O maracujá, nome popular atribuído ao fruto das diversas espécies do gênero Passiflora, da família Passifloraceae, é amplamente comercializado e consumido no mundo, sendo o Brasil um dos maiores produtores do fruto. Alguns estudos apontam possível toxicidade relacionada às espécies de Passiflora, principalmente P. incarnata. No entanto, há pouco conhecimento acerca das espécies P. edulis e P. alata, sobretudo em relação à polpa e sementes. Os extratos da polpa e das sementes dos frutos dessas duas espécies de \"maracujá\", Passiflora alata e Passiflora edulis, foram estudados com o objetivo de identificar alcaloides harmânicos, pelo preparo das amostras por extração por sorção em barra magnética recoberta com polidimetilsiloxano (SBSE-PDMS) e SBSE recoberta com polietilenoglicol silicone (SBSE-EG Silicone) e análise por cromatografia líquida de alta eficiência com detector por fluorescência (CLAE-Flu) e cromatografia líquida de ultra eficiência acoplada à espectrometria de massas sequencial (CLUE-EM/EM). A análise dos alcaloides harmana e harmina nos extratos da polpa de P. alata foi feita por meio do método de adição de padrão e mostrou menor quantidade destes alcaloides, em comparação com os resultados da análise dos extratos da polpa dos frutos de P. edulis, no trabalho de Pereira e colaboradores. As análises CLUE-EM e CLUE-EM/EM possibilitaram a identificação dos alcaloides nos extratos: nas sementes de P. alata, os alcaloides harmana, harmina, harmol, harmalol e harmalina foram identificados, enquanto que na polpa, harmana e harmina tiveram a confirmação da sua presença. Nos extratos da polpa dos frutos de P. edulis observou-se os alcaloides harmana, harmina e harmalina. E nas sementes de P. edulis harmina foi encontrada, porém há indícios da presença de harmana. A literatura sobre as barras de SBSE-EG Silicone Twister&®; não relata nenhum estudo relacionado ao seu uso para extração e concentração de alcaloides harmânicos nos extratos de P. alata e P. edulis. Por isso foi proposto inicialmente a aplicação do planejamento fatorial fracionário para otimização do método de extração, utilizando os padrões comerciais dos alcaloides harmana e harmina. O planejamento experimental revelou as variáveis principais e seus níveis de importância, e a partir destes resultados foi realizado o estudo cinético dos tempos de extração e de dessorção das barras de SBSE-EG Silicone. Porém, os resultados mostraram que as barras de SBSE-EG Silicone não são adequadas para a extração dos alcaloides harmana e harmina, uma vez que a recuperação obtida foi baixa, na ordem de 30%. / \"Maracujá\" is the popular name given to the fruit of several species of Passiflora genus, from Passifloraceae family, it is widely commercialized and consumed around the world, and Brazil is one of the largest producers of this fruit. Some studies pointed out the possible toxicity related to Passiflora species, mainly P. incarnata. Although, there is a lack of knowledge about the P. edulis and P. alata species, especially with regards to the pulp and seeds. The extracts of pulp and seeds from the \"maracujá\" species Passiflora alata and Passiflora edulis, were studied in order to identify harman alkaloids. The samples were prepared by extraction with sorptive stir bar coated with polydimethylsiloxane (SBSE-PDMS) and SBSE coated with polyethylene glycol silicon (SBSE-EG Silicone). The samples were analyzed by high performance liquid chromatography with fluorescence detector (HPLC-Flu), and ultra-high pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Harmane and harmine alkaloids in P. alata pulp extracts were analyzed using the standard addition method and the results showed a lower amount of these alkaloids, compared with the test results for the extracts from the P. edulis pulp in the work of Pereira et al. UHPLC-MS and UHPLC-MS/MS analysis enabled to identify the alkaloids amount present in the extracts. In the P. alata seeds extract the following alkaloids were identified harmane, harmine, harmol, harmalol and harmaline, while in the pulp extract, harmane and harmine were confirmed. In the extracts of P. edulis pulp the alkaloids identified were harmane, harmine and harmaline. And in the P. edulis seeds extract the harmine alkaloid was found, some indications of the presence of harmana were observed. The literature about SBSE-EG Silicone Twister® bars reports no study related to their use for extraction and concentration of harman alkaloids in P. alata and P. edulis. Thus, it was initially proposed the application of fractional factorial design to optimize the extraction method using commercial standards of harmane and harmine alkaloids. The experimental design revealed the main variables and their importance levels, and from these results kinetic studies were performed for the extraction and desorption times of SBSE-EG Silicone bars. However, the results showed SBSE-EG Silicone bars are not suitable for the extraction of harmane and harmine alkaloids, since the recovery obtained was low, on the order of 30%.

O maracujá, nome popular atribuído ao fruto das diversas espécies do gênero Passiflora, da família Passifloraceae, é amplamente comercializado e consumido no mundo, sendo o Brasil um dos maiores produtores do fruto. Alguns estudos apontam possível toxicidade relacionada às espécies de Passiflora, principalmente P. incarnata. No entanto, há pouco conhecimento acerca das espécies P. edulis e P. alata, sobretudo em relação à polpa e sementes. Os extratos da polpa e das sementes dos frutos dessas duas espécies de \"maracujá\", Passiflora alata e Passiflora edulis, foram estudados com o objetivo de identificar alcaloides harmânicos, pelo preparo das amostras por extração por sorção em barra magnética recoberta com polidimetilsiloxano (SBSE-PDMS) e SBSE recoberta com polietilenoglicol silicone (SBSE-EG Silicone) e análise por cromatografia líquida de alta eficiência com detector por fluorescência (CLAE-Flu) e cromatografia líquida de ultra eficiência acoplada à espectrometria de massas sequencial (CLUE-EM/EM). A análise dos alcaloides harmana e harmina nos extratos da polpa de P. alata foi feita por meio do método de adição de padrão e mostrou menor quantidade destes alcaloides, em comparação com os resultados da análise dos extratos da polpa dos frutos de P. edulis, no trabalho de Pereira e colaboradores. As análises CLUE-EM e CLUE-EM/EM possibilitaram a identificação dos alcaloides nos extratos: nas sementes de P. alata, os alcaloides harmana, harmina, harmol, harmalol e harmalina foram identificados, enquanto que na polpa, harmana e harmina tiveram a confirmação da sua presença. Nos extratos da polpa dos frutos de P. edulis observou-se os alcaloides harmana, harmina e harmalina. E nas sementes de P. edulis harmina foi encontrada, porém há indícios da presença de harmana. A literatura sobre as barras de SBSE-EG Silicone Twister&®; não relata nenhum estudo relacionado ao seu uso para extração e concentração de alcaloides harmânicos nos extratos de P. alata e P. edulis. Por isso foi proposto inicialmente a aplicação do planejamento fatorial fracionário para otimização do método de extração, utilizando os padrões comerciais dos alcaloides harmana e harmina. O planejamento experimental revelou as variáveis principais e seus níveis de importância, e a partir destes resultados foi realizado o estudo cinético dos tempos de extração e de dessorção das barras de SBSE-EG Silicone. Porém, os resultados mostraram que as barras de SBSE-EG Silicone não são adequadas para a extração dos alcaloides harmana e harmina, uma vez que a recuperação obtida foi baixa, na ordem de 30%. / \"Maracujá\" is the popular name given to the fruit of several species of Passiflora genus, from Passifloraceae family, it is widely commercialized and consumed around the world, and Brazil is one of the largest producers of this fruit. Some studies pointed out the possible toxicity related to Passiflora species, mainly P. incarnata. Although, there is a lack of knowledge about the P. edulis and P. alata species, especially with regards to the pulp and seeds. The extracts of pulp and seeds from the \"maracujá\" species Passiflora alata and Passiflora edulis, were studied in order to identify harman alkaloids. The samples were prepared by extraction with sorptive stir bar coated with polydimethylsiloxane (SBSE-PDMS) and SBSE coated with polyethylene glycol silicon (SBSE-EG Silicone). The samples were analyzed by high performance liquid chromatography with fluorescence detector (HPLC-Flu), and ultra-high pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Harmane and harmine alkaloids in P. alata pulp extracts were analyzed using the standard addition method and the results showed a lower amount of these alkaloids, compared with the test results for the extracts from the P. edulis pulp in the work of Pereira et al. UHPLC-MS and UHPLC-MS/MS analysis enabled to identify the alkaloids amount present in the extracts. In the P. alata seeds extract the following alkaloids were identified harmane, harmine, harmol, harmalol and harmaline, while in the pulp extract, harmane and harmine were confirmed. In the extracts of P. edulis pulp the alkaloids identified were harmane, harmine and harmaline. And in the P. edulis seeds extract the harmine alkaloid was found, some indications of the presence of harmana were observed. The literature about SBSE-EG Silicone Twister® bars reports no study related to their use for extraction and concentration of harman alkaloids in P. alata and P. edulis. Thus, it was initially proposed the application of fractional factorial design to optimize the extraction method using commercial standards of harmane and harmine alkaloids. The experimental design revealed the main variables and their importance levels, and from these results kinetic studies were performed for the extraction and desorption times of SBSE-EG Silicone bars. However, the results showed SBSE-EG Silicone bars are not suitable for the extraction of harmane and harmine alkaloids, since the recovery obtained was low, on the order of 30%.

Honey has been used to heal wounds since ancient times. There are many references in ancient literature that cite honey for its medicinal uses. It is used as an alternative agent to cure infections of wounds, burns, ulcers etc. Researchers have shown some of the antimicrobial properties of honey when used as an ointment. When applied to an affected area, it helps to promote the growth of healthy tissue. One of the factors on which the quality of the honey depends, is its geographical origin. Based on the location, honey types can vary as much as 100-fold from each other in color, aroma, viscosity, and antimicrobial properties. The important components in honey that play an essential part in healing wounds and contributing to the antimicrobial properties are enzymes. Their presence allows honey to kill various types of pathogenic bacteria, viruses, fungi etc. A higher antimicrobial effect is seen in monofloral honey (when a single plant species is the source of nectar), which is often more potent than other types of honey in terms of antibacterial activity. Resistance of pathogens to these antimicrobial actions has never been shown, which makes honey a more promising source of antimicrobial research. Presently, infections of burns and wounds are very challenging to treat, especially when they are caused by antibiotic-resistant bacteria. The purpose of this study was to examine the antimicrobial properties of honey from Utah and other locales, and to identify promising antimicrobial activities that could be useful in treating infections caused by resistant bacteria.

In recent years, the abuse of synthetic cathinones or ‘bath salts’ has become a major public health concern. Although these compounds were initially sold legally and labeled “not for human consumption”, the ‘bath salts’ are psychostimulants, with similar structures and pharmacologic mechanisms to cocaine, the amphetamines, and 3,4 methylendioxymethamphetamine (MDMA, Molly, or Ecstasy). The reported use of these substances by women of child-bearing age highlights the necessity of studies seeking to delineate risks of prenatal exposure. Three popular drugs of this type are methylone, mephedrone, and 3, 4-methylenedioxypyrovalerone (MDPV). Unfortunately, there is currently no information available on the teratogenicity of these compounds, or of the extent to which they cross the placenta. As such, the purpose of this study was to examine the pharmacokinetic profile of the ‘bath salts’ in a pregnancy model. Pregnant mice (E17.5 gestation) were injected intraperitoneally with a cocktail of 5mg/kg methylone, 10mg/kg mephedrone, and 3mg/kg (MDPV) dissolved in sterile saline. Maternal brain, maternal plasma, placenta, and fetal brain were collected at 30s, 1min, 5min, 10min, 15min, 30min, 1h, 2h, 4h, and 8h following injection. Methylone, mephedrone, and MDPV were extracted from tissue by solid phase extraction, and concentrations were determined using a previously validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Interestingly, all 3 cathinones reached measurable concentrations in the placenta, as well as the fetal brain; in fact, for MDPV, the maximal concentration (Cmax) was highest in fetal brain, while mephedrone's highest Cmax value was achieved in placenta. Additionally, the total drug exposure for all 3 compounds (as represented by area under the curve, AUC) was higher in fetal matrices (placenta and fetal brain) than in maternal matrices (maternal brain and plasma), and the half-lives for the drugs were longer. Given the extensive presence of methylone, mephedrone, and MDPV in the fetal brain following prenatal exposure, fetal risk is definitely a concern. As there are currently no prenatal studies available on the teratogenicity of these agents, pregnant patients should be informed about the potential risks that these substances may have.

De nos jours, environ 3000 huiles essentielles sont produites et utilisées dans le monde avec des champs d'application aussi variés que la cosmétique, la parfumerie l'agro-alimentaire, la pharmacie et l'aromathérapie. Ces huiles sont extraites des hespéridés ou des plantes aromatiques et médicinales. Étant donné que la culture de ces matières premières implique généralement l'application de pesticides, la présence de tels résidus dans les huiles essentielles ne peut pas être écartée. Compte tenu du nombre important de pesticides employés et des nombreux champs d'application des huiles essentielles, il apparaît nécessaire, afin d'assurer la santé du consommateur, de disposer d'une méthode d'analyse multi-résidus capable de doser les pesticides dans les huiles essentielles. L'état de l'art des méthodes d'analyse lié à cette problématique a révélé un manque évident de performance des méthodes actuelles aussi bien en termes de limite de détection que du nombre de pesticides analysés. Ce mémoire propose donc la mise au point d'une méthode d'analyse multi-résidus de pesticides dans les huiles essentielles par couplage de la chromatographie liquide et de la spectrométrie de masse, technologie la plus à même de répondre à la problématique au vue de la bibliographie. Après avoir mis en évidence les performances du nouveau mode d'acquisition Scheduled SRM, disponible sur le spectromètre de masse 4000 QTrap, pour la détection et la quantification de 250 pesticides dans un solvant déterminé, l'importance de considérer la nature de la matrice, aussi bien dans les méthodes de préparation que lors de l'analyse de l'échantillon, a ensuite été démontrée en étudiant deux huiles essentielles représentatives (lavandin et citron). Enfin, la méthode d'analyse multi-résidus LC-MS/MS a été appliquée à la recherche de pesticides dans des échantillons réels d'huiles essentielles. Elle a démontré sa capacité à détecter, quantifier et identifier les pesticides dans ces matrices au travers de l'utilisation d'un mode d'acquisition couplé SRM-EPI faisant appel aux spécificités du spectromètre de masse hybride et notamment de sa trappe d'ion linéaire. Ce travail a de plus révélé l'importance de disposer d'une telle méthode au regard du nombre de pesticides détectés dans les échantillons et de leurs concentrations relativement élevées. Celles-ci peuvent en effet atteindre des teneurs supérieures au milligramme par litre dans les huiles essentielles analysées.

Le foisonnement filamenteux est un problème récurant dans de nombreuses stations d'épuration à boues activées. L'objectif de ces travaux est d'améliorer la compréhension des mécanismes physiologiques et moléculaires impliqués dans la filamentation des microorganismes, afin de pouvoir orienter de futures stratégies de lutte contre le phénomène de bulking. Sphaerotilus natans, qui peut croître réversiblement sous forme monocellulaire ou filamenteuse, a été utilisée comme bactérie modèle pour cette étude. Différents types de cultures, ainsi que des suivis par cytométrie en flux et marquage au cFDA/SE, ont montré que les diverses souches de S. natans adoptent des morphologies différentes et que les filaments croissent par divisions cellulaires successives et non par un chaînage des bactéries. Une analyse par RT-QPCR a mis en évidence que l'expression du gène sthA augmente fortement après induction de la filamentation et reste ensuite à un niveau élevé. Une comparaison de l'expression protéique des formes monocellulaire et filamenteuse, par LC-MS-MS, a permis d'identifier des protéines impliquées dans la filamentation, et notamment dans la synthèse de la gaine. La concentration intracellulaire en ARNr, mesurée par RT-QPCR, varie durant la croissance de S. natans et d'autres microorganismes, entraînant une diminution importante de l'intensité du marquage FISH, mesurée par cytométrie en flux. L'utilisation de la technique FISH pour quantifier des microorganismes est donc remise en question, d'autant plus dans des matrices aussi complexes que les boues activées. Ces observations mettent également en doute l'hypothèse, émise en utilisant ce mode de quantification, d'une déstructuration des filaments consécutive à un retour à des conditions de culture plus favorables.

<p>Electrospray ionization (ESI) mass spectrometry (MS) in combination with liquid chromatography (LC) is an excellent tool for the identification of drug metabolites. Utilizing this hyphenated technique in combination with proper sample pretreatment, the metabolic pathways of the analgesic drug ketobemidone were investigated in human urine and rat microdialysate from blood and brain. Two novel phase I metabolites (ketobemidone N-oxide and meta-hydroxymethoxyketobemidone) and three novel phase II metabolites (glucuronic acid conjugates of ketobemidone, norketobemidone and hydroxymethoxyketobemidone) were identified in human urine. Further, norketobemidone and ketobemidone N-oxide were identified in rat microdialysate from brain after regional distribution of ketobemidone in striatum. This indicates that the brain itself has the possibility to metabolize ketobemidone. </p><p>Synthetic ketobemidone metabolites were used for comparison of retention times and tandem MS spectra with the possible metabolites recovered from the biological samples. The conjugated metabolites were identified by accurate mass measurements and tandem MS spectra of the aglycones. The accuracy of the estimated masses was better than 2.1 ppm for two out of three conjugates in presence of internal standard.</p><p>On-line micro-SPE was successfully used for trapping and desalting of the microdialysates. The small SPE pre-column made it possible to inject approximately 100 times more sample on the analytical column compared to injection without pre-column. Selective trapping was demonstrated for the polar catechol amine metabolite, dihydroxyketobemidone, which forms covalent complexes with phenylboronic acid (PBA). A fluorinated silica type stationary phase was the only column out of several tested that was able to separate ketobemidone and all relevant phase I metabolites. </p><p>Liquid chromatography and mass spectrometry are independently valuable tools in the field of analytical pharmaceutical chemistry. The present study showed that the combination of LC-MS, with its excellent selectivity and sensitivity, offers an outstanding tool in the qualitative analysis of drugs and metabolites in biological fluids. </p>

Electrospray ionization (ESI) mass spectrometry (MS) in combination with liquid chromatography (LC) is an excellent tool for the identification of drug metabolites. Utilizing this hyphenated technique in combination with proper sample pretreatment, the metabolic pathways of the analgesic drug ketobemidone were investigated in human urine and rat microdialysate from blood and brain. Two novel phase I metabolites (ketobemidone N-oxide and meta-hydroxymethoxyketobemidone) and three novel phase II metabolites (glucuronic acid conjugates of ketobemidone, norketobemidone and hydroxymethoxyketobemidone) were identified in human urine. Further, norketobemidone and ketobemidone N-oxide were identified in rat microdialysate from brain after regional distribution of ketobemidone in striatum. This indicates that the brain itself has the possibility to metabolize ketobemidone. Synthetic ketobemidone metabolites were used for comparison of retention times and tandem MS spectra with the possible metabolites recovered from the biological samples. The conjugated metabolites were identified by accurate mass measurements and tandem MS spectra of the aglycones. The accuracy of the estimated masses was better than 2.1 ppm for two out of three conjugates in presence of internal standard. On-line micro-SPE was successfully used for trapping and desalting of the microdialysates. The small SPE pre-column made it possible to inject approximately 100 times more sample on the analytical column compared to injection without pre-column. Selective trapping was demonstrated for the polar catechol amine metabolite, dihydroxyketobemidone, which forms covalent complexes with phenylboronic acid (PBA). A fluorinated silica type stationary phase was the only column out of several tested that was able to separate ketobemidone and all relevant phase I metabolites. Liquid chromatography and mass spectrometry are independently valuable tools in the field of analytical pharmaceutical chemistry. The present study showed that the combination of LC-MS, with its excellent selectivity and sensitivity, offers an outstanding tool in the qualitative analysis of drugs and metabolites in biological fluids.

Blumeria graminis f. sp. hordei is a biotroph pathogen that causes powdery mildew disease in barley. In this study, Pallas01 and Pallas03 barley lines having Mla1, Ml (Al2) and Mla6, Mla14 R-genes were inoculated with Bgh103(64/01) race of the Blumeria graminis f. sp. hordei having avirulence and virulence to Pallas01 and Pallas03, respectively. The proteins were isolated from the three biological replicates of 12, 24, and 48 hpi samples following the method in Rampitsch et al., 2006. These there biological replicates of three time points together with the mock inoculated plant proteins were separated on 2D-PAGE using IPG strips of 4-7 pH values as three technical replicates, resulting 108 gels. The gels were analyzed using PdQuest (Bio Rad) in order to assess up- or down-regulated protein spots by comparing against controls and the samples having resistance or susceptible responses with each other. According to the analysis, 36 proteins were found to be differentiated and among them 18 proteins were found up-regulated and 8 proteins were found down-regulated. The spots were manually v excised and subjected to the nano-LC-ESI-MS/MS analysis (Proteome Factory, Germany). The MASCOT algorithm was used for identification of the possible proteins. The experimental pI and MW values were used for selecting the differentiated proteins from the mass results. The relative abundance of each of the 38 identified polypeptides was calculated in terms of spot intensity. The majority of the most abundant proteins were found to be carbohydrate metabolism related. The relative distribution of the proteins into four main functional categories was taken into consideration. Statistical tests (Students&amp / #8223 / T-test) were carried among the identified proteins in order to reveal statistically significant proteins throughout the study. By making a WoLF PSORT search, subcellular localization of the proteins was predicted. Accordingly, most of the proteins were found to be located in cytoplasm or chloroplast.

Rhodobacter capsulatus is a versatile organism capable of growing on different growth conditions including photofermentation in the presence of carbon source, aerobic respiration, anaerobic respiration in the presence of an external electron acceptor such as DMSO. The photofermentative growth of R.capsulatus results in hydrogen production which stands out as an environmentally harmless method to produce hydrogen and accepted as one of the most promising process. Due to the serious problems such as as global climate change and environmental pollution caused by the fossil fuels, there is an increasing requirement for a clean and sustainable energy source. Furtherrmore, the ability of R.capsulatus to fix nitrogen, to use solar energy makes it a model to study various aspects of its metabolism. Thus the goal of this study is to increase the potential in biohydrogen production with the photofermentative bacteria and to investigate the proteins playing roles in different growth modes of the bacteria. In the present study, protein profiles of Rhodobacter capsulatus grown on respiratory, anaerobic respiratory and photofermentative growth modes were obtained. LC-MS/MS system is used to analyze the proteome as a high throughput technique. Physiological analysis such as HPLC for the analysis of the carbon source consumption, GC and analysis of pigments were carried out to state the environmental conditions. As a result, total of 460 proteins were identified with 17 proteins being unique to particular growth condition. Ratios of the proteins in different growth conditions were compared and important proteins were highlighted.