A lipoperoxidação gera diversas espécies carbonílicas altamente reativas dentre as quais se destacam acroleína (ACR), malondialdeído (MDA), 4-hidroxi-2-hexenal (HHE) e 4-hidroxi-2-nonenal (HNE). A principal via endógena de metabolização desses compostos é através de conjugação com glutationa por ação da glutationa-S-tranferase. Contudo, diversos trabalhos têm mostrado que dipeptídeos contendo histidina, tal como a carnosina (CAR), também podem formar conjugados com aldeídos e auxiliar na detoxificação desses compostos. Em nosso trabalho adutos de CAR com ACR, HHE, HHEd5, HNE e HNEd11 foram sintetizados, purificados e caracterizados. A reação da CAR com ACR foi estudada em detalhes. Resultados mostraram que a carnosina reage com acroleína formando 03 produtos principais: m/z = 265, m/z = 283 e m/z = 303, sendo este último mais estável e mais abundante. Dados de RMN H1, COSY e HSQC permitiram elucidar a estrutura dessa molécula (m/z = 303) e propor uma rota de reação. Em seguida, uma metodologia baseada em cromatografia líquida acoplada à espectrometria de massas do tipo \"Ion Trap\" (ESI+ HPLC/MS-MS) foi desenvolvida e validada para quantificação simultânea dos adutos sintetizados. Pelo método desenvolvido é possível quantificar com precisão 25 pmol de CAR-HHE, 1 pmol de CAR-ACR e 1 pmol de CAR-HNE com um coeficiente de variação de aproximadamente 10 % e acurácia de 98 % (HHEd5 e HNEd11 foram usados como padrão interno). Análise em urina de adultos não fumantes mostraram que os produtos sintetizados estão presentes na urina de humanos em concentrações de 3,6 ± 1,4; 2,3 ± 1,5 e 1,3 ± 0,5 nmol / mg de creatinina, respectivamente para CAR-ACR, CAR-HHE e CAR-HNE. Em ratos transgênicos SODG93A modelo para esclerose lateral amiotrófica (ELA), a suplementação da dieta dos animais com 35 ± 5 mg carnosina/animal/semana melhorou a manutenção do peso e a sobrevida dos animais. Análises dos adutos sintetizados em amostras de músculo sugerem que a metabolização de aldeídos esteja comprometida nesses animais e que a carnosina poderia funcionar como \"scavenger\" para esses compostos. Esses resultados comprovam que dipeptídeos de histidina atuam na detoxificação de compostos carbonílicos e participa de suas vias de excreção. Além disso, a caracterização da estrutura e desenvolvimento de método sensível de detecção abre a possibilidade de utilização desses adutos como biomarcadores de estresse redox e exposição a aldeídos. / Lipid peroxidation generates reactive carbonyl species, including 4-hydroxy-2-nonenal (HNE), acrolein (ACR), 4-hydroxy-2-hexenal (HHE) and malondialdehyde (MDA). One major pathway of aldehyde detoxification in vivo is through conjugation with glutathione catalyzed by glutathione-S-transferases or, alternatively, by conjugation with endogenous histidine containing dipeptides, such as carnosine (CAR). The reaction of CAR with ACR was investigated in an effort to assess its possible biological role. One stable adduct was isolated by reverse-phase HPLC and characterized on the basis of extensive spectroscopic measurements. The proposed reaction route for product formation involves the reaction of the CAR amino group with ACR via a Schiff base formation followed by dehydration and cyclization through Michael addition in the imidazole ring forming an instable compound with m/z = 265. The subsequent reaction with another molecule of ACR followed by cyclization gives rise to the final product with m/z = 303.A highly sensitive method involving HPLC-MS analysis was developed for the simultaneous accurate quantification of CAR- ACR, CAR-HHE and CAR-HNE adducts in human urinary samples from non-smoking adults. This methodology permits quantification of 10 pmol CAR-HHE and 1 pmol of CAR-ACR and CAR-HNE. Adduct levels in urine were 3.6 ± 1.4, 2.3 ± 1.5, 1.3 ± 0.5 nmol/mg of creatinine, respectively to CAR-ACR, CAR-HHE and CAR-HNE. In SODG93A transgenic rats model to amyotrophic lateral sclerosis (ALS), the food supplementation of the animals with 35 ± 5 mg carnosine/animal/week improve de body weight and the life span of the ALS treated group. Analysis of the synthesized adducts in muscle sample showed suggest than aldehyde metabolization is compromised in this animals and that may be carnosine work like a scavenger for these compounds. Our results indicate that carnosine adduction can be an important detoxification route of α,β -unsaturated aldehydes. Moreover, carnosine adducts quantification may be useful as redox stress indicator in vivo.

Submitted by Ricardo Cedraz Duque Moliterno (ricardo.moliterno@uefs.br) on 2015-08-03T22:07:54Z No. of bitstreams: 1 DISSERTA??O - RENATA VIEIRA SILVA.pdf: 1258695 bytes, checksum: 8741b07320d6f4c024d94e4aa2fd913a (MD5) / Made available in DSpace on 2015-08-03T22:07:54Z (GMT). No. of bitstreams: 1 DISSERTA??O - RENATA VIEIRA SILVA.pdf: 1258695 bytes, checksum: 8741b07320d6f4c024d94e4aa2fd913a (MD5) Previous issue date: 2015-03-19 / Fruits of Spondias purpurea L. (Anacardeacea), popularly known as seriguela,are rich with phenolic compounds, which can be applied in the production of phytocosmetic. This study aimed to analyze the chemical constituents of the metanolic crude extract of S. purpurea (EBMSP) by high performance liquid chromatography coupled with mass spectrometry (HPLC-ESI-MSn) and to evaluate this extract as principle active in photoprotective formulations. In addition, the flavonoid and phenolic content, sequestration activity of free radicals by DPPH method and antimicrobial activity by the inhibition zone method, were investigated. HPLC-ESI-MSnanalyses were possible to characterize five phenolic compounds, including phenolic acids, tannins and flavonols. The extract showed phenolic content and flavonoid values at 28.68 ? 0.046 mg gallic acid equivalent/g and at 2.64 ? 0.005 mg of flavonoid quercetin equivalent/g of extract, respectively. In vitro antioxidant activity showed percentage of inhibition of 74.41 to 250 ?g/mL, a high value compared with the rutin. The EBMSP demonstrated sunscreen activity, with solar protection factor (SPF) values> 6.0 at low dilutions and UVA protection equal to the reference standards benzophenone-3 and rutin. The best results among formulations 5, 10, 20 and 30% for UVA and UVB sunscreens profile corresponded to lotion 30%, however, no formulations showed synergism with the standard benzophenone-3. To evaluate the antimicrobial activity, suggests that the EBMSP has activity against Gram-negative micro-organisms Pseudomonas aeruginosa and Salmonella dublin, without action to strains of Escherichia coli. / Os frutos de Spondias purpurea L. (Anacardeacea), conhecidos popularmente como seriguela, s?o ricos em compostos fen?licos, que podem ser aplicados na produ??o de fitocosm?ticos. Este trabalho teve como objetivo analisar os constituintes qu?micos do extrato bruto metan?lico de S. purpurea(EBMSP) por cromatografia l?quida de alta efici?ncia acoplada ? espectrometria de massas (CLAE-ESI-EMn), avaliando sua utiliza??o como princ?pio ativo em formula??es fotoprotetoras. Em adi??o, o teor de fen?licos e de flavonoides, bem como a atividade de sequestro de radicais livres pelo m?todo de DPPH e atividade antimicrobiana pelo m?todo do halo de inibi??o, tamb?m foram verificados. A an?lise por CLAE-ESI-EMn possibilitou caracterizar cinco compostos fen?licos, entre eles ?cidos fen?licos, taninos e flavon?is. O extrato apresentou valores de teor de fen?licos e flavonoides de 28,68 ? 0,046 mg de equivalente a ?cido g?lico/g e 2,64 ? 0,005 mg de flavonoide equivalente a quercetina/g de extrato, respectivamente. A atividade antioxidante in vitro demonstrou percentual de inibi??o de 74,41 a 250?g/mL, valor elevadoquando comparado com o padr?o de rutina. A avalia??o do potencial fotoprotetor in vitro do EBMSP demonstrou atividade fotoprotetora, com valores de FPS>6,0 em baixas dilui??es, e prote??o UVA igual aos padr?es de benzofenona-3 e rutina. Os melhores resultados entre as formula??es de 5, 10, 20 e 30%, para o perfil fotoprotetor UVA e UVB, corresponderam ? lo??o de 30%, entretanto, nenhuma das formula??es obteve sinergismo com o padr?o de benzofenona-3. Para avalia??o da atividade antimicrobiana, sugere-se que o EBMSP possui atividade contra os micro-organismos Gram-negativos Pseudomonas aeruginosa e Salmonella dublin, sem a??o para cepas de Escherichia coli.

Les phytochélatines (PC) sont des polypeptides ayant la structure générale, (alpha-Glu-Cys)n-Gly, où n = 2 à 11. Leur synthèse est induite par un grand nombre de végétaux en réponse à une élévation de la concentration du milieu en métaux, en particulier le cadmium (ci-après, Cd). Le but de cette étude a été de développer un outil pour évaluer la biodisponibilité du Cd dans les eaux douces. Pour ce faire, une méthode analytique a été réalisée afin de déterminer les phytochélatines induites dans les algues C. reinhardtii. Celle-ci consiste à utiliser la chromatographie liquide couplée à la spectrométrie de masse en tandem (LCHP-SM/SM) "on-line". L’ionisation des molécules est celle faite par électronébulisation (IEN) (traduction de electrospray ionisation). L’objectif principal de ce mémoire est la validation de cette méthode : la détermination des courbes de calibration et des limites de détection et l'identification d'interférences potentielles. L’utilisation de dithiothreitol (DTT) à une concentration de 25 mM a été nécessaire à la conservation de la forme réduite des phytochélatines. En effet, suite à la validation de la méthode d’analyse des phytochélatines il a été démontré qu’elle représente un potentiel d’application. Ceci dans la mesure où l’induction des phytochélatines (PC2, PC3 et PC4) dans les algues C. reinhardtii a été possible à deux concentrations de Cd (1 x 10-7 M et 1 x 10 6 M) et ce, après plusieurs temps d'induction (1, 2, 4, et 6 h). Ainsi, l’étude de la stabilité des phytochélatines a été réalisée et toutes les températures examinées ont démontré une diminution des phytochélatines analysées par HPLC-ESI-MS/MS. Il se pourrait que la cause de la dégradation des phytochélatines soit physique ou chimique plutôt que bactérienne. Toutefois, un approfondissement au niveau de la purification de la solution d’extraction serait nécessaire à la mise au point de la dite méthode analytique afin de quantifier les phytochélatines dans l’algue C. reinhardtii. / Phytochelatins (PC) are polypeptides having the general structure, (alpha-Glu-Cys)n-Gly, where n = 2 to 11. Many plants respond to an elevated concentration of metals in environment, particularly Cd, by synthesizing PC. The purpose of this study was to develop a tool to assess the bioavailability of the Cd in fresh water by determining phytochelatins in algae, C. reinhardtii, by online HPLC-ESI-MS/MS. The gold of this work was the validation of the analytical method i.e. the determination of the calibration curves and the limits of detection. The addition of dithiothreitol (DTT), 25 mM, was found to be necessary to maintain the PC in their reduced form for analysis. It was shown that the liquid chromatography coupled to tandem mass spectrometry (HPLC-ESI-MS/MS) technique has excellent potential for PC analysis, however, it will still requires some more work with respect to sample purification. Furthermore, the stability of the PC was evaluated for different sample storage temperatures. At all temperatures studied, some degradation of PC was observed possibly due to physical rather than chemical or bacterial reasons. Finally, the induction of phytochelatins (PC2, PC3 and PC4) was observed in C. reinhardtii for two Cd concentrations (10-7 M and 10-6 M) and for several induction times (1, 2, 4, and 6 h).

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O feijÃo-de-corda tem grande importÃncia socioeconÃmica no Nordeste brasileiro. Entretanto, sua produÃÃo à baixa devido a diversos fatores biÃticos, como, por exemplo, o vÃrus do mosaico severo do caupà (CPSMV, gÃnero Comovirus), que apresenta grande destaque, por causar a virose que mais acomete essa cultura no paÃs. No estudo da interaÃÃo planta-vÃrus, diversos trabalhos mostram que o tratamento de sementes com o etil metanosulfonato (EMS, mutagÃnico quÃmico) resulta no fenÃtipo de resistÃncia em plantas que, anteriormente, apresentavam susceptibilidade à infecÃÃo por vÃrus do gÃnero Potyvirus. Por essa razÃo, o presente estudo teve como objetivo investigar as respostas de defesa bioquÃmicas e fisiolÃgicas das plantas de feijÃo-de-corda do genÃtipo (CE-31) susceptÃvel ao CPSMV (CPI) a partir de sementes tratadas com EMS (0,04% v/v), e avaliar se as plantas mutagenizadas (MCPI), produzidas a partir dessas sementes se tornaram resistentes ao CPSMV. Duas diferentes abordagens foram utilizadas neste trabalho: 1) anÃlises bioquÃmicas (enzimas antioxidantes e conteÃdo de H2O2, PR-proteÃnas e compostos secundÃrios) e fisiolÃgicas (parÃmetros fotossintÃticos e teor de clorofila); 2) abordagem proteÃmica quantitativa (LC-ESI-MS/MS), livre de marcaÃÃo, para identificar proteÃnas responsivas à infecÃÃo viral. Os resultados obtidos demonstram que as plantas MCPI sÃo capazes de induzir respostas bioquÃmicas (aumento de H2O2, induÃÃo de PR-proteÃnas e aumento no conteÃdo de compostos secundÃrios) e alteraÃÃes nos parÃmetros fisiolÃgicos (alta taxa fotossintÃtica e teor de clorofila) que, aparentemente, tÃm relaÃÃo com o fenÃtipo de resistÃncia das plantas mutagenizadas ao CPSMV. Na anÃlise proteÃmica, 99 proteÃnas foram identificadas como sendo diferenciais, das quais 68 aumentaram e 31 diminuÃram em abundÃncia nas plantas MCPI em relaÃÃo as plantas CPI. A anÃlise proteÃmica, mostrou diversas vias metabÃlicas (Metabolismo Redox, Energia e Metabolismo, FotossÃntese, Metabolismo de RNA e Defesa) envolvidas nas respostas de defesa das plantas MCPI frente a infecÃÃo viral. O tratamento das sementes com o EMS, resultou em plantas de feijÃo-de-corda com fenÃtipo de resistÃncia capazes de acionar mecanismos de defesa para impedir a infeÃÃo viral / Cowpea is an important crop that makes major nutritional contributions as a source of proteins and carbohydrates in the diet of many people worldwide. However, its production is impaired due to various stresses including those of biotic origins. Cowpea Severe Mosaic Virus (CPSMV) infects cowpeas leading to severe symptoms and low productivity. Several studies of plant-virus interaction show that seed treatment with Ethyl methanesulfonate (EMS, chemical mutagen), results in a resistant phenotype in plants, which was previously susceptibility, to virus infection of the Potyvirus genus. The aim of this study was to investigate some physiological and biochemical parameters of a susceptible cowpea cultivar (CPI) (CE-31, sin. Pitiuba) in comparison with its derived resistant mutagenized (MCPI), both infected with CPSMV. MCPI plantlets were obtained after treatment of CE-31 seeds with 0.04% EMS. Two different approaches were used in this study: 1) biochemical (antioxidant enzymes and H2O2 content, PR-proteins and secondary metabolites) and physiological analysis (photosynthetic parameters and chlorophyll content); and 2) Label free quantitative proteomic approach (LC-ESI-MS / MS) to identify proteins responsive to viral infection. Our results showed that MCPI had no symptoms of CPSMV infection and biochemical (high H2O2, PR-proteins and secondary compounds [phenolic and lignin]) and physiological responses (High photosynthesis index and chlorophyll content) is activated in MCPI plantlets after CPSMV inoculation. With regard to proteomic analysis, 99 proteins were differentially represented, where these 68 are up- and 31 down represented in MCPI compared to CPI. Regardless whether to CPI (susceptible) or MCPI (mutagenized resistant) plantlets, CPSMV induce changes in proteome profile that involve several biological process (energy and metabolism, photosynthesis, response to stress, oxidative burst, and scavenging). Moreover, these results suggest that the CPSMV responsive proteins in the MCPI represent a complex network involving in resistant mechanisms to CPSMV. Treatment of the susceptible CE-31 genotype seeds with the mutagenic agent EMS induced genomic alterations generating a cowpea mutagenized resistant to CPSMV by apparently inducing classical biochemical and physiological responses against infection

A lipoperoxidação gera diversas espécies carbonílicas altamente reativas dentre as quais se destacam acroleína (ACR), malondialdeído (MDA), 4-hidroxi-2-hexenal (HHE) e 4-hidroxi-2-nonenal (HNE). A principal via endógena de metabolização desses compostos é através de conjugação com glutationa por ação da glutationa-S-tranferase. Contudo, diversos trabalhos têm mostrado que dipeptídeos contendo histidina, tal como a carnosina (CAR), também podem formar conjugados com aldeídos e auxiliar na detoxificação desses compostos. Em nosso trabalho adutos de CAR com ACR, HHE, HHEd5, HNE e HNEd11 foram sintetizados, purificados e caracterizados. A reação da CAR com ACR foi estudada em detalhes. Resultados mostraram que a carnosina reage com acroleína formando 03 produtos principais: m/z = 265, m/z = 283 e m/z = 303, sendo este último mais estável e mais abundante. Dados de RMN H1, COSY e HSQC permitiram elucidar a estrutura dessa molécula (m/z = 303) e propor uma rota de reação. Em seguida, uma metodologia baseada em cromatografia líquida acoplada à espectrometria de massas do tipo \"Ion Trap\" (ESI+ HPLC/MS-MS) foi desenvolvida e validada para quantificação simultânea dos adutos sintetizados. Pelo método desenvolvido é possível quantificar com precisão 25 pmol de CAR-HHE, 1 pmol de CAR-ACR e 1 pmol de CAR-HNE com um coeficiente de variação de aproximadamente 10 % e acurácia de 98 % (HHEd5 e HNEd11 foram usados como padrão interno). Análise em urina de adultos não fumantes mostraram que os produtos sintetizados estão presentes na urina de humanos em concentrações de 3,6 ± 1,4; 2,3 ± 1,5 e 1,3 ± 0,5 nmol / mg de creatinina, respectivamente para CAR-ACR, CAR-HHE e CAR-HNE. Em ratos transgênicos SODG93A modelo para esclerose lateral amiotrófica (ELA), a suplementação da dieta dos animais com 35 ± 5 mg carnosina/animal/semana melhorou a manutenção do peso e a sobrevida dos animais. Análises dos adutos sintetizados em amostras de músculo sugerem que a metabolização de aldeídos esteja comprometida nesses animais e que a carnosina poderia funcionar como \"scavenger\" para esses compostos. Esses resultados comprovam que dipeptídeos de histidina atuam na detoxificação de compostos carbonílicos e participa de suas vias de excreção. Além disso, a caracterização da estrutura e desenvolvimento de método sensível de detecção abre a possibilidade de utilização desses adutos como biomarcadores de estresse redox e exposição a aldeídos. / Lipid peroxidation generates reactive carbonyl species, including 4-hydroxy-2-nonenal (HNE), acrolein (ACR), 4-hydroxy-2-hexenal (HHE) and malondialdehyde (MDA). One major pathway of aldehyde detoxification in vivo is through conjugation with glutathione catalyzed by glutathione-S-transferases or, alternatively, by conjugation with endogenous histidine containing dipeptides, such as carnosine (CAR). The reaction of CAR with ACR was investigated in an effort to assess its possible biological role. One stable adduct was isolated by reverse-phase HPLC and characterized on the basis of extensive spectroscopic measurements. The proposed reaction route for product formation involves the reaction of the CAR amino group with ACR via a Schiff base formation followed by dehydration and cyclization through Michael addition in the imidazole ring forming an instable compound with m/z = 265. The subsequent reaction with another molecule of ACR followed by cyclization gives rise to the final product with m/z = 303.A highly sensitive method involving HPLC-MS analysis was developed for the simultaneous accurate quantification of CAR- ACR, CAR-HHE and CAR-HNE adducts in human urinary samples from non-smoking adults. This methodology permits quantification of 10 pmol CAR-HHE and 1 pmol of CAR-ACR and CAR-HNE. Adduct levels in urine were 3.6 ± 1.4, 2.3 ± 1.5, 1.3 ± 0.5 nmol/mg of creatinine, respectively to CAR-ACR, CAR-HHE and CAR-HNE. In SODG93A transgenic rats model to amyotrophic lateral sclerosis (ALS), the food supplementation of the animals with 35 ± 5 mg carnosine/animal/week improve de body weight and the life span of the ALS treated group. Analysis of the synthesized adducts in muscle sample showed suggest than aldehyde metabolization is compromised in this animals and that may be carnosine work like a scavenger for these compounds. Our results indicate that carnosine adduction can be an important detoxification route of α,β -unsaturated aldehydes. Moreover, carnosine adducts quantification may be useful as redox stress indicator in vivo.

The median prevalence of smoking among women of childbearing age in the United States is 22.4%. Of women who identify themselves as smokers in the three months prior to conception, 55% quit during pregnancy; however, 40% of those who quit relapse and return to smoking within six months after delivery. Smoking has been identified as an important means of stress management among smokers in general, and though limited to the perinatal period, pregnancy-specific stress adds to a woman’s typical day-to-day stress burden. Little data exists as to the effect of SHS exposure on smoking status during pregnancy and the impact of SHS exposure on the maternal perception of stress is unknown. Due to limited evidence, a critical need exists to examine the relationships of perceived maternal stress, SHS exposure, and perinatal smoking status in order to better understand perinatal smoking behaviors. The purposes of this dissertation were to: 1) evaluate the literature examining the relationship between the variables of maternal stress, SHS exposure, and perinatal smoking status; 2) determine the reliability and validity of the Everyday Stressors Index (ESI) use in pregnant women; and 3) to investigate the impacts of maternal perception of everyday stress, and SHS exposure on perinatal smoking status. Evidence obtained from the critical review of the literature supported an association between psychosocial stress and smoking during pregnancy or postpartum. Little information regarding the role of SHS exposure on perinatal smoking status was discovered. Psychometric testing of the ESI demonstrated strong internal consistency reliability, and factor analysis yielded three factors capturing three important domains of everyday stress. SHS exposure emerged as the most significant predictor of smoking status. Persistent smokers/relapsers had the highest ESI scores, followed by quitters, and then nonsmokers. While ESI means decreased in all smoking status groups from the first to the third trimester, the magnitude of decrease was not predictive. A significant interaction effect of SHS exposure in the home and decrease in ESI score occurred in the quit group only with quitters 1.14 times more likely to experience a decrease in ESI score compared to smokers/relapsers.

Over the past ten years, the development of High Throughput (HT) synthetic chemistry techniques has allowed the rapid preparation of libraries of hundreds to thousands of compounds. These tools are now extensively used for drug and material discovery programmes. The subsequent development of analytical capabilities to carry out qualitative and quantitative assessment of the compounds generated by HT synthesis as well as their HT screening has led to a dramatic broadening of the scope of HT techniques, ranging from image based analysis techniques to mass spectrometry (MS). Based on the latter, a range of solid phase and solution phase analytical constructs was developed to enable the qualitative and quantitative assessment of mixtures of small compounds, using positive electrospray MS as the sole analytical tool. A version of the construct allowed HT reactivity profiling to be carried out on a range of ten carboxylic acids, ten aldehydes and ten isonitriles in the Ugi 4-component condensation reaction. The effect of various parameters such as the concentration of the monomers on the reactivity was investigated. The elaboration of a HT Hammett parameter assessment method was made possible by the development of an electrophilic version of the construct. The value of the Hammett value was afforded by means of combinatorial Hammett plots and values were successfully evaluated in a HT mode for around thirty anilines with substituents in the meta and para position of the aromatic ring. Finally, analytical constructs were used in an attempt to evaluate enzyme reaction kinetics via the labelling of peptides and small drug fragment with coded constructs, to afford affinity determinations between the enzyme (protease) and peptidic or fragment based substrates.

Over the past decades, electrospray ionization for mass spectrometry (ESI-MS) has become one of the most commonly employed techniques in analytical chemistry, mainly due to its broad applicability to polar and semipolar compounds and the superior selectivity which is achieved in combination with high resolution separation techniques. However, responsiveness of an analytical method also determines its suitability for the quantitation of chemical compounds; and in electrospray ionization for mass spectrometry, it can vary significantly among different analytes with identical solution concentrations. Therefore, we investigated the ESI-response behavior of 56 nitrogen-containing compounds including aromatic amines and pyridines, two compound classes of high importance to both, synthetic organic chemistry as well as to pharmaceutical sciences. These compounds are increasingly analyzed employing ESI mass spectrometry detection due to their polar, basic character. Signal intensities of the peaks from the protonated molecular ion (MH+) were acquired under different conditions and related to compound properties such as basicity, polarity, volatility and molecular size exploring their quantitative impact on ionization efficiency. As a result, we found that though solution basicity of a compound is the main factor initially determining the ESI response of the protonated molecular ion, other factors such as polarity and vaporability become more important under acidic solvent conditions and may nearly outweigh the importance of basicity under these conditions. Moreover, we show that different molecular descriptors may become important when using different types of instruments for such investigations, a fact not detailed so far in the available literature.

Lysophosphatidic acid (LPA) is a bioactive lipid with a plethora of biological functions, including roles in cell survival, proliferation, and migration. Although high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC ESI-MS/MS) technology has been used to measure the levels of LPA in human blood, serum and plasma, current methods cannot readily detect the minute levels of LPA from cell culture. In this study, a novel HPLC ESI-MS/MS method with enhanced sensitivity was developed which allows accurate measurements of LPA levels with a limit of quantitation at approximately 10 femtomoles. The method was validated by quantitation of LPA levels in the media of previously characterized cell lines ectopically expressing autotaxin. Autotaxin overexpression induced an increase in several subspecies of LPA while others remained unchanged. Lastly, this HPLC ESI-MS/MS method was validated via biological assays previously utilized to assay LPA production. Hence, this new HPLC ESI-MS/MS will allow researchers to measure in vitro LPA levels and also distinguish between specific LPA subspecies for the delineation of individual biological mechanisms.

Resumo: Diretamente sob influência do Canal de São Sebastião - onde está estabelecido o maior terminal petrolífero do Brasil e ocorre intensa movimentação de petroleiros -, a faixa litorânea do município de Ilhabela-SP é freqüentemente impactada por vazamentos de óleo. Daí, sua condição de ser, entre todos os municípios do litoral-norte paulista, aquele que mais é impactado por vazamentos. Trata-se de área com alta relevância ecológica por conter ricos e diversificados recursos biológicos, bem como por apresentar significativa atividade sócio-econômica que envolve o turismo e a pesca. Este estudo apresenta, em escala de detalhe (1:10.000 a 1:25.000), um conjunto de vinte e quatro cartas SAO de caráter operacional e uma de caráter tática para a face oeste da Ilha de São Sebastião. Contempla, ainda, mapas temáticos, quadros de recursos visuais e listagem das espécies encontradas no arquipélago. A elaboração de tais cartas seguiu as recomendações do MMA-Brasil. Ao indicar as áreas historicamente mais atingidas por vazamento de óleo e definir áreas que requerem proteção especial e faixas de sacrifício, este estudo passa, no seu conjunto, a constituir valioso documento a ser usado em planos de contingência e emergência referentes ao arquipélago. / Abstract: Directly influenced by the São Sebastião Channel - where the biggest petroliferous terminal is installed and where occurs the highest numbers of tankers coming alongside - the coast side of Ilha Bela city - SP is frequently damaged by oil spills. Therefore it is considered the most 'harmed by leaking' city in the coast. It is a high ecologically relevant area for its risks and diversified biological resources, as well as for its significant socio-economical activities that involves tourism and fishing. This article shows, in detail (1:10.000 to 1:25.000) a set of twenty-three ESI maps and also a tactical one, facing the west side of São Sebastião Island. It also shows thematic maps, visual resources and a species list from the archipelago. The process to create those maps was strict to the Brazilian Environmental Ministry recommendations. As it indicates the historical areas that were damaged the most for oil spills and as it defines the areas that require special attention and sacrifice areas, it has become, as a whole, a valuable document to be used in emergency and contingency plans for the archipelago. / Orientador: Dimas Dias-Brito / Coorientador: João Carlos Carvalho Milanelli / Banca: Décio Luis Semensatto Junior / Banca: Paulina Setti Riedel / v. 2 - mapas temáticos, quadros descritivos de recursos visuais, listagens de espécies da fauna / Mestre