Beta-N-methylamino-L-alanine (BMAA) is a non-protein amino acid produced by many cyanobacteria, and thought to induce neurotoxic effects through excitotoxicity, contributing to neurodegenerative diseases such as Amyotrophic Lateral Sclerosis/Parkinsonism-dementia complex (ALS-PDC) and Alzheimer’s. The ubiquitous nature of cyanobacteria, and evidence of biomagnification through our food web, creates a dire need for the development of an analytical platform that will provide accurate identification and quantification of BMAA amounts in our ecosystem and potential food supply. The present study evaluated the ability of a MALDI-ToF-MS method to detect and quantify BMAA in a variety of biological matrices. Through validation procedures, it was demonstrated that this MALDI-ToF-MS method provided comparable data to currently accepted analytical methods, specifically LC-MS/MS. Further, the development of said method reduced sample preparation and data acquisition time (1-2 seconds per sample), while providing high throughput analysis and eliminating the need for derivatization, chromatographic separation, and modification of amino acids.

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Les cyanobactéries ont une place très importante dans les écosystèmes aquatiques et un nombre important d’espèces considéré comme nuisible de par leur production de métabolites toxiques. Ces cyanotoxines possèdent des propriétés très variées et ont souvent été associées à des épisodes d’empoisonnement. L’augmentation des épisodes d’efflorescence d’origine cyanobactériennes et le potentiel qu’ils augmentent avec les changements climatiques a renchéri l’intérêt de l’étude des cyanobactéries et de leurs toxines. Considérant la complexité chimique des cyanotoxines, le développement de méthodes de détection simples, sensibles et rapides est toujours considéré comme étant un défi analytique. Considérant ces défis, le développement de nouvelles approches analytiques pour la détection de cyanotoxines dans l’eau et les poissons ayant été contaminés par des efflorescences cyanobactériennes nuisibles a été proposé. Une première approche consiste en l’utilisation d’une extraction sur phase solide en ligne couplée à une chromatographie liquide et à une détection en spectrométrie de masse en tandem (SPE-LC-MS/MS) permettant l’analyse de six analogues de microcystines (MC), de l’anatoxine (ANA-a) et de la cylindrospermopsine (CYN). La méthode permet une analyse simple et rapide et ainsi que la séparation chromatographique d’ANA-a et de son interférence isobare, la phénylalanine. Les limites de détection obtenues se trouvaient entre 0,01 et 0,02 μg L-1 et des concentrations retrouvées dans des eaux de lacs du Québec se trouvaient entre 0,024 et 36 μg L-1. Une deuxième méthode a permis l’analyse du b-N-méthylamino-L-alanine (BMAA), d’ANA-a, de CYN et de la saxitoxine (STX) dans les eaux de lac contaminés. L’analyse de deux isomères de conformation du BMAA a été effectuée afin d’améliorer la sélectivité de la détection. L’utilisation d’une SPE manuelle permet la purification et préconcentration des échantillons et une dérivatisation à base de chlorure de dansyle permet une chromatographie simplifiée. L’analyse effectuée par LC couplée à la spectrométrie de masse à haute résolution (HRMS) et des limites de détections ont été obtenues entre 0,007 et 0,01 µg L-1. Des échantillons réels ont été analysés avec des concentrations entre 0,01 et 0,3 µg L-1 permettant ainsi la confirmation de la présence du BMAA dans les efflorescences de cyanobactéries au Québec. Un deuxième volet du projet consiste en l’utilisation d’une technologie d’introduction d’échantillon permettant des analyses ultra-rapides (< 15 secondes/échantillons) sans étape chromatographique, la désorption thermique à diode laser (LDTD) couplée à l’ionisation chimique à pression atmosphérique (APCI) et à la spectrométrie de masse (MS). Un premier projet consiste en l’analyse des MC totales par l’intermédiaire d’une oxydation de Lemieux permettant un bris de la molécule et obtenant une fraction commune aux multiples congénères existants des MC. Cette fraction, le MMPB, est analysée, après une extraction liquide-liquide, par LDTD-APCI-MS/MS. Une limite de détection de 0,2 µg L-1 a été obtenue et des concentrations entre 1 et 425 µg L-1 ont été trouvées dans des échantillons d’eau de lac contaminés du Québec. De plus, une analyse en parallèle avec des étalons pour divers congénères des MC a permis de suggérer la possible présence de congénères ou d’isomères non détectés. Un deuxième projet consiste en l’analyse directe d’ANA-a par LDTD-APCI-HRMS pour résoudre son interférence isobare, la phénylalanine, grâce à la détection à haute résolution. La LDTD n’offre pas de séparation chromatographique et l’utilisation de la HRMS permet de distinguer les signaux d’ANA-a de ceux de la phénylalanine. Une limite de détection de 0,2 µg L-1 a été obtenue et la méthode a été appliquée sur des échantillons réels d’eau avec un échantillon positif en ANA-a avec une concentration de 0,21 µg L-1. Finalement, à l’aide de la LDTD-APCI-HRMS, l’analyse des MC totales a été adaptée pour la chair de poisson afin de déterminer la fraction libre et liée des MC et comparer les résultats avec des analyses conventionnelles. L’utilisation d’une digestion par hydroxyde de sodium précédant l’oxydation de Lemieux suivi d’une purification par SPE a permis d’obtenir une limite de détection de 2,7 µg kg-1. Des échantillons de poissons contaminés ont été analysés, on a retrouvé des concentrations en MC totales de 2,9 et 13,2 µg kg-1 comparativement aux analyses usuelles qui avaient démontré un seul échantillon positif à 2 µg kg-1, indiquant la possible présence de MC non détectés en utilisant les méthodes conventionnelles. / Cyanobacteria have a very important place in aquatic ecosystems and a significant number of species are considered harmful given their production of toxic metabolites. These cyanotoxins have various chemical proprieties and have often been associated with poisoning episodes. The frequency of cyanobacterial blooms is increasing and the study of cyanobacteria and their toxins is of increasing interest, especially considering the potential increase associated with climate changes. Given the chemical complexity of the cyanotoxins, the development of simple, sensitive and fast detection methods is an analytical challenge. Considering these issues, the development of new analytical approaches for the detection of cyanotoxins in water and fish samples contaminated with harmful cyanobacterial blooms have been proposed. A first approach consists of the use of an on-line solid phase extraction coupled to liquid chromatography and tandem mass spectrometry (SPE-LC-MS/MS) for the analysis of six microcystins (MCs), anatoxin-a (ANA-a) and cylindrospermopsin (CYN). This method allows a simple and rapid analysis and enables the chromatographic separation of ANA-a and its isobaric interference, phenylalanine. The detection limits ranged from 0.01 to 0.02 µg L-1 and concentrations in lake waters were found between 0.024 and 36 µg L-1. A second method consists of using manual solid phase extraction (SPE) coupled to high resolution mass spectrometry (HRMS) for the determination of b-N-methylamino-L-alanine (BMAA), ANA-a, CYN and saxitoxin (STX) in contaminated lake water. The analysis of two conformational isomers of BMAA was done to improve the selectivity. Dansyl chloride-based derivatization allows simplified chromatography. The detection limits were obtained between 0.007 and 0.01 µg L-1. The analysis of bloom water samples detected concentrations of cyanotoxins between 0.01 and 0.3 µg L-1 allowing the confirmation of the presence of BMAA in algal blooms in Québec. A second part of the project consists in the use of an alternative sample introduction technology for MS analysis. It enables ultra-fast analysis (< 15 seconds/sample) without the use of a chromatographic step, and is called laser diode thermal desorption (LDTD) coupled with atmospheric pressure chemical ionization (APCI). The first LDTD project consists of the analysis of total MCs via Lemieux oxidation in order to obtain a common moiety of all MCs existing congeners. This fraction, the MMPB, is analyzed after a liquid-liquid extraction step, with the LDTD-APCI-MS/MS. A value of 0.2 µg L-1 was obtained for detection limit and concentrations between 1 and 425 µg L-1 have been found in contaminated water samples. In addition, a comparison with a parallel analysis using MCs congeners’ standards suggested the possible presence of undetected MCs or isomers. A second project involves the direct analysis of ANA-a using LDTD-APCI-HRMS in order to solve the isobaric interference, phenylalanine, which is possible due to the high resolution detection. The LDTD offers no chromatographic separation and by using HRMS, we can distinguish ANA-a signals from those of phenylalanine. A value of 0.2 µg L-1 was obtained as detection limit and the method has been applied on water bloom samples with a positive concentration of 0.21 µg L-1. Finally, using the LDTD-APCI-HRMS combination, analysis of total MCs has been adapted to fish tissues to determine the unbound and bound MCs and compare the results with standard analysis. The use of digestion with sodium hydroxide prior to Lemieux oxidation followed by SPE purification yielded a detection limit of 2.7 µg kg-1. Total MCs concentrations were found between 2.9 and 13.2 µg kg-1 in real field-collected contaminated fish samples and comparison was made with standard analysis which yield a single positive sample with a concentration of 2 µg kg-1. This indicates the possible presence of undetected MCs using conventional analytical methods.

La neurotoxine β-N-méthyl-amino-l-alanine (BMAA) et ses isomères, notamment la N-(2- aminoéthyl glycine) (AEG), la β-amino-N-méthyl alanine (BAMA) et l'acide 2,4- diaminobutyrique (DAB), ont été détectés précédemment dans des échantillons de cyanobactéries. Cependant, il existe des rapports contradictoires concernant leur présence dans les eaux de surface. Dans cette étude, nous avons évalué l'impact de l'acide trichloracétique (TCA 0,1M) sur la détection des isomères de BMAA, par rapport aux protocoles préexistants. Une méthode instrumentale sensible a été utilisée pour l'étude, avec des limites de détection de l'ordre de 5-10 ng L-1. Des meilleures limites de détection plus élevés et des niveaux significativement plus importants (test des rangs signés de Wilcoxon appariés, p < 0,001) d'isomères de BMAA ont été observés dans les échantillons traités par le TCA, avec des augmentations relatives allant jusqu'à +725 % pour l'AEG et +1450 % pour le DAB, et des augmentations de concentration absolue allant jusqu'à +15 000 ng L-1 pour l'AEG et +650 ng L-1 pour le DAB. Nous avons également documenté les tendances de la présence des isomères de BMAA dans plusieurs lacs de différents pays tels que le Brésil, le Canada, la France, le Mexique et le Royaume-Uni. Les données obtenues au cours de cette étude (n = 390 provenant de 45 sites d'échantillonnage) indiquent des détections fréquentes des isomères AEG et DAB, avec des taux de détection de 30 % et 43 % et des niveaux maximums de 19 000 ng L-1 et 1 100 ng L-1, respectivement. En revanche, le BAMA a été trouvé dans moins de 8 % des échantillons d'eau, et la BMAA n'a été trouvée dans aucun échantillon. Ces résultats appuient les analyses des cyanobactéries libres, dans lesquelles la BMAA a souvent été détectée avec des concentrations inférieures de 2 à 4 ordres de grandeur à celles de l'AEG et du DAB. Les mesures saisonnières effectuées dans deux lacs impactés par des efflorescences ont indiqué des corrélations limitées entre les isomères de la BMAA et les microcystines totales ou la chlorophylle-a, ce qui mériterait une étude plus approfondie. / The neurotoxic alkaloid β-N-methyl-amino-l-alanine (BMAA) and related isomers, including N-(2-aminoethyl glycine) (AEG), β-amino-N-methyl alanine (BAMA) and 2,4-diaminobutyric acid (DAB), have been reported previously in cyanobacterial samples. However, there are conflicting reports regarding their occurrence in surface waters. In this study, we evaluated the impact of amending lake water samples with trichloroacetic acid (0.1M TCA) on the detection of BMAA isomers, compared with pre-existing protocols. A sensitive instrumental method was enlisted for the survey, with limits of detection in the range of 5-10 ng L-1. Higher detection limits ans significantly greater levels (paired Wilcoxon’s signed-rank tests, p < 0.001) of BMAA isomers were observed TCA-amended samples (method B) compared to samples without TCA (method A). The overall range of B/A ratios was 0.67-8.25 for AEG (up to +725 %) and 0.69-15.5 for DAB (up to +1450 %), with absolute concentration increases TCA-amended samples up to +15,000 ng L-1 for AEG and +650 ng L-1 for DAB. We also documented the trends in the occurrence of BMAA isomers for a large breadth of field-collected lakes from Brazil, Canada, France, Mexico, and the United Kingdom. Data gathered during this overarching campaign (overall n = 390 within 45 lake sampling sites) indicate frequent detections of AEG and DAB isomers, with detection rates of 30 % and 43 % and maximum levels of 19,000 ng L-1 and 1,100 ng L- 1, respectively. In contrast, BAMA was found in less than 8 % of the water samples, and BMAA not found in any sample. These results support analyses of free-living cyanobacteria, wherein BMAA was often reported at concentrations 2-4 orders of magnitude lower than AEG and DAB. Seasonal measurements conducted at two bloom-impacted lakes indicated limited correlations of BMAA isomers with total microcystins or chlorophyll-a, which deserves further investigation.

VILELA, Rivelino Cunha. Estudo de transições de fase em cristais de l-alanina + ácido oxálico. 2013. 113 f. Tese (Doutorado em Física) - Programa de Pós-Graduação em Física, Departamento de Física, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2013. / Submitted by Edvander Pires (edvanderpires@gmail.com) on 2015-10-29T21:51:24Z No. of bitstreams: 1 2013_tese_rcvilela.pdf: 12577577 bytes, checksum: 4dc32419c1bbdde79375612c04227181 (MD5) / Approved for entry into archive by Edvander Pires(edvanderpires@gmail.com) on 2015-11-03T19:48:22Z (GMT) No. of bitstreams: 1 2013_tese_rcvilela.pdf: 12577577 bytes, checksum: 4dc32419c1bbdde79375612c04227181 (MD5) / Made available in DSpace on 2015-11-03T19:48:22Z (GMT). No. of bitstreams: 1 2013_tese_rcvilela.pdf: 12577577 bytes, checksum: 4dc32419c1bbdde79375612c04227181 (MD5) Previous issue date: 2013 / In the present word we have studied the effect of temperature on the Raman spectra of crystals of L-alanine + oxalic acid, C3H8NO2+.C2HO4-. Raman spectroscopy measurements were performed on polycrystalline samples at different temperatures varying in the range from room temperature to T = 20 K; a tentative assignment of all normal modes was furnished. In order to help the understanding of the crystal behavior we have also obtained X-ray diffractograms and studied the dependence of lattice parameters through dilatometry as a function of temperature in the 290 K – 93 K range. The three different techniques allowed us to construct a picture of the material under low temperature conditions. As a consequence we have realized that L-alanine + oxalic acid crystal undergoes three phase transitions at low temperatures. The splitting of a band at 90 cm-1 and an anomaly in one of the lattice parameters are the signature for the first phase transition that is observed at 250 K. At 150 K it was observed the appearance of two new bands in the Raman spectrum and, simultaneously, it was observed change in the curves of a and c lattice parameters. Additionally, it was verified the appearance of new peaks in the X-ray diffractogram at the same temperature, characterizing the second phase transition. At a temperature even lower, at about 43 K, it was verified the occurrence of the third phase transition that has as main characteristic the splitting of two bands that are associated to the lattice modes. Changes in the modes associated with CH3 and NH3+ during the cooling is discussed. An important behavior of the crystal with the cooling process was the red shift of the band of lower frequency, similar to the soft-mode vibration of ferroelectric materials, although the frequency of the mode in L-alanine + oxalic acid does not goes to zero. Based on the results on Raman spectroscopy, dilatometry and X-ray diffraction, and on the possible symmetry sites occupied by the molecules through the O=CC group in the various phases, it is suggested the following sequence of phase transitions D24  C2h5  Cs3  C23, which should occur at 250 K, 150 K and 43 K. / Neste trabalho, estudou-se o efeito da temperatura nos espectros Raman de cristais de L-alanina + ácido oxálico, C3H8NO2+.C2HO4-. Foram realizadas medidas de espectroscopia Raman em policristais a diferentes temperaturas no intervalo compreendido entre a temperatura ambiente e a temperatura de 20 K, sendo fornecida uma identificação tentativa para todos os modos normais de vibração observados. Para auxiliar o entendimento do comportamento do cristal também foram obtidos os difratogramas de raios-X bem como estudada a dependência dos parâmetros de rede em função da temperatura através de dilatometria no intervalo entre 290 K e 93 K. As três técnicas utilizadas em conjunto permitiram mostrar o comportamento estrutural do material em baixas temperaturas. Deste quadro foi possível inferir que os cristais de L-alanina + ácido oxálico apresentam três diferentes transições de fase durante o resfriamento. Em 250 K o aparecimento de um dubleto em 90 cm-1 e a anomalia num dos parâmetros de rede apontam para a ocorrência da primeira transição de fase. Em 150 K surgem pelo menos duas novas bandas no espectro Raman, ao mesmo tempo em que ocorrem bruscas mudanças de inclinação nas curvas que representam as dimensões dos eixos a e c do cristal. Também se verifica que, de forma semelhante ao que ocorre com os espectros Raman, aparecem novos picos no difratograma de raios-X em torno desta temperatura, caracterizando assim a segunda transição de fase. A uma temperatura ainda mais baixa, em torno de 43 K, foi verificada a ocorrência da terceira transição de fase, que tem como principal característica a separação de dois modos Raman associados a modos da rede. Mudanças nos ambientes dos grupos CH3 e do NH3+ durante o resfriamento são discutidas. Um importante aspecto apresentado pelos espectros Raman com o resfriamento da amostra foi o deslocamento da banda de mais baixa energia para menores valores de frequências, semelhantemente ao que ocorre com vibrações do tipo soft-mode em materiais ferroelétricos, embora a frequência do modo no cristal de L-alanina + ácido oxálico não tenha ido à zero. Baseado nos resultados acima e nos possíveis sítios de simetria ocupados pelas moléculas através do grupo O=CC nas diversas fases, sugere-se a seguinte sequência de transições de fase D24 para C2h5 para Cs3 para C23, que aconteceriam, respectivamente, nas temperaturas de 250 K, 150 K e 43 K.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Neste trabalho, estudou-se o efeito da temperatura nos espectros Raman de cristais de L-alanina + Ãcido oxÃlico, C3H8NO2+.C2HO4-. Foram realizadas medidas de espectroscopia Raman em policristais a diferentes temperaturas no intervalo compreendido entre a temperatura ambiente e a temperatura de 20 K, sendo fornecida uma identificaÃÃo tentativa para todos os modos normais de vibraÃÃo observados. Para auxiliar o entendimento do comportamento do cristal tambÃm foram obtidos os difratogramas de raios-X bem como estudada a dependÃncia dos parÃmetros de rede em funÃÃo da temperatura atravÃs de dilatometria no intervalo entre 290 K e 93 K. As trÃs tÃcnicas utilizadas em conjunto permitiram mostrar o comportamento estrutural do material em baixas temperaturas. Deste quadro foi possÃvel inferir que os cristais de L-alanina + Ãcido oxÃlico apresentam trÃs diferentes transiÃÃes de fase durante o resfriamento. Em 250 K o aparecimento de um dubleto em 90 cm-1 e a anomalia num dos parÃmetros de rede apontam para a ocorrÃncia da primeira transiÃÃo de fase. Em 150 K surgem pelo menos duas novas bandas no espectro Raman, ao mesmo tempo em que ocorrem bruscas mudanÃas de inclinaÃÃo nas curvas que representam as dimensÃes dos eixos a e c do cristal. TambÃm se verifica que, de forma semelhante ao que ocorre com os espectros Raman, aparecem novos picos no difratograma de raios-X em torno desta temperatura, caracterizando assim a segunda transiÃÃo de fase. A uma temperatura ainda mais baixa, em torno de 43 K, foi verificada a ocorrÃncia da terceira transiÃÃo de fase, que tem como principal caracterÃstica a separaÃÃo de dois modos Raman associados a modos da rede. MudanÃas nos ambientes dos grupos CH3 e do NH3+ durante o resfriamento sÃo discutidas. Um importante aspecto apresentado pelos espectros Raman com o resfriamento da amostra foi o deslocamento da banda de mais baixa energia para menores valores de frequÃncias, semelhantemente ao que ocorre com vibraÃÃes do tipo soft-mode em materiais ferroelÃtricos, embora a frequÃncia do modo no cristal de L-alanina + Ãcido oxÃlico nÃo tenha ido à zero. Baseado nos resultados acima e nos possÃveis sÃtios de simetria ocupados pelas molÃculas atravÃs do grupo O=CC nas diversas fases, sugere-se a seguinte sequÃncia de transiÃÃes de fase D24 para C2h5 para Cs3 para C23, que aconteceriam, respectivamente, nas temperaturas de 250 K, 150 K e 43 K. / In the present word we have studied the effect of temperature on the Raman spectra of crystals of L-alanine + oxalic acid, C3H8NO2+.C2HO4-. Raman spectroscopy measurements were performed on polycrystalline samples at different temperatures varying in the range from room temperature to T = 20 K; a tentative assignment of all normal modes was furnished. In order to help the understanding of the crystal behavior we have also obtained X-ray diffractograms and studied the dependence of lattice parameters through dilatometry as a function of temperature in the 290 K â 93 K range. The three different techniques allowed us to construct a picture of the material under low temperature conditions. As a consequence we have realized that L-alanine + oxalic acid crystal undergoes three phase transitions at low temperatures. The splitting of a band at 90 cm-1 and an anomaly in one of the lattice parameters are the signature for the first phase transition that is observed at 250 K. At 150 K it was observed the appearance of two new bands in the Raman spectrum and, simultaneously, it was observed change in the curves of a and c lattice parameters. Additionally, it was verified the appearance of new peaks in the X-ray diffractogram at the same temperature, characterizing the second phase transition. At a temperature even lower, at about 43 K, it was verified the occurrence of the third phase transition that has as main characteristic the splitting of two bands that are associated to the lattice modes. Changes in the modes associated with CH3 and NH3+ during the cooling is discussed. An important behavior of the crystal with the cooling process was the red shift of the band of lower frequency, similar to the soft-mode vibration of ferroelectric materials, although the frequency of the mode in L-alanine + oxalic acid does not goes to zero. Based on the results on Raman spectroscopy, dilatometry and X-ray diffraction, and on the possible symmetry sites occupied by the molecules through the O=CC group in the various phases, it is suggested the following sequence of phase transitions D24 &#61664; C2h5 &#61664; Cs3 &#61664; C23, which should occur at 250 K, 150 K and 43 K.

This master thesis deals with the development, optimization and validation of an analytical method for determination of neurotoxin -N-methylamino-L-alanine in pond water and cyanobacteria using LC/MS. Firstly, basic parameters of the analytical method developed within authors´s bachelor thesis were verified. Following parameters were selected for verification: selection of suitable MRM transitions, voltage applied to S-lens and F-lens and standardized collision energy. Secondly, the system suitability testing was performed. Thirdly, the analytical method was successfully validated. Then, the testing and optimization of solid phase extraction for analysis of water samples were carried out. The pH of sample solution and composition of elution solution were chosen for the optimization. In addition, the trichloroacetic acid extraction with acid hydrolysis for cyanobacterial samples was carried out too. Finally, both solid phase extraction and trichloroacetic acid extraction were evaluated and applied to the analysis of real samples.

The electrical properties of proteins in solution are important for their structure and function. Computational biophysics studies of proteins need accurate parameters to ensure that numerical simulations match physical reality. Past work in this eld has compared the electrical properties of proteins obtained from dielectric spectroscopy to numerical simulations of proteins in water with adjustment of pKa values to try to capture the inevitable changes in electrical conformation that will occur in a complex structure such as a folded protein. However, fundamental veri cation of the charge parameters of the amino acid building blocks in common molecular dynamics software packages with electrical experiments needs to be performed to have increased con dence in the results from numerical simulations. The aim of this thesis is to start from a fundamental building block, the single amino acid alanine, and to compare numerical simulations of this amino acid in water using parameters from commonly used charge structures in CHARMM, GROMOS, and OPLS, with electrical parameters obtained from dielectric spectroscopy experiments in the GHz range. To this end, multiple molecular dynamics simulations were performed to accurately determine how these different charge structures yield different dielectric increments. Additionally, a commercial RF dielectric measurement probe was modi ed to perform measurements on solutions containing alanine at different concentrations. Using regression, the dielectric increment of alanine is readily determined and compared with the numerical simulations. The results indicate that the CHARMM and OPLS parameters seem to adequately capture the charge con guration of alanine in solution, while the GROMOS parameters produce a dielectric increment but do not seem to adequately capture the charge con guration of alanine in solution. These studies lay the foundation for future studies of additional amino acids in solution as well as a stepping stone for larger simulations of the electrical properties of fully solvated proteins in solution.