Dissertations / Theses on the topic 'Mass spectometry'

Thesis (Ph. D.)--West Virginia University, 2001.
Title from document title page. Document formatted into pages; contains xiii, 165 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 157-165).

Signal Transduction is mediated by protein complexes whose spatial- and temporal-distribution, composition and function within cells are often regulated by different post-translational modifications (PTM). As PTMs add or subtract a specific mass difference to a protein, mass spectrometry becomes very amenable for modification analysis. These modifications have conventionally been monitored by fragmenting the modified protein or peptide by collision induced dissociation (CID) within the mass spectrometer, and then screening for the characteristic neutral fragment or fragment ion (marker ion), which is particular to the modification in question. Unfortunately, there are two major issues with respect to the traditional mass spectrometric analysis of PTMs: (1) as there are over 300 known types of modifications, the characteristic fragmentation of only a fraction of these modifications has been studied and (2) the traditional mass spectrometric approaches can only monitor these modifications sequentially, and thus comprehensive modification analysis would be unfeasible considering the breadth of PTMs. The following work aims to address these issues by (1) analyzing PTMs that have never been characterized mass spectrometrically and (2) developing a multiplexed technique for comprehensive PTM monitoring by simultaneously screening for all known characteristic fragments. With respect to the first issue, the characteristic fragmentation of lipid modifications and HNO-induced modifications was investigated. The most prevalent indicator(s) of the modification within the mass spectra are as follows: fragmentation of N-terminal myristoylated peptides produced marker ions at 240 and 268 Th, fragmentation of cysteine farnesylated peptides produced a marker ion at 205 Th and a neutral fragment of 204 Da, and fragmentation of cysteine palmitoylated peptides produced a neutral fragment of 272 Th. For HNO-induced modifications, fragmentation of the sulfinamide- and sulfinic acid-modified peptides produced a neutral fragment of 65 Da and 66 Da, respectively. With respect to the second issue, a multiplexed technique for monitoring modifications that fragment as neutral losses, termed Multiple Neutral Loss Monitoring (MNM), has been developed, successfully validated, and then shown to be the most sensitive approach for PTM analysis. MNM, combined with a second multiplexed approach, targeted Multiple Precursor Ion Monitoring, has been used to provide a comprehensive PTM analysis.

The present PhD thesis was focused on the development and application of chemical methodology (Py-GC-MS) and data-processing method by multivariate data analysis (chemometrics). The chromatographic and mass spectrometric data obtained with this technique are particularly suitable to be interpreted by chemometric methods such as PCA (Principal Component Analysis) as regards data exploration and SIMCA (Soft Independent Models of Class Analogy) for the classification. As a first approach, some issues related to the field of cultural heritage were discussed with a particular attention to the differentiation of binders used in pictorial field. A marker of egg tempera the phosphoric acid esterified, a pyrolysis product of lecithin, was determined using HMDS (hexamethyldisilazane) rather than the TMAH (tetramethylammonium hydroxide) as a derivatizing reagent. The validity of analytical pyrolysis as tool to characterize and classify different types of bacteria was verified. The FAMEs chromatographic profiles represent an important tool for the bacterial identification. Because of the complexity of the chromatograms, it was possible to characterize the bacteria only according to their genus, while the differentiation at the species level has been achieved by means of chemometric analysis. To perform this study, normalized areas peaks relevant to fatty acids were taken into account. Chemometric methods were applied to experimental datasets. The obtained results demonstrate the effectiveness of analytical pyrolysis and chemometric analysis for the rapid characterization of bacterial species. Application to a samples of bacterial (Pseudomonas Mendocina), fungal (Pleorotus ostreatus) and mixed- biofilms was also performed. A comparison with the chromatographic profiles established the possibility to: • Differentiate the bacterial and fungal biofilms according to the (FAMEs) profile. • Characterize the fungal biofilm by means the typical pattern of pyrolytic fragments derived from saccharides present in the cell wall. • Individuate the markers of bacterial and fungal biofilm in the same mixed-biofilm sample.

Burkholderia pseudomallei is the causative agent of Melioidosis, an endemic disease in South East Asia, and is classified as a category B biological agent. Currently, there is no licensed vaccine for this disease; the mortality rate is high due to the incorrect diagnosis and the pathogen insusceptibility to general antibiotics. A mass spectrometry based proteomic approach has been applied in order to identify the proteins that are responsible for pathogenicity.Methods were developed for the proteomic analysis of Burkholderia species using B. vietnamiensis G4, an opportunistic pathogen as the model organism. Both gel-based (LC-MS/MS) and gel-free MudPIT (LC/LC-MS/MS) approaches have been applied for the analysis of the proteins extracted from four different cellular fractions of these bacteria. More than 1200 proteins were identified from these analyses, including many proteins previously identified as virulence factors of these bacteria. Similar methodologies were applied to build a proteome map of non-pathogenic B. thailandensis E264 to use as a reference for the pathogenic studies. Additionally, proteomes of two B. thailandensis strains isolated from two geographical locations were compared to investigate the differences in protein expression of these organisms.Proteins identified from pathogenic B. pseudomallei were compared with the non-pathogenic B. thailandensis and opportunistic pathogen B. vietnamiensis proteins. Many species specific proteins were identified from this proteomic analyses; those proteins can be used as antigen targets to selectively identify these pathogenic bacteria in a complex biological matrix using affinity capture methods.Ticks are vectors that can transmit disease causing pathogens one host to another. Knowing the pathogen reservoir is important in order to control disease spread in the environment. Application of mass spectrometric methods to identify the host blood components from tick vectors was investigated using tick nymphs which feed only once in their life cycle. Using mass spectrometry based proteomics; host specific proteins like hemoglobin and immunoglobulin were identified from a single tick nymph analysis. Additional studies have examined the fatty acid profiles of rabbit and sheep blood fed tick nymphs using SPALDI mass spectrometry. Different fatty acid profiles were obtained for these tick nymphs, but further investigations are required to validate these findings.

Direct solid sampling is an area of analytical research that has generated a large amount of interest in recent years. Two analysis systems offering fast and nondestructive methods of determining the elemental composition of substances, without requiring complicated sample preparation procedures, are laser ablation inductively coupled plasma mass spectroscopy (LA-ICPMS) and radio frequency glow discharge mass spectroscopy (rf-GDMS). A Cetac LSX-200 laser system coupled to a LECO Renaissance ICPMS was utilized to analyze coal and ash samples prepared by incorporation into a lithium borate matrix to form a disk. In addition, a VG 9000 Glow Discharge Mass Spectrometer (GDMS) with Nier-Johnson reverse ion optic geometry, equipped with a radio frequency source (rf-source), was used for the determination of nonconductors or insulators in addition to the normal metals and semiconductors previously determined by dc-source analysis. Further addition of a pulse generator to the rf-source resulted in a variable duty cycle, allowing greater ionization efficiency without the risk of catastrophic damage to the sample. The results of this research indicate that the LA-ICPMS system can be used to directly determine the composition of ash samples, with further method development, and that the Prf-GDMS system can be used successfully to analyze nonconductive solid samples including bone tissue.

Thesis (MScMedSc (Pathology. Medical Microbiology))--Stellenbosch University, 2008.
The genus Mycobacterium is a group of acid–fast, aerobic, slow- growing organisms which include more than 90 different species. A member of this genus, Mycobacterium tuberculosis, belonging to the Mycobacterium tuberculosis complex (MTB), is the causative agent of tuberculosis (TB). This disease is currently considered a global emergency, with more than 2 million deaths and over 8 million new cases annually. TB is the world’s second most common cause of death after HIV/AIDS. About one-third of the world’s population is estimated to be infected with TB. This catastrophic situation is further compounded by the emergence of Multi Drug Resistant tuberculosis (MDR-TB) and in more recent times, Extensive Drug Resistant tuberculosis (XDR-TB). Early diagnosis is critical to the successful management of patients as it allows informed use of chemotherapy. Also, early diagnosis is also of great importance if the menace of MDR-TB and XDR-TB is to be curbed and controlled. As MTB is highly infectious for humans, it is of paramount importance that TB be diagnosed as early as possible to stop the spread of the disease. Traditional conventional laboratory procedures involving microscopy, culture and sensitivity tests may require turnaround times of 3-4 weeks or longer. Tremendous technological advancement over the years such as the advent of automated liquid culture systems like the BACTEC® 960 and the MGITTM Tube system, and the development of a myriad of molecular techniques most of which involves nucleic acid amplification (NAA) for the rapid identification of mycobacterial isolates from cultures or even directly from clinical specimens have contributed immensely to the early diagnosis of tuberculosis. Most of these NAA tests are nevertheless fraught with various limitations, thus the search for a rapid, sensitive and specific way of diagnosing tuberculosis is still an active area of research. The search has expanded to areas that would otherwise not have been considered ‘conventional’ in diagnostic mycobacteriology. One of such areas is mass spectrometry. This study joins the relatively few studies of its kind encountered in available literature to establish the ground work for the application of mass spectrometry, specifically Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-ToF MS) in the field of diagnostic mycobacteriology. This is an area which is in need of the speed, sensitivity and specificity that MALDI-ToF technique promises to offer. Since this technology is still in its infancy, the use of utmost care in the preparation of reagents, and the handling and storage of the organisms used to generate reference mass spectra for the database cannot be overemphasized. Similarly, the optimization of certain crucial experimental factors such as inactivating method and choice of matrix is of paramount importance. The main aim of this thesis was to generate a database of reference mass spectra fingerprints of selected (repository) Mycobacterium species. This necessitated the standardization of an experimental protocol which ensured that experimental factors and the various instrument parameters were optimized for maximum spectra generation and reproducibility. A standard operating procedure (SOP) for generating the database of reference mass spectra finger print of selected Mycobacterium species was developed and used to investigate the ability of the database to differentiate between species belonging to the same clinical disease complex as well as the nontuberculosis complex. The findings of this study imply that if the defined protocol is followed, the database generated has the potential to routinely identify and differentiate (under experimental conditions) more species of Mycobacterium than is currently practical using PCR and its related techniques. It is therefore a realistic expectation that when the database is clinically validated and tested in the next phase of the study, it will contribute immensely to the diagnosis of tuberculosis and other mycobacterioses. It will also aid in the identification of emerging pathogens particularly amongst the non-tuberculous mycobacteria.

This thesis evaluates the ability of ¹H nuclear magnetic resonance (NMR) spectroscopic and ultra performance liquid chromatography-mass spectrometry (UPLC-MS) strategies to extract latent biochemical information from complex biofluid matrices from animal models and humans. ¹H NMR spectroscopy was applied to urine samples to investigate time-dependent biochemical changes in acid-base balance studies of metabolic alkalosis and metabolic acidosis and in a study of caloric restriction in order to establish normal physiological variation in organic acids including tricarboxylic acid (TCA) cycle intermediates. A method for profiling organic acids in human urine was developed, optimised and validated using UPLC-MS and then applied to targeted and untargeted metabolic profiling. A combined approach using both techniques was then applied in order to assess the relative efficiencies and merits of ¹H NMR spectroscopy and UPLC-MS for diagnosis of unknown inborn errors of metabolism (IEM) diseases using human urine samples in a blind fashion. Both NMR and UPLC-MS analytical platforms detected and identified all of the IEM correctly and revealed complementary biochemical information from IEM and controls. This evaluation was further extended and scaled up to assess the capability of the technologies for extracting latent biological information from urine samples collected in a large human population study. Chemometric methods such as Principal Component Analysis (PCA) and Orthogonal Projection to Latent Structure Discriminant Analysis (O-PLS-DA) were used to interpret and compare the two spectroscopic data types. These statistical methodologies allowed the detection of population specific biomarkers and the establishment of key metabolic phenotype differences in selected populations. In particular, both analytical approaches were able to discriminate Icelandic participants from other populations. The methods developed as part of this project should prove useful in widespread metabonomic applications in pharmaceutical and disease related areas.

Thesis (MSc.) (Physics) --University of Limpopo, 2007
Molecular dynamics simulations have been carried out in order to examine the mechanism of diffusion of molecules in amorphous polymer matrix. PDMS model was folded in to a periodic cell, generated by rotational isomeric state (RIS) method at a prescribed temperature and density. Molecular dynamics was used to study transport properties of cyclic PDMS oligomers (hexa-methylcyclotrisiloxane (D3), octa-methylcyclotetrasiloxane (D4) and deca-methylcyclopentasiloxane (D5) using Dreiding and COMPASS force fields. Diffusion coefficients were calculated from the Einstein relation. Only D3 penetrant reached the long time limit from which the Einstein relation is satisfied. Analysis of displacement versus time for all the penetrants in PDMS matrix indicates that the penetrant motion is characterized by relatively long periods interspersed with fairly long and small jumps. Transport of solvent molecules occurs by jumps between individual sections of free volume (cavity/hole) through temporarily open channels.
The National Research Foundation (NRF) and Eskom

Orientador: Fabiana Ferreira de Souza
Resumo: Os objetivos deste estudo foram validar a sonda fluorescente (MitoStatus Red) para análise de potencial mitocondrial em espermatozoide de cães por citometria de fluxo, associada a análise de integridade de membrana plasmática e do acrossomo (experimento I). Além disso, foi conduzida análise proteômica (abordagem shotgun) do plasma seminal de cães, sua correlação com a congelabilidade do sêmen e capacidade de ligação das células espermáticas em membrana perivitelina da gema de ovo (TL) (experimento II). No experimento I foram utilizado 10 ejaculados, com motilidade espermática >75% e as concentrações de 20 nM, 50 nM, 100 nM e 200 nM da sonda MitoStatus Red foram comparadas na citometria de fluxo. Concluiu-se que 20 nM foi a concentração ideal, sem danos as estruturas celulares. No experimento II foram utilizados 10 animais, 2 ejaculados/animal (n = 20). Os ejaculados foram avaliados por cinética espermática, citometria de fluxo (potencial mitocondrial, integridade de membrana plasmática e de acrossomo) e TL. A análise proteômica do plasma seminal foi avaliada por espectrometria de massas. Os ejaculados foram divididos em 3 clusters (cluster Low, cluster Medium, cluster Hight) de acordo com os parâmetros espermáticos pré e pós-descongelação. Os grupos foram correlacionados com as proteínas encontradas no plasma seminal. Concluiu-se que existe relação entre presença ou ausência de proteínas do plasma seminal de acordo com a qualidade espermática.
Mestre

Au cours de son cycle de vie, le principe actif se retrouve en solution dans différentes situations : dans desformes pharmaceutiques liquides, dans l’organisme et dans les eaux usées. Or, par rapport à l’état solide, lamise en solution du principe actif l’expose davantage à des facteurs susceptibles de conduire à sa dégradation.Les transformations modifient sa structure chimique et donc potentiellement ses activités pharmacologiques ettoxicologiques.L’objectif de ce travail de thèse est de présenter une méthodologie et des études visant à prédire le devenir ensolution de principes actifs et les impacts potentiels consécutifs à leur dégradation.Trois principes actifs ont été sélectionnés pour la réalisation de ce travail. Ils ont en commun de présenter,d’une part, une activité pharmacologique élevée corrélée à une toxicité potentielle de leurs produits dedégradation et, d’autre part, l'absence de données sur leurs comportements en solution. Dans tous les cas,bien que le contexte soit singulier pour chaque molécule, l’approche méthodologique suivie intègre aussi biendes travaux expérimentaux que des études ab initio et in silico.La première étude porte sur le devenir de l’apixaban, principe actif actuellement commercialisé sous formeorale solide, en solution aqueuse. Les données expérimentales ont mis en évidence des groupementschimiques du principe actif pouvant contribuer à son instabilité. L’approche ab initio a permis d’expliquer larégio-spécificité de la réaction d’hydrolyse dépendamment du pH. À partir de la structure des produits dedégradation caractérisés, l’étude de leur potentiel toxique a été réalisée par approche in silico. Ces donnéesconcourent à la démarche d'analyse et évaluation des risques déployée lors de développements de formespharmaceutiques liquides ou des situations particulières impliquant la mise en solution de l'apixaban aumoment de l'administration.De telles approches ont également été employées pour caractériser les mécanismes de photodégradation del’argatroban et évaluer le potentiel toxique des produits de dégradation. Les processus initiant laphotodégradation ont fait l’objet d’études complémentaires reposant sur des calculs d’énergies. Cesconnaissances pourront apporter le rationnel nécessaire au choix de procédés capables de réduire laphotodégradation de l’argatroban et son impact sur les patients. Elles pourront également servir à anticiper lessituations d’écarts pouvant mettre en jeu le rapport bénéfice risque du médicament telles que le mésusage oula modification de la forme pharmaceutique administrée.Enfin, dans un contexte autre que le contexte pharmaceutique, une étude de dégradation du pémétrexed parphotocatalyse via un procédé d'oxydation avancée a été réalisée. Il s'agit d'un procédé particulièrement étudiépour sa capacité à réduire l’empreinte environnementale de composés organiques en accélérant leurdégradation. Le choix de ce principe actif utilisé comme anticancéreux a été justifié par son caractère toxiqueet rémanent dans les eaux de surface, ce qui en fait un produit à haut risque environnemental. Ce travail amontré que des produits de plus faible masse résultant de la transformation photocatalytique du pémétrexedsont malheureusement plus toxiques et encore plus rémanents que la molécule mère elle-même. Ces résultatscontribuent donc à souligner que les procédés d'oxydation avancée, bien qu'efficaces pour l'élimination despolluants médicamenteux, sont à évaluer au regard de l'existence d'un risque accru pour l'environnementavant toute perspective d'utilisation à grande échelle.Les approches et les résultats présentés dans cette thèse pourront être employés pour d’autres études visant àprédire, prévenir et réduire l’impact de la dégradation du principe actif sur le patient et l’environnement
During its life cycle, an active substance is in solution for various reasons: in a liquid pharmaceutical form, in the body and in wastewater. However, compared to the solid state, the active substance in solution exposes it more to factors likely to cause its degradation. The transformations modify its chemical structure and thus potentially its pharmacological and toxicological activities.The objective of this thesis is to present a methodology and studies aiming to predict the fate in solution of active substances and the potential impacts following their degradation.Three active ingredients have been selected for this work. They have in common, on the one hand, a high pharmacological activity correlated to a potential toxicity of their degradation products and, on the other hand, the fact that there is little information on their behaviour in solution. In all cases, although the context is specific to each molecule, the methodological approach followed integrates both experimental work and ab initio and in silico studies.The first study concerns the fate of apixaban, an active substance currently marketed in solid oral form, in aqueous solutions. The experimental data made it possible to highlight chemical groups of the active ingredient that could contribute to its own instability. The ab initio approach explained the regio-specificity of the hydrolysis reaction as a function of pH. Based on the structure of the characterized degradation products, their toxic potential was studied using an in silico approach. These data contribute to the risk analysis and evaluation process deployed at different stages of development of liquid pharmaceutical forms or in particular situations involving the solution of apixaban at the time of administration.Such approaches have also been used to characterize the photodegradation mechanisms of argatroban and assess the toxic potential of degradation products. The processes that initiate photodegradation were also addressed by calculating the energies potentially involved. This knowledge provides a rational basis for the choice of processes and formulations to limit photodegradation of argatroban and its impact on patients. They also make it possible to anticipate situations where the benefit/risk ratio of the medicinal product may be modified, such as incorrect handling or modification of the pharmaceutical form administered.Finally, in a context other than the pharmaceutical context, a study of degradation of pemetrexed by photocatalysis via an advanced oxidation process was carried out. This process is particularly studied for its ability to reduce the environmental footprint of organic compounds by accelerating their degradation. The choice of this active substance as an anti-cancer agent was justified by its toxic and persistent nature in surface waters, making it a product with a high environmental risk. This work has shown that products of lower mass produced by photocatalytic transformation of pemetrexed are unfortunately more toxic and even more persistent than the parent molecule itself. These results underline the fact that advanced oxidation processes, although effective in removing drug pollutants, must be evaluated because of an increased risk to the environment before any prospect of large-scale use.The approaches and results presented in this thesis can be used for other studies to predict, prevent and reduce the impact of active ingredient degradation on the patient and the environment

Advances in chemical and bio-technology have helped boost modern agriculture with increased food productivity and consequently, impacted the environment leading to more saline and drought prone arable lands. Mandate of world food security heavily depends on crop improvement and developing strategies to increase abiotic stress tolerance. The use of rhizobacteria and their excreted compounds is a contextually safe and viable option. Lipo-chito-oligosaccharide (LCO) from Bradyrhizobium japonicum 532C and Thuricin 17 (Th17) from Bacillus thuringiensis NEB17 are bacterial signal compounds promoting plant growth in legumes and non-legumes. The effect of these compounds at proteome level under unstressed and salt stressed conditions on Arabidopsis thaliana was studied using root drench method. The phytohormones in Arabidopsis thaliana rosettes were differential expressed at 24 h treatment. At the proteome level, > 2-fold changes in the activation of the carbon and energy metabolism pathway proteins in both LCO and Th17 were observed in comparison to control. At 250 mM NaCl stress, the control plants under osmotic-shock shut down most of the carbon-metabolism and up-regulated the energy-metabolism and antioxidant pathways, while the LCO and Th17 with salt stress retained some of the Light harvesting complex, Photosystem I and II proteins along with the up-regulation of energy and antioxidant pathways suggesting that the rosettes were able to amend the salt stress better when treated with LCO and Th17.Soybean (Absolute RR) germination for salt tolerance suggested that LCO and Th17 helped seeds germinate the best at 100 mM NaCl. The proteome suggested efficient and speedier partitioning of storage proteins, up-regulation of carbon, nitrogen and energy metabolisms in LCO and Th17 seeds in comparison with controls both under optimal and salt stress. These findings suggest that the Arabidopsis rosettes and the soybean germinating seeds alter their proteome based on bacterial signals and on stress. The specificity of this response plays a crucial role in the plants life cycle, and understanding this response is of importance in commercial application.
Les progrès de la chimie et de la biotechnologie ont contribué à stimuler l'agriculture moderne et la productivité accrue de nourriture. La conséquence en est l'impact sur l'environnement rendant les terres arables plus sujettes à la salinité et à la sécheresse. La sécurité alimentaire mondiale dépend fortement de l'amélioration des cultures et du développement de stratégies visant à accroître la tolérance aux stress abiotiques. L'utilisation de rhizobactéries et de leurs composés excrétés est dans ce contexte une option sécuritaire et viable.Le lipo-chito-oligosaccharide (LCO) du Bradyrhizobium japonicum 532C et le Thuricin 17 (Th17) du Bacillus thuringiensis NEB17 sont des composés de signaux de bactéries favorisant la croissance des plantes dans les légumineuses et les non-légumineuses. L'effet de ces composés au niveau du protéome d'Arabidopsis thaliana, sans stress et avec le stress du sel, a été étudié en utilisant la méthode par trempage de racine. Les phyto-hormones dans les rosettes d'Arabidopsis thaliana ont été exprimées de manière différentielle lors du traitement de 24h. Au niveau du protéome, des changements 2 fois supérieurs en comparaison au témoin ont été observés dans l'activation des protéines de la voie métabolique énergétique du carbone, pour LCO et pour Th17. Sous un stress produit par 250 mM de NaCl, les plants témoins sous choc osmotique ont diminué la plupart du métabolisme du carbone, et régulé à la hausse les voies du métabolisme énergétique et antioxydant ; tandis que LCO et Th17 sous stress salin ont conservé une partie du complexe de capture de la lumière, les protéines de Photosystem I et II, et régulé positivement les voies énergétiques et antioxydants. Cela suggère que les rosettes furent en mesure de modifier positivement le stress salin lorsque traitées avec LCO et Th17.La germination des graines de soja (Absolute RR) pour la tolérance au sel, a montré que LCO et Th17 aident les graines à germer de manière optimale avec 100 mM de NaCl. Pour les graines avec LCO et Th17 et par rapport aux témoins à la fois sous stress optimal et salin, le protéome suggère: une séparation des protéines de stockage éfficace et plus rapide, et la régulation à la hausse des métabolismes du carbone, de l'azote et énergétiques.Ces résultats démontrent que les rosettes d'Arabidopsis et les graines de soja en germination modifient leur protéome selon les signaux bactériens et le stress. La spécificité de cette réponse joue un rôle crucial dans le cycle de vie des plantes, et la compréhension de cette réponse est d'importance dans son application commerciale.

Thesis (Ph. D.)--Cleveland State University, 2009.
Abstract. Title from PDF t.p. (viewed July 29, 2009). Includes bibliographical references (p. 105). Available online via the OhioLINK ETD Center and also available in print.

The work in this thesis has been clearly divided into three parts: In the first part, efforts were directed to obtaining a trace level quantification method for halogenated flame retardants using high-volume injections and gas chromatography coupled to GC-MS/MS tandem mass spectrometry. In the second part, the potential of the GC-MS / MS technique was evaluated together with the new atmospheric pressure chemical ionization source (APCI) for the quantification of PBDEs, NBFRs (including hexabromocyclododecane (HBCD)) and DL-PCBs at trace levels in food and environmental samples. In the third part, two untargeted analytical methodologies by GC-MS were developed as complementary tools to the PANEL TEST for the classification of olive oil samples according to their quality.
El trabajo en la presente tesis se ha dividido claramente en tres partes: En la primera parte, los esfuerzos se dirigieron a la obtención de un método de cuantificación a nivel de trazas para retardantes de llama halogenados utilizando inyecciones de gran volumen y cromatografía de gases acoplada a espectrometría de masas en tándem GC-MS/MS. En la segunda parte, se evaluó el potencial de la técnica GC-MS/MS junto con la nueva fuente de ionización química a presión atmosférica (APCI) para la cuantificación de PBDEs, NBFRs (incluido el hexabromociclododecano (HBCD)) y DL-PCBs a niveles de traza en alimentos y muestras ambientales. En la tercera parte, se desarrollaron dos metodologías de análisis no dirigido por GC-MS como herramientas complementarias de la PRUEBA DE PANEL para la clasificación del aceite de oliva de acuerdo a su calidad.

Orientador: Lucia Maria Valente Soares
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Um bom número de espécies de fungos pode, sob condições especiais, produzir metabólitos secundários tóxicos conhecidos como micotoxinas. Cerca de 20 grupos de toxinas são conhecidos hoje, porém, algumas têm recebido atenção especial devido à sua toxicidade e ampla ocorrência natural: aflatoxinas, zearalenona, ocratoxina A, fumonisinas e tricotecenos. Os tricotecenos constituem um grupo de cerca de mais de uma centena de compostos caracterizados pela presença do sistema 12,13-epoxi-tricotec-9-eno em suas estruturas. A maior parte dos tricotecenos conhecidos foi isolada apenas em laboratório, porém, alguns deles foram obtidos e caracterizados como contaminantes naturais. Dentre estes podem ser citados o desoxinivalenol (DON), o nivalenol (NIV), a toxina T-2 (T2), o diacetoxiscirpenol (DAS) e menos freqüentemente os derivados 3-acetil-desoxinivalenol (3-Ac-DON) e 15-acetildesoxinivalenol (15-Ac-DON), a fusarenona-X (FX) e a toxina HT-2 (HT2). Entre todos, o DON é o de maior ocorrência em alimentos e rações animais, porém é também o menos tóxico. Apesar do milho (Zea Mays) ser um dos substratos mais susceptíveis a este tipo de contaminação, há poucos dados sobre o milho brasileiro. Brasil é o terceiro colocado quanto à produção mundial de milho, colhendo ao redor de 30 milhões de toneladas por ano e, portanto, um cereal com grande impacto na economia brasileira. Estes fatos apontam a necessidade de avaliar a extensão e o tipo de contaminação de tricotecenos em milho nacional. Por outro lado, a similaridade das estruturas químicas dos tricotecenos exige o uso de cromatografia com alto poder de resolução para sua separação como é como é o caso da cromatografia à gás. As diferentes toxicidades para os diversos membros do grupo requer, por sua vez, que a identidade de cada toxina seja confirmada por um sistema de alta confiabilidade como a espectrometria de massas. o presente trabalho visou avaliar a incidência de tricotecenos em milho plantado no estado de São Paulo e em produtos de milho comercializados na cidade de São Paulo. Dentre os tricotecenos, DON e NIV foram escolhidos devido a maior freqüência com que são encontrados em todo o mundo. As toxinas DAS, HT2 e T2 foram incluídas devido às suas maiores toxicidades e ocorrência natural comprovada em alimentos e rações. Para alcançar os objetivos descritos foi inicialmente avaliado método para determinação simultânea destes 5 tricotecenos por cromatografia à gás associada à espectrometria de massas. O método analítico após otimização e avaliação apresentou limites de detecção variando de 20 a 60 ng/g para DON, de 10 a 40 ng/g para NIV, de 20 a 120 nglg para DAS, de 20 a 50 ng/g para HT2 e de 20 a 100 ng/g para T2, de acordo com a as matrizes testadas (milho em grão, milho verde em lata, farinha de milho, canjica, fubá e flocos de milho). Da mesma forma, as recuperações variaram de 83 a 113% para DON, de 84 a 115% para NIV, de 69 a 123% para DAS, de 82 a 155% para HT2 e de 71 a 96% para T2. Foram analisadas 80 amostras de milho produzido em duas cooperativas do estado de São Paulo, tendo sido encontradas 11 amostras contendo NIV e uma com DON em nível de traços. Além disso, cinco amostras apresentaram NIV em níveis variando de 51 a 106 ng/g e uma delas co-contaminada com 71 ng/g de DON. Foram também analisadas 78 amostras de produtos de milho comercializados na cidade de São Paulo, sendo que uma delas, farinha de milho, apresentou traços de DON e NIV e outra, quirera, apresentou 555 ng/g e 767 ng/g de toxinas T2 e HT2, respectivamente. Os resultados obtidos nos levantamentos realizados nos anos de 2000 a 2002, mostram baixa ocorrência de tricotecenos, não havendo risco para a população no consumo destes produtos. Devido à sazonal idade da contaminação por micotoxinas em alimentos, conclusões mais amplas só serão possíveis após vários anos de observação da presença dessas toxinas em milho plantado no estado de São Paulo e produtos de milho comercializados na cidade de São Paulo
Abstract: A great number of the fungal species, under specific conditions, can produce toxic secondary metabolites known as mycotoxins. About 20 groups of such toxins are known today but some have received special attention due to their toxicity and widespread natural occurrence: aflatoxins, zearalenone, ochratoxin A, fumonisins and trichothecenes. The trichothecenes constitute a group of more than a hundred compounds characterized by the presence of the system 12,13-epoxy-trichotec-9-en in their structures. However, most of the known trichothecenes have been isolated only in laboratory conditions. Just a few have been isolated and characterized as natural contaminants. Among them, deoxynivalenol (DON), nivalenol (NIV), toxin T-2 (T2) , diacetoxyscirpenol (DAS) and less frequently the derivatives 3-acetyldeoxynivalenol (3-Ac-DON), 15-acetyl-deoxynivalenol (15-Ac-DON), fusarenone X (FX) and the toxin HT-2 (HT2). DON is the trichothecene most frequently found in foods and feeds and it's also the less toxic of this family. Despite the fact that com is one of the substrates more susceptible to this type of contamination, there is little information on the occurrence of trichothecenes in Brazilian com. Brazil ranks third in world production of com, harvesting about 30 million tons annualy, and com is a grain with great impact in Brazilian economy. These facts point to the need to evaluate the extension and the type of trichothecene contamination in national corn. On the other hand, the similarity of the trichothecene chemical structures requires the use of a high resolution gas chromatographic method for their separation, The differing toxicities for the members of this group of toxins requires, by their turn, that the identity of each toxin be confirmed by a system capable of great degree of reliability such as mass spectrometry. The present work aimed at evaluating the occurrence of trichothecenes in com p/anted in the State of São Pau/o and in com products commercialized in the city of São Paulo. DON and NIV were chosen for this investigation due to their worldwide occurrence. DAS, HT2 and T2 were inc/uded due to their high toxicity and their natural occurrence in foods and feeds. In order to reach the objectives of the present work, a gas chromatography/ mass spectrometry method for simultaneous determination of these tive trichothecenes was optimized and evaluated. Afier optimization the method presented detection limits ranging from 20 to 60 ng/g for DON, from 10 to 40 ng/g for NIV, 20 to 120 ng/g for DAS, 20 to 50 for HT2 and from 20 to 100 ng/g for T2, varying according to the tested matrices (dried com, canned sweet com, com grits, com flour, hominy sweet meal, com meal and com flakes). Similarly, the recoveries ranged from 83 to 113% for DON, 84 to 115% for N/V, 69 to 123% for DAS, 82 to 155% for HT2 and 71 to 96% for T2. A survey was conducted in samp/es of com grown in São Paulo State and also in com-based products commercia/ized in the city of São Paulo. Eighty samples of com from two cooperatives located in the State of São Paulo were analysed. E/even samples presented traces of N/V and another sample had traces of DON. Besides that, tive samples showed levels of N/V ranging trom 51 to 106 ng/g and one of them was co-contaminated with 71 ng/g of DON. Seventy-eight samp/es of com-based products commercialized in Sao Paulo city were analyzed. Among these samples, on/y one sample of com flour, presented DON and N/V. Another sample of com grits , had the toxins T2 an HT2 at the leve/s of 555ng/g and 767 ng/g, respectively. The resu/ts of the two surveys during the years 2000 to 2002, indicated a low occurrence of trichothecenes, and as a consequence a low risk for the consumers. Due to the well known variability in mycotoxins contamination of foods from year to year, only after several years of surveyng corn planted in the State of São Paulo and com products commercialized in the city of São Paulo, it will be possible to have a complete picture of the contamination of trichothecenes in com and com products
Doutorado
Doutor em Ciência de Alimentos

Grande parte do desenvolvimento científico nas análises em alimentos está relacionado à identificação e quantificação de resíduos de drogas veterinárias e contaminantes ambientais em matéria-prima. Dentre as substâncias mais amplamente estudadas como resíduos em alimentos estão os praguicidas. Cada vez mais as legislações e normas reguladoras buscam garantir que os alimentos levados aos consumidores estejam apropriados à ingestão, diminuindo o risco negligenciável à saúde, através da exigência de níveis cada vez menores dos limites máximos de resíduo, e muitas vezes, banindo diversas substâncias com altos e médios graus de toxicidade. Para tal, tem sendo intensificado o desenvolvimento de métodos precisos, exatos, robustos e baratos para a identificação e quantificação de praguicidas, de forma que tanto os órgãos públicos como as empresas privadas possam realizar o monitoramento dos alimentos oferecidos pelos produtores e fornecedores, e assim identificar possíveis fontes de exposição à substâncias que levem risco através da ingestão diária. Os indivíduos que apresentam maior suscetibilidade aos efeitos nocivos causados pela presença de praguicidas em níveis tóxicos nos alimentos são crianças e idosos, o que exige cuidados e limites ainda mais rígidos. Para garantir que os alimentos infantis fornecidos no mercado se mantenham dentro das condições adequadas para a ingestão, a sua produção deve ser controlada e monitorada. O presente trabalho visa avaliar a presença de praguicidas em Solanum tuberosum (batata), Daucus carota (cenoura) e Arracacia xanthorrhiza (mandioquinha) destinadas para a produção de baby-food e produtos similares, cultivados na área de São José do Rio Pardo, no estado de São Paulo, e disponíveis no varejo da cidade de São Paulo, através da técnica QuEChERS e identificação e quantificação por LC-MS/MS. Através da otimização dos parâmetros de espectrometria de massas e validação do método de extração QuEChERS para 216 compostos, foram identificados quais praguicidas possuíam a capacidade de serem detectados e quantificados com a performance adequada por cromatografia líquida acoplada a espectrometria de massas em tandem nas commodities estudadas. Amostras de batata, cenoura e mandioquinha de fornecedores da área de São José do Rio Pardo e fontes de varejo da cidade de São Paulo foram analisadas para identificar a presença dos analitos validados. Para as amostras investigadas, somente a commodity cenoura apresentou praguicidas quantificados acima do valor da Capacidade de Detecção (CCβ), sendo eles o Linurom, o Tebuconazol e o Triciclazol. Realizada a Avaliação de Risco, verificou-se que a exposição aos praguicidas nas commodities, mesmo nos casos de maior concentração, não era evidenciada como um risco, tendo em vista que os mesmos obtiveram resultados de 0,25% (Linurom), 0,1% (Tebuconazol) e 0,03% (Triciclazol) dos valores de suas Ingestões Diárias Aceitáveis (IDA).
Great part of the scientific development on food analysis is related to the identification and quantification of veterinary drugs and environmental contaminants residues in raw materials. Among the most studied residue substances in food are the pesticides compounds. Increasingly, legislations and regulatory standards seek to ensure that the food served to consumers are appropriate to the intake, reducing a negligible health risk, by requiring each time lower maximum residue levels, and often banning various substances with high and medium levels of toxicity. With this purpose, the development of accurate, robust and cheap methods has been intensified for the identification and quantification of pesticides, so that both public bodies and private companies are able to carry out the monitoring of food and feed offered by the producers and suppliers, and by doing so, identify possible sources of exposure to substances that lead to risk by daily intake. The individuals who are more susceptible to adverse effects caused by the presence of pesticides in toxic levels in food are children and the elderly, and because of this fact, the requirement for extensive care and limits even stricter is higher. In the attempt to ensure that children\'s food and feed market supplies fall within the right conditions for ingestion, its production should be controlled and monitored. The present study aims to evaluate the presence of pesticides in Solanum tuberosum (potato), Daucus carota (carrot) and Arracacia xanthorrhiza (mandioquinha) in samples grown in the area of São José do Rio Pardo, in the State of São Paulo, and directed to baby-food and similar products production, and in samples available at the market retail in the city of São Paulo, through the QuEChERS technique, and identification and quantification by LC-MS/MS. The pesticides molecules which had the ability to be detected and quantified with adequated performance by liquid chromatography coupled to tandem mass spectrometry in the studied commodities were identified by optimizing the mass spectrometer parameters and validating the QuEChERS extraction method of 216 compounds. Potato, carrot and mandioquinha samples, provided by suppliers in the area of Sao Jose do Rio Pardo and retail sources from São Paulo, were analyzed for the presence of the validated pesticides. Among the investigated samples, only the carrot commodity presented quantified pesticides above the value of Detection Capability (CCβ), namely Linuron, the Tebuconazole and Tricyclazole. Held the chemical risk assessment, it was found that the exposure to pesticides by the commodities, even in cases of higher concentrations, was not shown as a risk, given that they achieved results of 0.25% (Linuron), 0, 1% (Tebuconazole) and 0.03% (Tricyclazole) of the Acceptable Daily Intakes (ADI) values.

Mestrado em Biologia Molecular e Celular
A heterogenidade da natureza molecular dos tumores de bexiga tem dificultado o estabelecimento de abordagens no campo da medicina de precisão, revelando-se a necessidade de terapias mais eficientes e novas ferramentas de detecção não-invasivas. Contudo, têm-se denotado um desenvolvimento no estudo da carcinogénese de bexiga e na progressão do tumor, acompanhado de profundas alterações na glicosilação de proteínas que, dada a sua superfície celular e a natureza secretada, apresenta um potencial elevado na melhoria da gestão da doença. Segundo esta abordagem foi efectuado um estudo sobre tumores de bexiga de diferentes naturezas clinicopatológicas para O-glicanos de cadeia curta, regularmente encontrados na maioria dos tumores sólidos, recorrendo-se à imunohistoquímica. O estudo incluiu os antígenos Tn e T e os seus homólogos sialilados sialil-Tn (STn) e sialil-T (ST), geralmente associados com um mau prognóstico. Explorou-se ainda a sialilação da natureza dos antigénios T, especificamente as sialoformas sialil-3-T (S3T) e sialil-6-T (S6T), com base em combinações de tratamentos enzimáticos. Observou-se uma predominância de sialoglicanos, em comparação com as glicoformas neutras (antígenos Tn e T) em tumores de bexiga. Em particular, o antigénio STn foi associado ao estado avançado da doença e invasão muscular. Os antígenos S3T e S6T foram detectados pela primeira vez em tumores de bexiga, estando ausentes no urotélio normal, permitindo destacar a natureza específica em tumores. Verificou-se também a sobreexpressão dos glicanos em lesões avançadas, especialmente nos casos com invasão muscular.As análises glicoproteómicas dos tumores avançados de bexiga permitiram identificar diversas glicoproteínas-chave associadas ao cancro (MUC16, CD44, integrinas), denotando uma glicosilação alterada.As glicoformas da MUC16 STN positivas, características do cancro de ovário, encontram-se num subconjunto de tumores de bexiga em estado avançado, com um pior prognóstico. Em suma, os tumores de bexiga apresentam severas alterações no O-glicoma e no Oglicoproteoma devendo ser abordados de forma abrangente com o objectivo de desenvolver ferramentas de diagnóstico não invasivas e terapias dirigidas. As glicoformas aberrantes de MUC16 apresentam potencial como biomarcadores de mau prognóstico. Este trabalho estabeleceu um guia para a descoberta de glicobiomarcadores no cancro de bexiga, que pode ser utilizado para a estratificação dos pacientes e, por fim, levar à descoberta de novos alvos terapêuticos.
The heterogeneous molecular nature of bladder tumours has hampered the establishment of precision medicine approaches, more efficient therapeutics and novel non-invasive detection tools. Still, it has been long described that bladder carcinogenesis and tumour progression is accompanied by profound alterations in protein glycosylation which, given its cell surface and secreted nature, holds tremendous potential for disease management improvement. Therefore, we have screened series of bladder tumours of different clinicopathological natures for short-chain O-glycans, found in most solid tumours, by immunohistochemistry. These included the Tn and T antigens and their sialylated counterparts sialyl-Tn (STn) and sialyl-T(ST), generally associated with poor prognosis. We have also explored the nature of T antigens sialylation, namely the sialyl-3-T(S3T) and sialyl-6-T(S6T) sialoforms, based on combinations of enzymatic treatments. We observed a predominance of sialoglycans over neutral glycoforms (Tn and T antigens) in bladder tumours. In particular, the STn antigen was associated with high-grade disease and muscle invasion, in accordance with our previous observations.The S3T and S6T antigens were detected for the first time in bladder tumours, but not in healthy urothelia, highlighting their cancer-specific nature. These glycans were also overexpressed in advanced lesions, especially in cases showing muscle invasion. Glycoproteomic analyses of advanced bladder tumours identified several key cancer-associated glycoproteins (MUC16, CD44, integrins) carrying altered glycosylation. Particular interest was devoted to MUC16 STn+-glycoforms, characteristic of ovarian cancers, which were found in a subset of advanced stage bladder tumours facing worst prognosis. In summary, bladder tumours present severe O-glycome and O-glycoproteome alterations that should be comprehensively addressed envisaging novel non-invasive diagnostic tools and targeted therapeutics. Furthermore, abnormal MUC16 glycoforms holds potential as surrogate biomarkers of poor prognosis. Finally, this work established a roadmap for glycobiomarker discovery in bladder cancer, which may be used for patient stratification and ultimately lead to novel therapeutic targets.

Streptococcus gordonii is a primary coloniser of the tooth surface where it efficiently ferments carbohydrates at pH levels above 6.0. By not being able to maintain the pH of dental plaque to a level required for enamel dissolution, the dominance of S. gordonii in dental plaque is considered a sign of a healthy oral cavity. However, upon entering the bloodstream and encountering a rise in pH, S. gordonii may become pathogenic, being one of the major causative organisms associated with infective endocarditis. Proteome analyses of S. gordonii grown at steady state in a chemostat allowed the phenotypic changes associated with alterations in pH levels characteristic of these two environments to be determined. As an initial starting point to this study, a two-dimensional electrophoresis (2- DE) reference map of S. gordonii grown at pH 7.0 was produced. Although only 50% of the S gordonii genome was available in an annotated form during the course of this study, the closely related Streptococcus pneumoniae genome (with which S. gordonii shares 97.24% DNA sequence homology) had been completed in 2001. The use of both of these databases allowed many of the S. gordonii proteins to be identified by mass spectrometry. Four hundred and seventy six protein spots, corresponding to 250 different proteins, or 12.5% of the S. gordonii proteome, were identified, giving rise to the first comprehensive proteome reference map of this oral bacterium. Of the 250 different proteins, 196 were of cellular origin while 68 were identified from the extracellular milieu. Only 14 proteins were common to both compartments. Of particular interest among the 54 uniquely identified extracellular proteins was a homologue of a peptidoglycan hydrolase that has been associated with virulence in S. pneumoniae. Among the other proteins identified were ones involved in transport and binding, energy metabolism, translation, transformation, stress response and virulence. Twelve cell envelope proteins were identified as well as 25 others that were predicted to have a membrane association based on the presence of at least one transmembrane domain. The study also confirmed the existence of 38 proteins previously designated as �hypothetical� or with no known function. Mass spectral data for over 1000 protein spots were accumulated and archived for future analysis when sequencing of the S. gordonii genome is finally completed. Following the mapping of the proteome of S. gordonii, alterations in protein spots associated with growth of the bacterium at pH intervals of 0.5 units in the pH range 5.5 - 7.5 were determined. Only 16 protein spots were shown to be significantly altered in their level of expression despite the range of pH studied. Among the differentially expressed proteins was a manganese-dependent inorganic pyrophosphatase (PpaC), which regulates expression of adhesins required for coaggregation. The expression of PpaC was highest at pH 6.5 - 7.0, the pH of a healthy oral cavity, indicating that PpaC may play an important part in dental plaque formation. Another differentially expressed protein was the heat-inducible transcription repressor (HrcA). Alterations in HrcA were consistent with its role as a negative repressor in regulating heat-shock proteins at low pH, even though no changes in the level of heat-shock proteins were observed as the pH declined. This result gave rise to the hypothesis that the possible reason cariogenic bacteria, such as Streptococcus mutans, can out compete S. gordonii at low pH might simply be due to their ability to manipulate their proteome in a complex manner for survival and persistence at low pH, unlike S. gordonii. This may imply some prevailing level of genetic regulation that is missing in S. gordonii.

Streptococcus gordonii is a primary coloniser of the tooth surface where it efficiently ferments carbohydrates at pH levels above 6.0. By not being able to maintain the pH of dental plaque to a level required for enamel dissolution, the dominance of S. gordonii in dental plaque is considered a sign of a healthy oral cavity. However, upon entering the bloodstream and encountering a rise in pH, S. gordonii may become pathogenic, being one of the major causative organisms associated with infective endocarditis. Proteome analyses of S. gordonii grown at steady state in a chemostat allowed the phenotypic changes associated with alterations in pH levels characteristic of these two environments to be determined. As an initial starting point to this study, a two-dimensional electrophoresis (2- DE) reference map of S. gordonii grown at pH 7.0 was produced. Although only 50% of the S gordonii genome was available in an annotated form during the course of this study, the closely related Streptococcus pneumoniae genome (with which S. gordonii shares 97.24% DNA sequence homology) had been completed in 2001. The use of both of these databases allowed many of the S. gordonii proteins to be identified by mass spectrometry. Four hundred and seventy six protein spots, corresponding to 250 different proteins, or 12.5% of the S. gordonii proteome, were identified, giving rise to the first comprehensive proteome reference map of this oral bacterium. Of the 250 different proteins, 196 were of cellular origin while 68 were identified from the extracellular milieu. Only 14 proteins were common to both compartments. Of particular interest among the 54 uniquely identified extracellular proteins was a homologue of a peptidoglycan hydrolase that has been associated with virulence in S. pneumoniae. Among the other proteins identified were ones involved in transport and binding, energy metabolism, translation, transformation, stress response and virulence. Twelve cell envelope proteins were identified as well as 25 others that were predicted to have a membrane association based on the presence of at least one transmembrane domain. The study also confirmed the existence of 38 proteins previously designated as �hypothetical� or with no known function. Mass spectral data for over 1000 protein spots were accumulated and archived for future analysis when sequencing of the S. gordonii genome is finally completed. Following the mapping of the proteome of S. gordonii, alterations in protein spots associated with growth of the bacterium at pH intervals of 0.5 units in the pH range 5.5 - 7.5 were determined. Only 16 protein spots were shown to be significantly altered in their level of expression despite the range of pH studied. Among the differentially expressed proteins was a manganese-dependent inorganic pyrophosphatase (PpaC), which regulates expression of adhesins required for coaggregation. The expression of PpaC was highest at pH 6.5 - 7.0, the pH of a healthy oral cavity, indicating that PpaC may play an important part in dental plaque formation. Another differentially expressed protein was the heat-inducible transcription repressor (HrcA). Alterations in HrcA were consistent with its role as a negative repressor in regulating heat-shock proteins at low pH, even though no changes in the level of heat-shock proteins were observed as the pH declined. This result gave rise to the hypothesis that the possible reason cariogenic bacteria, such as Streptococcus mutans, can out compete S. gordonii at low pH might simply be due to their ability to manipulate their proteome in a complex manner for survival and persistence at low pH, unlike S. gordonii. This may imply some prevailing level of genetic regulation that is missing in S. gordonii.

The aim of the project described in this thesis was to develop a system in house that would be capable of providing a technique to enhance the reliability of detection of threat agents such as compounds used for chemical warfare and explosives. This was to be done by using a combination of an ion mobility spectrometer (IMS) in tandem with a quadrupole mass spectrometer (QMS). When meeting these requirements, the latest electronics and software were incorporated in the instrumentation to maximise sensitivity and flexibility. By attaching a QMS to an IMS, an extra dimension in specificity is gained whereby a more positive identification of a compound is made based on m/z values, thereby providing further information on the ion-molecule processes taking place in the IMS. Flexibility in operation was achieved by using the graphical programming language LabVIEW for the software aspects, allowing program development and modification to be made more quickly than would be the case than if a procedural language such as C++ had been used. A special ‘pulse to analogue’ converter developed during the project provided increased sensitivity and resolution over earlier systems in regard to obtaining selected mass mobility spectra. Proof-of-principle measurements are provided that demonstrate the capabilities of the newly developed IMS-QMS system in both positive and negative ion modes of operation, with some results obtained that are consistent with those from previous investigations. Data obtained for various chemicals not previously investigated are also provided.

Les travaux présentés dans ce mémoire concernent l’étude des plasmas d’argon créés dans une cavité résonnante micro-ondes fonctionnant à la pression atmosphérique et leur application au nettoyage de surface. Tout d’abord, une étude des enjeux du nettoyage de surfaces industrielles est présentée ainsi qu’un état de l’art des solutions existantes et leurs limitations, mettant en évidence l’intérêt des plasmas comme alternative, notamment ceux fonctionnant en cavité résonnante micro-ondes à pression atmosphérique dont les particularités sont présentées. Dans le cas de l’argon, ces décharges présentent la particularité de ne pas être homogènes mais constituées de un ou plusieurs filaments de faibles diamètres, dépendant des conditions expérimentales. L’étude de la filamentation de ces décharges est l’objet du second chapitre où il a été mis en évidence les corrélations, dans le cas d’un filament unique, entre ses dimensions, sa température et la puissance dissipée et qu’il existait un seuil de puissance au-delà duquel la filamentation apparaissait. Une modélisation électromagnétique simple a été réalisée permettant de décrire l’influence des paramètres principaux de la décharge sur la filamentation. Le troisième chapitre présente les résultats de la caractérisation d’un filament d’argon par absorption laser en plasma continu et pulsé. L’effet de l’addition d’oxygène y est également présenté. Le dernier chapitre concerne l’étude de l’application des post-décharges micro-ondes à la pression atmosphérique créées dans des mélanges argon-azote et argon-oxygène au nettoyage de surface. On y étudie notamment l’interaction de ces post-décharges avec des molécules organiques modèles (acide stéarique et 1-octadécène). L’analyse de surface avec des techniques d’analyse d’extrême surface par spectrométrie de masse (ToF-SIMS et FTMS) a permis d’améliorer notre compréhension des mécanismes de nettoyage
The present work deals with the study of argon microwave plasmas generated in resonant cavity at atmospheric pressure and their application to surface cleaning. First, a study of the aim of surface cleaning of industrial surfaces is presented, followed by a state of the art of existing solutions and their limitations, showing the interest of plasmas as an alternative, especially atmospheric pressure microwave resonant cavity plasmas. In the case of argon, these plasmas have the particularity to be inhomogeneous and constituted of one or many small diameter filaments, depending on experimental conditions. The study of the filamentation of these discharges is the subject of the second chapter. In the case of one filament, correlations have been evidenced between its size, its temperature and the dissipated power. A simple electromagnetic simulation allowed us to describe the influence of the main plasmas parameters on the filamentation process. The third chapter presents results from the characterisation of a single argon filament by the mean of diode laser absorption in continuous and pulsed plasma mode. The effect of oxygen addition is also studied. The last chapter deals with the study of the use of atmospheric pressure microwave post-discharges in argon-nitrogen or argon-oxygen mixtures for surface cleaning application. We studied the interaction of such post-discharges with model organic molecules (stearic acid and 1-octadecene). Surface analyses by the mean of extreme surface analysis techniques based on mass spectrometry (ToF-SIMS and FTMS) allow us to improve our understanding of cleaning mechanisms

Programmed cell death in animals, plants and protists is in part regulated by a variety of proteases, including cysteine aspartyl proteases, (caspases, paracaspases and metacaspases), cathepsins, subtilisin-like serine proteases, vacuolar processing enzymes and the proteasome. The role of different proteases in the cell death responses of the fungi is however largely unknown. A greater understanding of the fungal cell death machinery may provide new insights into the mechanisms and evolution of PCD and potentially reveal novel targets for a new generation of antifungal drugs. The role of a metacaspase encoding gene, MCA1, in the cell death response of the human pathogen Candida albicans pathogen has been investigated by functional analysis. MCA1 deletion not only alters the sensitivity of cells to a number of cell death stimuli, it also enhances virulence in an insect model. C. albicans shows altered cell and colony morphology on Lee’s medium. Evidence is presented to suggest that these functions appear to be dependent upon active mitochondria. In this study it has also been shown that key caspase substrates may be conserved between humans and the yeasts Saccharomyces cerevisiae and Candida albicans. Many substrates, particularly those which are essential, have retained their caspase cleavage motifs. 14 protease mutants displayed altered activity against caspase 1, 3, 6 or 8 substrates during acetic acid-induced PCD and caspase 1-like activity appeared to be particularly associated with PCD. Using a novel bioinformatic analysis of experimental LC-MS/MS data, changes in the degradation patterns of the proteome (destructome) following acetic acid-induced cell death have been investigated in wild-type yeast. In addition, potential native substrates of the yeast Mca1 have also been identified. The future challenge is to characterise the destructome of different proteases under a range of cell death conditions. In this way it may be possible to identify key components of the cell death machinery and their substrates and so reveal the most promising targets for future therapeutics.

The low efficiency of wastewater treatment plants to achieve the complete removal of micropollutants, including pharmaceuticals, has motivated the development of alternative water technologies to improve their efficiency, sustainability, and operational costs. However, even when the complete elimination of these contaminants is attained, they can be transformed into new and unknown intermediates which might be even more persistent and toxic than their parent compounds. In this doctoral thesis, the development of advanced suspect screening methodologies has been applied for the identification of the transformation products generated along biological and physical and/or chemical treatments. Additionally, their potential environmental effects were evaluated using in silico methods and in vitro bioassays in treated effluents. Finally, their removal efficiency was investigated in combined water treatment technologies. This doctoral thesis demonstrates that multidisciplinary research is needed to properly evaluate the best water treatment technology to use
La baja eficiencia de las plantas de tratamiento de aguas residuales para lograr la completa eliminación de microcontaminantes, incluidos los fármacos, ha motivado el desarrollo de tecnologías alternativas para mejorar su eficiencia, sostenibilidad y costos operativos. Sin embargo, incluso cuando la eliminación completa de estos contaminantes es alcanzada, éstos pueden transformarse en intermedios nuevos y desconocidos que podrían ser más persistentes y tóxicos que sus compuestos originales. En esta tesis doctoral, se han desarrollado metodologías analíticas avanzadas para la identificación de los productos de transformación generados a lo largo de tratamientos biológicos y físicos y/o químicos. Además, se han evaluado sus posibles efectos ambientales mediante métodos in silico y bioensayos in vitro en efluentes tratados. Por último, se ha investigado su eficiencia de eliminación en tecnologías de agua combinadas. Esta tesis doctoral demuestra que la investigación multidisciplinaria es necesaria para evaluar la mejor tecnología de tratamiento de agua a utilizar
Programa de Doctorat en Ciència i Tecnologia de l'Aigua

The detection of endogenous anabolic androgenic steroids (EAAS) is one of the most difficult analytical challenges in the doping control field. The main problem for their detection is to distinguish between normally endogenous concentrations and those observed after the exogenous administration of an EAAS. The screening methods for EAAS are currently based on the determination of the steroid profile and the application of the athlete biological passport. The inclusion of new steroid metabolites can improve the screening capabilities of the steroid profile. Thus, the objective of the thesis is to elucidate and characterize new testosterone metabolites that can be implemented to the current steroid profile and to evaluate their usefulness for doping control analysis. Four unreported testosterone metabolites were detected and characterized by using liquid chromatography coupled to tandem mass spectrometry approaches. These compounds were demonstrated to come from degradation of cysteine conjugates. The formation of these conjugates implies an addition of a double bond as a phase I metabolism followed by conjugation with glutathione and the subsequent transformation to cysteine conjugates in urine. In order to determinate the usefulness of the cysteinyl compounds for doping control purposes, a quantitative method for the indirect determination of these compounds was developed and validated. Using this method, reference population limits were established by the analysis of 174 urine samples. Additionally, different factors that can potentially influence the excretion of these compounds were evaluated. Finally, the usefulness of these cysteinyl metabolites for the detection of EAAS misuse was evaluated by the analysis of samples collected after different EAAS administration. The use of these metabolites seems to improve in some cases the detection capabilities of the current marker used in routine analysis.
La detecció d’esteroides androgènics anabolitzants endògens (EAAE) és un dels reptes analítics més difícils en la lluita contra el dopatge. El problema més important per a la seva detecció és distingir entre concentracions endògenes i aquelles que s’observen després de l’administració exògena d’un EAAE. Els mètodes de cribatge per a la detecció d’EAAE estan basats en la determinació del perfil esteroïdal i la introducció d’aquest en el passaport biològic de l’atleta. La inclusió de nous metabòlits d’esteroides pot ajudar a millorar les capacitats de cribatge del perfil esteroïdal. Per tant, l’objectiu d’aquesta tesis és detectar i caracteritzar nous metabòlits d’EAAE que puguin implementar-se en l’actual perfil esteroïdal i l’avaluació de la seva utilitat en la lluita contra el dopatge. Quatre metabòlits desconeguts de la testosterona van ser detectats i caracteritzats mitjançant la utilització de la cromatografia líquida acoblada a l’espectrometria de masses en tàndem. L’origen d’aquests compostos es va demostrar que provenia de la degradació de conjugats amb cisteïna. La formació d’aquests conjugats implica l’addició d’un doble enllaç com a reacció metabòlica de fase I acompanyat per la conjugació amb glutationa i la subseqüent degradació d’aquesta a cisteïna en orina. Per tal de poder veure la seva aplicació en el camp del dopatge, es va desenvolupar i validar un mètode per la quantificació indirecta d’aquests compostos en orina. Utilitzant aquest mètode es van establir límits de referència basats en l’anàlisi de 174 mostres de orina. Addicionalment, diferents factors descrits que poden afectar l’excreció en orina d’aquests compostos també van ser estudiats en detall. Finalment, es va avaluar la utilitat d’aquests metabòlits conjugats amb cisteïna per a la detecció de l’abús d’EAAE mitjançant l’ anàlisis de mostres després de l’administració de diferents EAAE. L’ús d’aquests metabòlits va millorar (en alguns casos) els temps de detecció comparant-los amb els actuals marcadors utilitzats en rutina.

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.

The production of engineered nanomaterials (ENMs) has grown in last years due to their special physicochemical properties in respect to their bulk counterparts, derived from their small size (1-100 nm). Among the existing ENMs, silver nanoparticles (AgNPs) are widely used in commercial products due to their antibacterial properties. This fact favours their release in the environment, increasing the concern about the hazards that these emerging contaminants might pose for living organisms and human health. Therefore, to understand their effects, it is of paramount importance their characterisation and quantification in environmental and biological samples by adequate analytical methodologies. The research presented in this thesis focuses on the application and improvement of existing analytical methodologies and on the development of new analytical procedures to acquire information about their mobility and bioavailability in soils and also their accumulation, biotransformation and toxicity in edible plants
La producció de nanomaterials (NMs) ha augmentat recentment degut a les seves propietats fisicoquímiques especials respecte als seus metalls homòlegs, derivats de la seva petita mida (1-100 nm). Entre els NMs existents, les nanopartícules de plata (AgNPs) són àmpliament utilitzades en productes comercials per les seves propietats antibacterianes. Aquest fet afavoreix la seva alliberació al medi ambient, augmentant la preocupació pels perills que poden suposar pels organismes vius i la salut humana. Per tant, per entendre els seus efectes, és de vital importància la seva caracterització i quantificació en mostres ambientals i biològiques emprant metodologies analítiques adequades. La recerca presentada en aquesta tesi es centra en l’aplicació i millora de metodologies analítiques existents i en el desenvolupament de nous procediments analítics per obtenir no només informació sobre la mobilitat i la biodisponibilitat de les AgNPs en els sòls, sinó també sobre la seva acumulació, biotransformació i toxicitat en plantes comestibles

During this work has been developed an innovative methodology for continuous and in situ gas monitoring (24/24 h) of fumarolic and soil diffusive emissions applied to the geothermal and volcanic area of Pisciarelli near Agnano inside the Campi Flegrei caldera (CFc). In literature there are only scattered and in discrete data of the geochemical gas composition of fumarole at Campi Flegrei; it is only since the early ’80 that exist a systematic record of fumaroles with discrete sampling at Solfatara (Bocca Grande and Bocca Nuova fumaroles) and since 1999, even at the degassing areas of Pisciarelli. This type of sampling has resulted in a time series of geochemical analysis with discontinuous periods of time set (in average 2-3 measurements per month) completely inadequate for the purposes of Civil Defence in such high volcanic risk and densely populated areas. For this purpose, and to remedy this lack of data, during this study was introduced a new methodology of continuous and in situ sampling able to continuously detect data related and from its soil diffusive degassing. Due to its high sampling density (about one measurement per minute therefore producing 1440 data daily) and numerous species detected (CO2, Ar, 36Ar, CH4, He, H2S, N2, O2) allowing a good statistic record and the reconstruction of the gas composition evolution of the investigated area. This methodology is based on continuous sampling of fumaroles gases and soil degassing using an extraction line, which after undergoing a series of condensation processes of the water vapour content - better described hereinafter - is analyzed through using a quadrupole mass spectrometer

The gas phase ion-molecule chemistry of organometallic ions CpFe+ (1) and C₅H₆Fe+ (2), shown below, has been examined by Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. [MODEL] These ions, generated by electron impact ionization from organometallic neutral precursors ferrocene and cyclohexadienone iron tricarbonyl, were allowed to react with neutral methanol, ethanol, n-, i-propanol, and n-, i-, f-butanol. Selected isotopologues of employed alcohols were used to infer the reaction mechanism. Abundances of ionic reactants and products were monitored as a function of reaction time to obtain kinetic data. A general trend was observed that the reactivity of ions 1 and 2 with alcohols increases with the increasing length of the alkyl chain. The most important difference in the gas phase chemistry of these two ions was noted for the reactions with methanol. CpFe+ forms only an ion-neutral complex without any bond activation. C₅H₆Fe+ was found to dehydrogenate methanol by a competitive insertion into C-H and H-0 bonds. Taking the ratio of ADO collision rates and the measured reaction rates it was found that ion 2 has a twofold increase in reaction efficiency as compared to ion 1. For the reactions of CpFe+ with higher alcohols the major processes observed were dehydration and loss of alkene. Dehydrogenation of alcohols was a minor reaction. In all C₅H₆Fe+/alcohol systems except for methanol, reductive elimination of water and alkene was always accompanied by loss of a hydrogen molecule. Experiments with labeled alcohols have shown that one of the hydrogen atoms in the eliminated neutrals originates on a cyclopentadiene ring. This was rationalized by proposing that the reacting form of C₅H₆Fe+ ion has a CpFe+-H structure, formed by an intramolecular 6-hydrogen transfer that results in a metal-hydrogen bond.

碩士
國立臺灣大學
化學系研究所
85
A study is described for the determination of nitro-substituted polycyclic aromatic hydrocarbons ( Nitro-PAHs ) in diesel engine exhaust which employs gas chromatographycombined with highresolution mass spectrometry. Because of omplicated matrix and exetremely low concentration, silica gel was used for fractionation of diesel exhaust extracts toseparate Nitro-PAHs from other compounds At 1000 resolution, 1-Nitropyrene was identified easily by retention time and compound characteristic ions. However, 3000 resolution wasneededto identified2-Nitronaphthalene and 9-nitroanthracene. When Nitro-PAHs and PAHs were collected together, MS mustbe operated at 5000 resolution to achieve similar result.