In this study, we collected 18 plasma samples from NPC patients, and 10 plasma samples from healthy person for plasma proteomic analysis. We classified these samples into 3 groups: treatment, pretreatment and recurrent. Two-dimensional electrophoresis (2-DE) and MALDI-TOF were performed followed by comparative and statistic analysis. In conclusions, we totally identified 30 proteins in Nasopharyngeal carcinoma (NPC) plasma, and found 10 proteins with expression level down regulated (p<0.001). These proteins were characterized to be Serotransferrin, Vitamin D-binding protein (VDB), alpha-1 antitrypsin (AAT), Haptoglobin, Apolipoprotein B fragment, Syntaxin-7, Apolipoprotein, A-I, PRO1779, Transthyrin, MDN1 protein, respectively. Consequently, we established a protocol to remove high abundant proteins (e.g., albumin, immunoglobin etc.) in plasma. We are especially interested in ATT and VDB. Western blotting assay was performed to confirm ATT and VDB expression. Furthermore, the quantity of ATT and VDB were measured by ELISA to obtain the threshold value of these proteins. Finally, we want to realize the relationship between these down regulation proteins and clinical parameters in NPC malignancy and tumor progression. Since there are few protein expression research of NPC in clinical studies, our works will provide insights in NPC studies for tumor progression with potential to elevate treatment efficiency.

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Peanut skin, a low-value agriculture waste product, has drawn lots of research interest in recent years, due to its high content of A-type proanthocyanidins. A-type proanthocyanidins have been believed to contribute to cranberries' anti-UTI (urinary tract infection) effect. In this study, we compared the A-type proanthocyanidins in cranberry and peanut skin crude extracts using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Many similarities were found in the proanthocyanidin composition of cranberries and peanut skins. New oligomeric and polymeric proanthocyanidins in peanut skins, including heteroproanthocyanidins and proanthocyanidins with sugar moieties or galloyl esters, were tentatively identified. Solid phase extraction (SPE) and HPLC fractionation largely improved MALDI-TOF's ability to detect proanthocyanidins with high degrees of polymerization (DP). By analyzing the identified compounds in each fraction, we were also able to find some interesting elution pattern of the proanthocyanidins on the SPE cartridges and on the HPLC column. For example, the elution order on both the SPE cartridges and the diol phase column generally followed the DP. A-type proanthocyanidins tended to elute earlier than the B-type. Prodelphinidins retained much longer than other proanthocyanidins with the same DP. These findings may help researcher to identify future research directions and develop new separation methods to facilitate the identification of bioactive components in proanthocyanidin-rich plant extracts. / Ph. D.

The aim of this study was to optimize the sample preparation and methods for analysis of oligosaccharides of hyaluronic acid with MALDI- TOF-MS. The analysis was carried out on an Autoflex speed MADLI-TOF- MS instrument with both linear and reflectron mode. Matrices used in this study were 2,5-DHB and Super-DHB and type of matrix was chosen depending on the size of the analyzed oligosaccharides. The application of sample and matrix on the target that gave the most homogenous crystallization was sandwich and the laser power in the MALDI was kept at 65 %. Since it is known that salts and buffers interfere with the analysis, sample clean-up such as solid phase extraction (SPE) in pipette tips and dialysis was performed. SPE worked best for low mass oligosaccharides and provided high intensity and little noise. With SPE a concentration of the analyte could be done which was the advantage over dialysis. Dialysis worked well for larger oligosaccharides and mixtures of different sized oligosaccharides. Another way of using MALDI for biomolecule analysis is with TLC-MALDI. A fast and accurate separation was achieved and analysis could be done directly from the plate. The optimized methods were evaluated according to linearity and precision, LOD, mass accuracy and matrix stability. The linearity and precision was good in a higher concentration range (50 μg/mL and higher), but the test for limit-of-detection (LOD) indicated that concentrations from 20-30 μg/mL could be analyzed with no interference from the background. The mass accuracy was within the acceptable limits according to Bruker Daltonics when a mass calibration was done for each analyzed sample. The stability of the matrix in solution was difficult to study because of the day-to-day variation in intensities given by the MALDI-TOF-MS technique.

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Metronidazole is an essential component of the triple therapy regimen against Helicobacter pylori infection. The development of resistance towards metronidazole results in failure to eradicate H. pylori completely. The main aim of the investigation was to understand further the mechanism(s) of resistance in H. pylori. The investigation focussed upon studying the role and function of NADH oxidase in metronidazole resistance. The NADH oxidase levels were shown to be significantly higher in metronidazole susceptible strains than in resistant strains. The purification and characterisation of the enzyme responsible for the oxidation of NADH resulted in isolation of a protein shown to be catalase. The results suggest that NADH oxidase activity in susceptible strains is a function of a bifunctional catalase rather than that of a distinct enzyme. This was confirmed by isolation of catalase from E. coli cells containing cloned H. pylori catalase and demonstration that catalase and NADH activities co-purified. The catalase activity of the purified protein from the bacterial strains used was retained but the oxidation of NADH was not significant in the resistant strain. The base sequence of the catalase gene from the susceptible strain was determined and shown to be 99% identical to that from the cloned gene. The comparison of the derived amino acid sequence of catalase from H. pylori and Proteus mirabilis showed that the heme-binding site is highly conserved. The amino acids in the NAD(P)H binding site are conserved in both strain NCTC 11639 (Mtz s ) and the genomic strain ATCC 26695 (Mtz s) of H. pylori but show significant differences compared with P. mirabilis. A three-dimensional model of catalase from a metronidazole-susceptible H. pylori strain showed stearic hindrance around the NAD(P)H binding site and a substitution of an acidic for a basic residue within the phosphate binding site. Both effects could result in NAD(P)H being less tightly bound and, hence able to leave the catalase in exchange for NADH. Other substitutions may account for the ability of the modified binding site to oxidise NADH. The oxidation of NADH aids in the activation of metronidazole, which damages DNA. The absence of NADH oxidase activity in resistant strains results in the inability of the enzyme to activate metronidazole leading to resistance. The finding that this NADH oxidase activity is a function of a modified catalase in susceptible strains suggests a novel mechanism of metronidazole resistance in H. pylori.

A criptococose é uma doença invasiva capaz de apresentar-se de forma fatal podendo acometer pacientes imunocompetentes e imunocomprometidos. Os agentes etiológicos Cryptococcus neoformans var. grubii e C. neoformans var. neoformans apresentam distribuição cosmopolita, sendo as excretas de pombos o seu principal reservatório. Com o advento de terapias imunossupressoras e a pandemia de HIV, observou-se o aumento significativo de casos de pacientes com criptococose. Atualmente, o diagnóstico é baseado na apresentação clínica, na observação microscópica de líquor corado com tinta da Índia e/ou no isolamento em cultura. Neste trabalho desenvolveu-se um ELISA para detecção de anticorpos contra C. neoformans var. grubii em soro de pacientes utilizando como antígeno um extrato protéico total de uma linhagem clínica isolada de um paciente com criptococose (HC6). Foram testados através de ELISA 40 amostras de soros de pacientes com criptococose, sendo 67,5% positivos e 32,5% falsos negativos. Como controles negativos foram testados 82 amostras de soros de indivíduos hígidos, dos quais 26,82% apresentaram resultados positivos para os testes realizados. Para testar a reatividade cruzada, foram utilizadas 10 amostras de pacientes com histoplasmose (20% de reatividade cruzada), 9 amostras de pacientes com paracoccidioidomicose (66,6% de reatividade cruzada), 9 amostras de pacientes com candidose (13,3% de reatividade cruzada) e 7 amostras de pacientes com aspergilose (14,28% de reatividade cruzada). Visando solucionar o problema da reatividade cruzada, identificamos proteínas antigênicas de C. neoformans var. grubii por eletroforese bidimensional seguida por western blot e espectrometria de massa (MALDI-TOF MS). Das 75 amostras analisadas, quatro foram identificadas: uma proteína hipotética, 2 isoformas de HSPs 70 e uma catalase-2. As proteínas identificadas apresentaram baixa similaridade com ortólogas de outros fungos patogênicos, sendo, dessa forma, possíveis alvos para a padronização do ELISA e diagnóstico da criptococose. / Cryptococosis is an invasive and potentially fatal disease. Cryptococcus neoformans is the etiological agent, which can affect both immunocompromised and immunocompetent individuals. C. neoformans var. grubii and C. neoformans var. neoformans are cosmopolitan and their major natural reservoir is the excrement from pigeons. With the advent of immunosupressor therapies and the pandemic HIV infection, a significant augmentation of cryptococosis cases in humans was observed. Nowadays cryptococcosis diagnosis is based on the clinical presentation, India ink sample preparation methods and/or in vitro culture isolation. In this work we had developed an ELISA to detect antibodies against C. neoformans var. grubii in serum from patients with cryptococcosis using as antigens a whole cell protein extract from a clinical cell line isolate (HC6). Sera from 40 patients with cryptococcosis were tested by ELISA. From these, 67.5% were positives and 32.5% were false-negatives. As a negative control 82 samples from health subjects were also tested, from these 26.82% were positives. To test cross-reactivity, samples from 10 patients with histoplasmosis (20% cross-reactivity), 9 from patients with paracoccidioidomicosis (66.6% cross-reactivity), 9 from patients with candidosis (13.3% cross-reactivity) and 7 from patients with aspergilosis (14.28% cross-reactivity) were tested. To solve the cross-reactivity problem, we searched immunogenic proteins which were specific to C. neoformans var. grubii applying two-dimensional polyacrylamide gel electrophoresis (2DE-PAGE) followed by western blot and mass spectrometry (MALDI-TOF MS). From the 75 sample analyzed, four were identified: one as a hypothetic protein, two HSPs 70 isoforms and the protein catalase 2. These proteins showed low similarity with orthologues from other pathogenic fungi, and are potential targets to further of the standardizing cryptococosis diagnosis by ELISA.

A criptococose é uma doença invasiva capaz de apresentar-se de forma fatal podendo acometer pacientes imunocompetentes e imunocomprometidos. Os agentes etiológicos Cryptococcus neoformans var. grubii e C. neoformans var. neoformans apresentam distribuição cosmopolita, sendo as excretas de pombos o seu principal reservatório. Com o advento de terapias imunossupressoras e a pandemia de HIV, observou-se o aumento significativo de casos de pacientes com criptococose. Atualmente, o diagnóstico é baseado na apresentação clínica, na observação microscópica de líquor corado com tinta da Índia e/ou no isolamento em cultura. Neste trabalho desenvolveu-se um ELISA para detecção de anticorpos contra C. neoformans var. grubii em soro de pacientes utilizando como antígeno um extrato protéico total de uma linhagem clínica isolada de um paciente com criptococose (HC6). Foram testados através de ELISA 40 amostras de soros de pacientes com criptococose, sendo 67,5% positivos e 32,5% falsos negativos. Como controles negativos foram testados 82 amostras de soros de indivíduos hígidos, dos quais 26,82% apresentaram resultados positivos para os testes realizados. Para testar a reatividade cruzada, foram utilizadas 10 amostras de pacientes com histoplasmose (20% de reatividade cruzada), 9 amostras de pacientes com paracoccidioidomicose (66,6% de reatividade cruzada), 9 amostras de pacientes com candidose (13,3% de reatividade cruzada) e 7 amostras de pacientes com aspergilose (14,28% de reatividade cruzada). Visando solucionar o problema da reatividade cruzada, identificamos proteínas antigênicas de C. neoformans var. grubii por eletroforese bidimensional seguida por western blot e espectrometria de massa (MALDI-TOF MS). Das 75 amostras analisadas, quatro foram identificadas: uma proteína hipotética, 2 isoformas de HSPs 70 e uma catalase-2. As proteínas identificadas apresentaram baixa similaridade com ortólogas de outros fungos patogênicos, sendo, dessa forma, possíveis alvos para a padronização do ELISA e diagnóstico da criptococose. / Cryptococosis is an invasive and potentially fatal disease. Cryptococcus neoformans is the etiological agent, which can affect both immunocompromised and immunocompetent individuals. C. neoformans var. grubii and C. neoformans var. neoformans are cosmopolitan and their major natural reservoir is the excrement from pigeons. With the advent of immunosupressor therapies and the pandemic HIV infection, a significant augmentation of cryptococosis cases in humans was observed. Nowadays cryptococcosis diagnosis is based on the clinical presentation, India ink sample preparation methods and/or in vitro culture isolation. In this work we had developed an ELISA to detect antibodies against C. neoformans var. grubii in serum from patients with cryptococcosis using as antigens a whole cell protein extract from a clinical cell line isolate (HC6). Sera from 40 patients with cryptococcosis were tested by ELISA. From these, 67.5% were positives and 32.5% were false-negatives. As a negative control 82 samples from health subjects were also tested, from these 26.82% were positives. To test cross-reactivity, samples from 10 patients with histoplasmosis (20% cross-reactivity), 9 from patients with paracoccidioidomicosis (66.6% cross-reactivity), 9 from patients with candidosis (13.3% cross-reactivity) and 7 from patients with aspergilosis (14.28% cross-reactivity) were tested. To solve the cross-reactivity problem, we searched immunogenic proteins which were specific to C. neoformans var. grubii applying two-dimensional polyacrylamide gel electrophoresis (2DE-PAGE) followed by western blot and mass spectrometry (MALDI-TOF MS). From the 75 sample analyzed, four were identified: one as a hypothetic protein, two HSPs 70 isoforms and the protein catalase 2. These proteins showed low similarity with orthologues from other pathogenic fungi, and are potential targets to further of the standardizing cryptococosis diagnosis by ELISA.

abstract: The ability of microalgae to be mass cultivated and harvested for production of pharmaceuticals, nutraceuticals, and biofuels has made microalgae a focal point of scientific investigation. However, negative impacts on production are essentially inevitable due to the open design of many microalgae mass culture systems. This challenge generates a need for the consistent monitoring of microalgae cultures for health and the presence of contaminants, predators, and competitors. The techniques for monitoring microalgae cultures are generally time-intensive, labor-intensive, and expensive. The scope of this work was to evaluate the use of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) as a viable alternative for the characterization of microalgae cultures. The studies presented here evaluated whether MALDI-TOF MS can be used to: 1) differentiate microalgae at the species and strain levels, 2) characterize simple mixtures of microalgae, 3) detect changes in a single microalgae culture over time, and 4) characterize growth phases of microalgae cultures. This research required the development of a MALDI-TOF MS microalgae analysis protocol for organism characterization. The results yielded in this research showed that MALDI-TOF MS was just as accurate, if not more so, than molecular techniques for the identification of microalgae at the species and strain levels during its logarithmic growth phase. Additionally, results suggest that MALDI-TOF MS is sensitive enough to characterize simple mixtures and detect changes in cultures over time. The data presented here suggests the next logical step is the development of protocols for the near-real time health monitoring of microalgae cultures and detection of contaminants using MALDI-TOF MS. / Dissertation/Thesis / Masters Thesis Biology 2016

A criptococose é uma doença invasiva capaz de apresentar-se de forma fatal podendo acometer pacientes imunocompetentes e imunocomprometidos. Os agentes etiológicos Cryptococcus neoformans var. grubii e C. neoformans var. neoformans apresentam distribuição cosmopolita, sendo as excretas de pombos o seu principal reservatório. Com o advento de terapias imunossupressoras e a pandemia de HIV, observou-se o aumento significativo de casos de pacientes com criptococose. Atualmente, o diagnóstico é baseado na apresentação clínica, na observação microscópica de líquor corado com tinta da Índia e/ou no isolamento em cultura. Neste trabalho desenvolveu-se um ELISA para detecção de anticorpos contra C. neoformans var. grubii em soro de pacientes utilizando como antígeno um extrato protéico total de uma linhagem clínica isolada de um paciente com criptococose (HC6). Foram testados através de ELISA 40 amostras de soros de pacientes com criptococose, sendo 67,5% positivos e 32,5% falsos negativos. Como controles negativos foram testados 82 amostras de soros de indivíduos hígidos, dos quais 26,82% apresentaram resultados positivos para os testes realizados. Para testar a reatividade cruzada, foram utilizadas 10 amostras de pacientes com histoplasmose (20% de reatividade cruzada), 9 amostras de pacientes com paracoccidioidomicose (66,6% de reatividade cruzada), 9 amostras de pacientes com candidose (13,3% de reatividade cruzada) e 7 amostras de pacientes com aspergilose (14,28% de reatividade cruzada). Visando solucionar o problema da reatividade cruzada, identificamos proteínas antigênicas de C. neoformans var. grubii por eletroforese bidimensional seguida por western blot e espectrometria de massa (MALDI-TOF MS). Das 75 amostras analisadas, quatro foram identificadas: uma proteína hipotética, 2 isoformas de HSPs 70 e uma catalase-2. As proteínas identificadas apresentaram baixa similaridade com ortólogas de outros fungos patogênicos, sendo, dessa forma, possíveis alvos para a padronização do ELISA e diagnóstico da criptococose. / Cryptococosis is an invasive and potentially fatal disease. Cryptococcus neoformans is the etiological agent, which can affect both immunocompromised and immunocompetent individuals. C. neoformans var. grubii and C. neoformans var. neoformans are cosmopolitan and their major natural reservoir is the excrement from pigeons. With the advent of immunosupressor therapies and the pandemic HIV infection, a significant augmentation of cryptococosis cases in humans was observed. Nowadays cryptococcosis diagnosis is based on the clinical presentation, India ink sample preparation methods and/or in vitro culture isolation. In this work we had developed an ELISA to detect antibodies against C. neoformans var. grubii in serum from patients with cryptococcosis using as antigens a whole cell protein extract from a clinical cell line isolate (HC6). Sera from 40 patients with cryptococcosis were tested by ELISA. From these, 67.5% were positives and 32.5% were false-negatives. As a negative control 82 samples from health subjects were also tested, from these 26.82% were positives. To test cross-reactivity, samples from 10 patients with histoplasmosis (20% cross-reactivity), 9 from patients with paracoccidioidomicosis (66.6% cross-reactivity), 9 from patients with candidosis (13.3% cross-reactivity) and 7 from patients with aspergilosis (14.28% cross-reactivity) were tested. To solve the cross-reactivity problem, we searched immunogenic proteins which were specific to C. neoformans var. grubii applying two-dimensional polyacrylamide gel electrophoresis (2DE-PAGE) followed by western blot and mass spectrometry (MALDI-TOF MS). From the 75 sample analyzed, four were identified: one as a hypothetic protein, two HSPs 70 isoforms and the protein catalase 2. These proteins showed low similarity with orthologues from other pathogenic fungi, and are potential targets to further of the standardizing cryptococosis diagnosis by ELISA.