, their functions remain unknown. The secretome is the set of proteins secreted by the parasite and, to explore this extract (ESA) composition in N. caninum, two complementary approaches were used. Firstly proteins present in ESA were identified by mass spectrometry. In the second approach, a relative quantification was performed on the proteomes of ethanol stimulated/non stimulated tachyzoites, expecting that the secreted proteins would be down regulated at the stimulated parasite. Both approaches were performed with high resolution mass spectrometry techniques (nanoLC-MS/MS), reaching a high number of identifications: 615 proteins iv in ESA and 2011 quantified proteins. The comparison between both approaches allowed the recognition of the most likely secreted proteins. An interaction network was predicted, involving the differentially expressed proteins. These results, associated with the information of up regulated proteins, allowed the investigation of proteins potentially involved with the secretion metabolism regulation. The findings from our two studies add up knowledge about N. caninum and demonstrate to be useful in guiding the search and selection for new targets for therapeutic development against neosporosis.

O preparo da amostra é uma das etapas mais importantes em toda a análise química. O isolamento e a concentração dos componentes da amostra são cruciais e busca-se sempre que essas etapas sejam as mais simples e consumam o mínimo possível de tempo e reagentes. Nos últimos anos, um tipo de material tem se mostrado bastante útil para análises químicas a partir de fluidos biológicos, os polímeros molecularmente impressos (MIPs). Os MIPs são sintetizados por reações de polimerização, na presença de uma molécula molde (template). A molécula molde se liga aos monômeros funcionais do polímero durante a reação de polimerização e permanece ligada à superfície das cadeias poliméricas quando a reação se completa. Terminada a polimerização, realiza-se a completa lavagem das moléculas molde, assim, restam na superfície polimérica cavidades tridimensionais complementares à molécula empregada como molde. Essas cavidades permitem a ligação reversível e preferencial da molécula molde ou outras com estrutura química semelhante. A Cannabis sativa é a droga ilícita mais consumida em todo o mundo e nos últimos anos muita atenção tem se voltado a seus efeitos toxicológicos no corpo humano e a aplicações medicinais. Nesta dissertação, foi sintetizado um MIP com a molécula molde catequina para a extração e posterior análise por LC-MS/MS dos canabinóides &Delta;9-tetrahidrocanabinol (THC), 11-hidroxi-&Delta;9-tetrahidrocannabinol (THC-OH) e 11-nor-&Delta;9-tetrahidrocannabinol-9-ácido carboxílico (THC-COOH) em amostras de urina. O MIP produzido foi empacotado em microdispositivo e empregado no preparo das amostras de urina por microextração por sorvente empacotado (MEPS). O método desenvolvido apresentou boa linearidade (valores de r de 0,977 para o THC e 0,994 para THC-OH e THC-COOH). Os limites de detecção e de quantificação foram respectivamente de 5 ng mL-1 e 20 ng mL-1, para os compostos THC e THC-OH, na faixa linear de 25 a 250 ng mL-1. Para o composto THC-COOH os limites de detecção e quantificação alcançados foram de 1 ng mL-1 e 5 ng mL-1, respectivamente, na faixa linear de 5 a 170 ng mL-1. O método apresentou valores razoáveis de precisão entre 3,2% (THC-COOH) e 25,1% (THC) e de exatidão, que variou entre -18,4 e 17,4 (ambos para o THC). O MIP empregado no preparo da amostra mostrou-se mais seletivo e específico do que materiais normalmente empregados para a extração dos canabinoides das amostras de urina, além de a técnica de extração por MEPS apresentar baixo consumo de solventes e amostra para a extração dos analitos e posterior análise por LC-MS/MS. / The sample preparation is one of the most important steps in every chemical analysis. The isolation and concentration of the sample components are crucial and it is always sought that these steps are simple and consume the lowest amount of time and reagents. In the recent years, a type of material has proved to be very useful for chemical analyzes of biological fluids, the molecularly imprinted polymers (MIPs). MIPs are synthesized by polymerization reactions in the presence of a template molecule. The template molecule binds to the functional monomers of the polymer during the polymerization reaction and remains bonded to the surface of the polymeric chains after the reaction is complete. After the polymerization is finished, the complete washing of the template molecules is carried out, thus, three-dimensional cavities, complementary to the molecule used as a template, remain on the polymer surface. These cavities allow the reversible and preferential bonding of the template molecule or others with similar chemical structure. Cannabis sativa is the most commonly consumed illicit drug in the world and in recent years much attention has focused on its toxicological effects on human body and for medical applications. In this master dissertation, a MIP was synthesized with the catechin molecule as template, for extraction and subsequent analysis by LC-MS/MS of the cannabinoids &Delta;9-tetrahydrocannabinol (THC), 11-hydroxy-&Delta;9-tetrahydrocannabinol (THC-OH), and 11-nor-&Delta;9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in urine samples. The MIP produced was packed in a microdevice and used in the preparation of the urine samples by microextraction by packed sorbent (MEPS). The developed method showed good linearity (r values of 0.977 for THC and 0.994 for THC-OH and THC-COOH). The detection and quantification limits were respectively 5 ng mL-1 and 20 ng mL-1 for THC and THC-OH in the linear range from 25 to 250 ng mL-1. For the compound THC-COOH the limits of detection and quantification achieved were 1 ng mL-1 and 5 ng mL-1, respectively, in the linear range from 5 to 170 ng mL-1. The method presented reasonable values of precision, between 3.2% (for THC-COOH) and 25.1% (for THC) and displayed accuracy ranging from -18.4 to 17.4 (both for THC). The MIP used in the sample preparation was more selective and specific than other materials usually employed for the extraction of the cannabinoids from the urine samples. The MEPS technique also showed low consumption of solvents and sample for sample preparation, extraction of analytes and subsequent analysis by LC-MS/MS.

Immunoassays basieren auf der Anwendung von Antikörpern, welche selektiv den zu messenden Analyten binden. Die Richtigkeit der erhaltenen Ergebnisse hängt maßgeblich von der Selektivität der Antikörper ab und kann durch Interferenzen gestört werden. In dieser Arbeit wurde eine Methode entwickelt, bei der die Probe mittels Hochleistungsflüssigkeitschromatographie (LC) in Fraktionen aufgetrennt wird und diese Fraktionen anschließend mittels Enzyme-linked Immunosorbent Assay (ELISA) vermessen werden. Dieses Verfahren wurde als LC-ELISA bezeichnet. Das erhaltene Profil aus im ELISA gemessener Analytkonzentration in Abhängigkeit von der Elutionszeit wurde als LC-ELISAgramm bezeichnet und bietet die Möglichkeit, Interferenzen zu erkennen, welche beim ELISA unentdeckt bleiben. Als Modellanalyten für die zu untersuchenden ELISAs dienten Sulfamethoxazol (SMX), Carbamazepin (CBZ) und Estron (E1). Dabei wurden verschiedene Umweltmatrices wie Oberflächenwasser und Abwässer mit dem jeweiligen ELISA vermessen. Es wurde ein Ansatz zur Unterscheidung von spezifischen und unspezifischen Interferenzen in Umweltproben aufgezeigt. Durch diesen Ansatz und Anwendung der sauren Hydrolyse der Probe war es möglich, einen bisher unbekannten SMX-Metaboliten zu detektieren und dessen wahrscheinliche Kreuzreaktivität mit 460 ± 150 % abzuschätzen. Es wurde zudem ein neuer Tracer in einer linearen 13-Stufen-Synthese entwickelt, wobei neuartig die Konjugation der Peroxidase an der N1-Position des SMX erfolgte. / Immunoassays are based on the use of antibodies that selectively bind the analyte. The trueness of the results obtained depends to a great extent on the selectivity of the antibodies and can be affected by interferences. In this study, a method was developed in which the sample is separated into fractions by using high-performance liquid chromatography (LC) and these fractions are measured using an enzyme-linked immunosorbent assay (ELISA). This method was referred to as LC-ELISA. The profile obtained from the measured analyte concentration by ELISA as a function of the elution time was referred to as LC-ELISAgram and offers the possibility to detect interferences which otherwise remain undetected during the ELISA. Sulfamethoxazole (SMX), carbamazepine (CBZ) and estrone (E1) were used as model analytes for the ELISA and LC-ELISA measurements. Various environmental matrices such as surface water and wastewater were examined for their interference in the respective ELISA. The good quantification properties of the validated LC-ELISA have been used to demonstrate an approach to distinguish between specific and non-specific interferences from environmental samples. By this approach and application of acidic hydrolysis of the sample, it was possible to detect a previously unknown metabolite of SMX and estimate its cross-reactivity to probably 460 ± 150%. Furthermore, a new tracer was developed in a linear 13-step synthesis, which resulted in the novel conjugation of the peroxidase at the N1-position of SMX. The new hapten was also used for the synthesis of a novel immunogen.