Zugl.: Clausthal, Techn. Univ., Diss., 2007

Mainz, Universiẗat, Diss., 2002.

Frankfurt (Main), Universiẗat, Diss., 2001.

Kiel, Universiẗat, Diss., 2001.

Matrix Assisted Laser Desorption / Ionization (MALDI) Mass Spectrometry (MS) has emerged as an important tool in the field of proteomics mainly because it is simple, quick and efficient. The identification and quantitation of biomarkers, protein targets for drugs, and metabolites are some of the important fields in proteomics research. Although MALDI MS is an important tool in proteomics research there are drawbacks of the technique that need further development in order for the approach to be used in clinical laboratories. One major limitation of MALDI MS is the generally poor reproducibility of ion signal intensities, which negatively impacts the quantitation of peptides and protein by MALDI MS. A considerable amount of research has been performed in an effort to improve the ion signal reproducibility in MALDI MS. However, many of the approaches developed have introduced specific drawbacks with respect to the traditional dried-droplet sample preparation technique, negating many of the advantages of the MALDI MS approach. This project has focused on the development of a novel approach to quantify peptides by MALDI MS while preserving traditional known advantages of the technique. The studies performed show that an approach in which the ion signal base widths are manipulated to match that of a reference ion signal, through adjustments in desorption laser intensity, leads to much higher reproducibility in the integrated ion signal intensities. A standard curve acquired using the constant ion signal base width approach showed lower average RSDs (< 10.00% vs.> 39.00%) and improved R2 values (> 0.9600 vs. < 0.809) as compared to the conventional constant desorption laser intensity approach. Subsequent work also revealed that the peptide hydrophobic / hydrophilic properties influenced the applicability of the quantitation approach to mixtures of peptides. Specifically, the data revealed that peptides with differing hydrophobic / hydrophilic properties appear to co-crystallize with the MALDI matrix differently leading to an inability to use a hydrophobic peptide signal to quantitate a hydrophilic peptide, and vice versa. This latter conclusion was further supported in similar studies performed on the mixture of peptides resulting from tryptic digestion of the protein bovine serum albumin.

No description available.

Urinary tract infection (UTI) is a very common infection in humans and a majority is caused by Escherichia coli. UTI are commonly treated empirically. However, empiric treatment has become more problematic due to increased antibiotic resistance to commonly used antibiotic agents. It is therefore desirable with short turnover times for antimicrobial susceptibility testing and species identification to improve antibiotic treatment at an early stage. Matrix Assisted Laser Desorption/Ionisation Time of Flight (MALDI-TOF) can provide species identification faster than former routine methods. This study compared direct and standard susceptibility testing using disk diffusion on Enterobacteriaceae (EB) from urine samples. The possibility to standardise the inoculum for direct susceptibility testing via a pellet obtained by a series of centrifugations was also evaluated, as well as direct species identification with MALDI-TOF from the pellet. Results from direct susceptibility testing from urine samples with EB, performed either directly from the urine or with a standardised inoculum, correlated well to those obtained with standardised susceptibility testing using EUCAST disk diffusion methodology with few errors, of which most were associated with Proteus mirabilis. The concept of standardising the inoculum for direct susceptibility testing to 0.5 McFarland was labour intensive and did not improve the results further. However, direct species identification from the urine pellet using MALDI-TOF showed good correlation to routine identification. Of 238 samples, an EB was correctly identified in 148 samples using MALDI-TOF.

Die klassische Identifizierung von obligat anaeroben Bakterien ist mit einem hohen Labor- und Zeitaufwand verbunden. Um festzustellen, ob die Identifizierung mittels Matrix-unterstützter Laser-Desorption/Ionisation und Massenspektrometrie mit Flugzeitanalysator (Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MALDI-TOF MS) ein Verfahren ist, um obligate Anaerobier eindeutig zu identifizieren, wurde mit der vorliegenden Arbeit die Identifizierung von unterschiedlichen Spezies der B. fragilis Gruppe mittels MALDI-TOF MS untersucht. Hierfür wurden 105 obligate Anaerobier der B. fragilis Gruppe aus der Stammsammlung des Institutes für Medizinische Mikrobiologie und Infektionsepidemiologie der Universität Leipzig untersucht. Es fanden sich für die untersuchten Erreger Spektren mit sehr guter Auflösung. Eine Identifizierung und Differenzierung war eindeutig möglich. Unter Verwendung dieser Daten wurde eine Referenzdatenbank erstellt. Die erhaltenen Ergebnisse wurden mittels einer verblindeten Studie überprüft, wobei 52 von 53 (98,1%) der untersuchten Stämme eindeutig identifiziert werden konnten. Dies schließt ebenfalls die Identifizierung und Differenzierung von 15 Metronidazol-sensiblen/ Enterotoxin-negativen, 8 Metronidazol-resistenten/ Enterotoxin-negativen und 8 Metronidazol-sensiblen/ Enterotoxin-positiven B. fragilis Stämmen ein. Die Identifizierung mittels MALDI-TOF MS ist somit eine zuverlässige Methode zur Identifizierung von obligaten Anaerobiern der B. fragilis Gruppe. Weiterhin finden sich Hinweise, dass ein Nachweis von Resistenz-, Virulenz- und Pathogenitätsfaktoren mittels MALDI-TOF MS bei diesen Erregern möglich ist.

Die klassische Identifizierung von obligat anaeroben Bakterien ist mit einem hohen Labor- und Zeitaufwand verbunden. Um festzustellen, ob die Identifizierung mittels Matrix-unterstützter Laser-Desorption/Ionisation und Massenspektrometrie mit Flugzeitanalysator (Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MALDI-TOF MS) ein Verfahren ist, um obligate Anaerobier eindeutig zu identifizieren, wurde mit der vorliegenden Arbeit die Identifizierung von unterschiedlichen Spezies der B. fragilis Gruppe mittels MALDI-TOF MS untersucht. Hierfür wurden 105 obligate Anaerobier der B. fragilis Gruppe aus der Stammsammlung des Institutes für Medizinische Mikrobiologie und Infektionsepidemiologie der Universität Leipzig untersucht. Es fanden sich für die untersuchten Erreger Spektren mit sehr guter Auflösung. Eine Identifizierung und Differenzierung war eindeutig möglich. Unter Verwendung dieser Daten wurde eine Referenzdatenbank erstellt. Die erhaltenen Ergebnisse wurden mittels einer verblindeten Studie überprüft, wobei 52 von 53 (98,1%) der untersuchten Stämme eindeutig identifiziert werden konnten. Dies schließt ebenfalls die Identifizierung und Differenzierung von 15 Metronidazol-sensiblen/ Enterotoxin-negativen, 8 Metronidazol-resistenten/ Enterotoxin-negativen und 8 Metronidazol-sensiblen/ Enterotoxin-positiven B. fragilis Stämmen ein. Die Identifizierung mittels MALDI-TOF MS ist somit eine zuverlässige Methode zur Identifizierung von obligaten Anaerobiern der B. fragilis Gruppe. Weiterhin finden sich Hinweise, dass ein Nachweis von Resistenz-, Virulenz- und Pathogenitätsfaktoren mittels MALDI-TOF MS bei diesen Erregern möglich ist.

A química de produtos naturais da família Asteraceae tem sido foco de estudo do Núcleo de Pesquisa em Produtos Naturais e Sintéticos (NPPNS) da FCFRP-USP que relatou diversas novas moléculas, com destaque para a classe das lactonas sesquiterpênicas. Para essas substâncias já foram atribuídas diversas atividades farmacológicas tais como antioxidante, anti-inflamatória, antimicrobiana, analgésicas e tripanossomicida. A atividade antitumoral da lactona sesquiterpênica goyazensolido (GOYA) foi avaliada no NPPNS e esse estudo revelou uma atividade farmacológica interessante paras as linhagens de tumor cutâneo e cerebral. Com isso, viu-se nesta classe de substâncias uma oportunidade para explorar o potencial antitumoral assim como sua distribuição e metabolismo. Para compreender melhor a distribuição dessa substância na pele, o modelo in vitro de penetração utilizando células de Franz e pele de orelha de porco como membrana foi aplicado. Para esse estudo um método de geração de imagem MALDI-MSI para avaliação da distribuição do GOYA na pele, assim como, um método por UPLC-MS/MS foram desenvolvidos. Para o desenvolvimento e validação do método de MALDI-MS as substâncias doxorrubicina e minoxidil, com estudos de penetração já estabelecidos, foram utilizadas. Para avaliação da distribuição de GOYA em sistema nervoso central (SNC) um modelo em insetos, utilizando gafanhotos foi aplicado. Nesse experimento os gafanhotos receberam a dose de 500 ?M de GOYA, e foram coletadas amostras de hemolinfa, fezes e cérebro nos tempos de 15 e 30 minutos, assim como amostras de gafanhoto total. Para determinação de GOYA nas amostras de gafanhoto um método quantitativo por UPLC-MS/MS e um método de geração de imagem por DESI-MSI foram desenvolvidos. Com os estudos de penetração cutânea pudemos concluir que MALDI-MSI foi capaz de confirmar a distribuição de minoxidil nas amostras de pele, no entanto, não se mostrou uma técnica eficaz para determinação de doxorrubicina. A técnica de MALDI-MSI, em adição ao método de UPLC-MS/MS foi capaz de revelar que o GOYA não penetrou na pele estando acumulado na sua camada superior, provavelmente no estrato córneo. Nos ensaios de distribuição em SNC foi possível observar através do método de UPLC-MS/MS que o GOYA está presente no cérebro, hemolinfa e fezes do gafanhoto. Com isso podemos concluir que o modelo utilizado é um bom modelo de predição de permeação a barreira-hematoencefálica, bem como para estudos de metabolismo. Conclui-se também que o método desenvolvido para essa finalidade foi adequado. A técnica de DESI-MS apesar de não gerar resultados positivos para permeação cerebral revelou a presença de GOYA no intestino do animal no tempo de 30 minutos, o que caracteriza uma rápida eliminação de GOYA do organismo / The Natural Products\' Chemistry has been the focus of the \"Núcleo de Pesquisa em Produtos Naturais e Sintéticos\"(NPPNS) at School of Pharmaceuthical Science of Ribeirão Preto, University of São Paulo (FCFRP- USP). NPPNS reported a variety of unknown molecules from Asteraceae family, highlighting the sesquiterpene lactones (STL). STL showed important pharmacological activities, such as antiinflammatory, antimicrobial, analgesic and trypanossomicide. The STL goyazensolide (GOYA) exhibit antitumor activity for skin and central nervous system (CNS) cancer cell lines . With that the research group saw in this class of compounds a chance to explore the antitumor potential as well as unveil its distribution and metabolism. For a better understanding of the distribution of this compound in the skin, the Franz cell in vitro model using ear pig skin was applied. For that, a MALDI-MSI method was developed to assess the distribution of GOYA in the skin, along with a UPLC-MS/MS method, to confirm the results. In order to develop and validate a MALDI-MSI method, doxorubicin and minoxidil, known substances in the cutaneous penetration studies, were used. An insect model with locust was applied for the investigation of GOYA distribution in CNS. The locust received a 500 ?M GOYA dose and hemolymph, brain and feces samples were collected in 15 and 30 minutes, as well as the entire locust. In order to assess GOYA in the locust samples, an UPLC-MS/MS method was developed, and for distribution in the entire locust a DESI-MSI was developed. The MALDI-MSI method developed for cutaneous penetration study was able to confirm the results for minoxidil experiments and allowed us to see the distribution of this compound in the skin. Unfortunately for doxorubicin MALDI-MSI by the is source analythe dissociation. The MALDI-MSI and the UPLC-MS/MS was able to show that GOYA does not permeate the skin, but is in the skin, probably interacting with the stratum corneum barrier. In the CNS studies we could see through the UPLC-MS/MS method that GOYA is present in the brain, hemolymph and feces in the in vivo model. With that we can conclude that the in vivo insect model is a good alternative for the metabolism and blood-brain-barrier studies. Also we can conclude that, although the DESI-MSI technique was not suitable for CNS permeation studies, it can be applied for metabolism studies, as it revealed the presence of GOYA in the intestine with a 30 minutes experiment, what characterizes a fast distribution and elimination of GOYA in the living organism