A otimização e padronização de métodos moleculares são fundamentais para evitar distorções nos resultados causadas por processamento de ácidos nucleicos da abundância relativa de micro-organismos de uma amostra. Nos estudos sobre identificação microbiana, a etapa de validação é, na maioria dos casos, negligenciada. As principais etapas em que podem ocorrer vieses capazes de alterar a informação sobre a composição da comunidade microbiana são a extração e a RT-PCR. O rRNA é a molécula ideal para identificar os micro-organismos metabolicamente ativos por estar em maior quantidade dentro da célula. A inexistência de um protocolo de extração de RNA gold standard e de kits comerciais específicos para cada tipo de amostra ambiental pode comprometer as etapas posteriores à extração. O protocolo de extração de RNA adaptado neste trabalho mostrou alta eficácia na lise celular e na remoção de contaminantes co-extraidos, garantindo a integridade e a qualidade do rRNA extraído de amostras contendo altas concentrações de contaminantes. Devido as diferentes características químicas das amostras ambientais, o uso deste protocolo adaptado pode preservar a informação sobre os indivíduos metabolicamente ativos de uma comunidade microbiana. Foram utilizados consórcios artificiais na validação da etapa de PCR. O efeito sinérgico do uso de aditivos, da Taq polimerase de alto rendimento, do programa de sub-ciclagem e da limitação do programa de amplificação em 10 ciclos, mantiveram a proporção dos moldes dos consórcios analisados próximo ao esperado. Após a padronização da técnica de ARDRA, foi definido que há necessidade de entre 500 e 550 UFC para uma cobertura completa da diversidade de lodo ativado. O uso de enzimas combinadas MspI/HaeIII e HhaI/RsaI em uma mesma reação double digest proporcionou a separação dos clones de lodo ativado utilizando menos etapas de ARDRA. E o critério de corte em 3% do total agrupamentos, possibilitou selecionar os perfis mais representativos sem a perda de grupos importantes para a análise das bibliotecas. Foi concluído que, o uso de um protocolo inadequado de extração de RNA de amostras complexas pode gerar extratos contendo contaminantes co-extraidos que interferem na atividade da Taq polimerase e nas enzimas de restrição. Após o sequenciamento de nova geração, foi observado que o uso de diferentes iniciadores de PCR e do pré-processamento manual para os dados obtidos, foram essenciais para ampliar a cobertura observada para a amostra de lodo e melhorar a resolução dos resultados e a profundidade de identificação taxonômica, respectivamente. / The optimization and standardization of molecular methods are fundamental to avoid distortions in the results caused by nucleic acid processing of the relative abundance of microorganisms in a sample. In the studies on microbial identification, the validation step is, in most cases, neglected. The main steps in which biases capable of altering the composition of the microbial community can occur are extraction and RT-PCR. The rRNA is the ideal molecule to identify metabolically active microorganisms because they are in the greatest amount within the cell. The lack of a gold standard RNA extraction protocol and specific commercial kits for each type of environmental sample may compromise the post-extraction steps. The RNA extraction protocol adapted in this work showed high efficacy in cell lysis and in the removal of co-extracted contaminants, guaranteeing the integrity and quality of the rRNA extracted from samples containing high concentrations of contaminants. Due to the different chemical characteristics of the environmental samples, the use of this adapted protocol can preserve the information about the metabolically active individuals of a microbial community. Artificial consortia were used to validate the PCR step. The synergistic effect of the use of additives, the high yield Taq polymerase, the sub-cycling program and the limitation of the amplification program in 10 cycles, kept the proportion of the molds of the consortia analyzed close to the expected. After standardization of the ARDRA technique, it was defined that there is a need for between 500 and 550 CFU for a complete coverage of the diversity of activated sludge. The use of MspI / HaeIII and HhaI / RsaI combined enzymes in the same double digest reaction provided the separation of activated sludge clones using fewer ARDRA steps. And the criterion of cut in 3% of the total groupings, allowed to select the most representative profiles without the loss of important groups for the analysis of the libraries. It was concluded that the use of an inadequate RNA extraction protocol from complex samples can generate extracts containing co-extracted contaminants that interfere with Taq polymerase activity and restriction enzymes. After the sequencing of the new generation, it was observed that the use of different PCR primers and manual preprocessing for the obtained data were essential to increase the coverage observed for the sludge sample and to improve the resolution of the results and the depth of taxonomic identification, respectively.

The Cretaceous Mowry Shale is an organic-rich, siliceous marine shale, and as such is a known source rock in the Western United States. Studies have documented that total organic carbon (TOC) in the Wind River Basin, Wyoming increases to the southeast. These studies cover large areas with limited sample sets. In this study, over 250 samples were collected near Lander, Wyoming to address spatial heterogeneity of TOC within the Mowry Shale at a much finer scale than previously examined. Samples were collected along five vertical sections at three localities, and following correlation of the vertical sections, which was strongly aided by the presence of regional bentonite horizons, samples were collected laterally from the same unit at regular 25-foot intervals. These samples were analyzed using pyrolysis and x-ray diffraction techniques. Average TOC values are fairly consistent within the study area (1.65%, with a range of 2.10% to 1.15%). Average Tmax values for vertical and lateral samples is 433 °C with a standard deviation of 7.25 °C suggesting immature to very early oil window thermal maturity. Kerogen types are determined to be dominantly type III, suggesting a dominance of terrestrial input, becoming slightly more mixed type II/III to the southeast. Redox-sensitive trace metals such as uranium, thorium, vanadium, chromium, cobalt, and molybdenum values all suggest a slightly oxygenated sediment water interface during time of deposition. These pyrolysis and trace metal data suggest that the study area was in a prograding proximal marine/prodeltaic depositional environment during Upper Mowry time with influences from higher energy bottom flows. Lateral homogeneity of strata and the low variability in geochemical character across the study area suggest that the local basin in the study area was not segmented by structural or oceanographic conditions. While efforts were made to collect unaltered outcrop samples (digging back into what appeared to be unfractured, unaltered rock), alteration or weathering of organic material is a concern for source rock evaluation of near-surface outcrops. In order to address this concern, a 5-foot-deep trench was dug back into the outcrop at the target horizon in one locality. Samples were taken at regular three-inch intervals from this trench as it was excavated to determine the effect of weathering on TOC in the study area. Based on pyrolysis results, TOC was affected by weathering only along fracture sets (several samples intersected fractures in the shallow subsurface) and did not appreciably increase from the surface to a depth of five feet. Due to the impermeable nature of shale rock, decreases of TOC due to weathering appear to be limited to the immediate surface of samples and along fracture sets.

Sample preparation is an essential component of analytical methods in chemistry. It is not only necessary but also presents an opportunity to increase the effectiveness of the method significantly. There are various commercially available technologies for sample preparation, including numerous variations of LLE, SPE, and SPME. However, these technologies all present significant deficiencies, including the inability to extract directly from complex samples such as whole milk. Instrumental analysis has been improved greatly in the last two decades but still is not applicable to complex samples without sample preparation. This work presents the theory of FPSE, including the synthesis of sol-gel sorbents, coating of FPSE cellulose substrates and the mechanism of retention. Original research data presented herein introduce a comprehensive view on possible applications of FPSE in forensic chemistry and otherwise. Five distinct FPSE-based methods were rigorously developed for analysis of targeted pollutant residues. These methods were validated and compare to leading methods published in peer-reviewed literature quite favorably. Four of the methods were coupled to HPLC-UV and designed for trace or ultra-trace analysis of PAHs, BTEX, substituted phenols and nitroaromatic explosives, respectively. An additional FPSE-based method was developed and validated for direct analysis of BPA and five estrogenic EDCs in commercially purchased whole milk. This latter was coupled to both HPLC-UV and HPLC(QQQ)-MS/MS. The applicability of FPSE(PTHF) media was also tested for screening of aqueous samples and subsequent storage of analytes on the sorbent. My study included simultaneous extraction of a mixture of eight forensically significant compounds with various physicochemical properties and effective storage of each compound in frozen and ambient conditions for 32 weeks. These findings suggest that the storage ability of FPSE media can be extended as long as necessary, which is very significant in forensic laboratories where evidence often needs to be stored in a costly manner that may not be as effective in maintaining the chemical composition of the sample.

PCR und Fluoreszenz - DNA - Fragment - Analyse zum Nachweis einer monoklonalen B-Zell-Population zur Diagnostik der kutanen B-Zell Lymphome (CBCL) Der Nachweis einer monoklonalen B-Zell Population mittels PCR hat sich seit ca. zehn Jahren ergänzend zu Klinik und Histopathologie in der Diagnostik der kutanen B-Zell Lymphome etabliert. Zu diesem Zweck wurden Primer für die IgH Framework-Regionen (FR1, 2, 3), die Leader-Sequenz und für die JH-Region sequenziert. Alle Primervarianten führen zur Amplifikation der hochvariablen CDR-3 Region, welche für jede B-Zelle spezifisch ist. Die Kapillarelektrophorese mit fluoreszenzmarkierten PCR-Produkten an automatischen Sequenziergeräten (z. B. Genescan ABI Prism 310) ermöglicht eine exakte Größenbestimmung des jeweiligen Fragmentes und ist daher in diesem Zusammenhang die geeignetste Methode. Zunächst wurden alle relevanten Primer mit der Simulationssoftware Oligo hinsichtlich ihrer Bindungseigenschaften geprüft. Danach wurden ausgewählte Primer-Sets an 58 in Paraffin eingebetteten und an 5 Kryoproben von sicher diagnostizierten CBCL-Patienten getestet. Die fluoreszenzmarkierten Produkte wurden mit dem Sequenziergerät ABI Prism 310 analysiert. Die ungeschachtelte FR3/JH-PCR konnte nur in 30% und zusammen mit der halbgeschachtelten FR3/JH-PCR nur in 37% der Fälle klonale B-Zellen nachweisen. Die Detektionsrate erhöhte sich auf 54% unter Einbeziehung der geschachtelten FR1/JH-PCR und schließlich auf 60% mit einer zusätzlichen geschachtelten FR2/JH-PCR. Das Auftreten von Pseudoklonen (variierende Größe des klonalen Peaks bei Wiederholung der PCR) bei den geschachtelten PCR machte eine Prüfung auf Reproduzierbarkeit der Ergebnisse unbedingt erforderlich. Die höchste Rate an Pseudoklonen zeigte die FR2/JH-PCR. Aufgrund der schlechten Qualität der IgH Leader-PCR konnten mit in Paraffin-eingebetteten Proben keine Amplifikate erzeugt werden. Zusammenfassend ist zu sagen, daß gemeinsame Verwendung der in dieser Arbeit entwickelten PCR eine Sensitivitätssteigerung von 30% auf 60% ermöglichen. / Clonality detection in cutaneous B-cell lymphomas (CBCL) using immunoglobulin heavy chain gene PCR assays and fluorescence PCR-fragment analysis on automated DNA sequencer Detection of clonally expanded immunoglobulin heavy chain (Igh) gene rearrangements by PCR and subsequent electrophoresis is increasingly used in the diagnosis of cutaneous B-cell lymphomas (CBCL). To this end, primers for the three Igh framework regions (FR1,2, 3), the leader sequence and the Jh region are applied, all amplifying the highly variable IgH third complementary region (CDR3) Recently, fluorescence PCR-fragment analysis on automated DNA sequencers (GeneScan analysis, GSA), providing an exact sizing has been applied as appropriate seperation technique in this context. We have evaluated all Igh primers hitherto known by a PC primer analysis program. Then, fluorescently labeled products generated from DNAs of 58 paraffin embedded and 5 frozen lesional skin biopsies of confirmed CBCL cases using the primer sets selected, were analysed by GSA on the ABI 310 Prism instrument. Single round or seminested FR3/JH-PCR showed clonal B-cells only in 30 or 37% of cases, respectively. This fraction was increased to 54% including a nested FR1/JH-PCR, and, to 60% applying a supplementary nested FR2/JH-PCR. However false clonal results, indicated by peaks of varying sizes from repeated PCR, have been received by nested PCR, mostly by FR2/JH. Obviously due to their poor quality, the IgH leader-PCR has not yielded amplification products from paraffin-derived DNAs. Our data show that the FR3/JH-PCR only is not sufficient for detecting B-cell clonality in CBCL, but following inclusion of additional IgH-PCR, an increase of detection rate up to 60% is possible. A substantial number of cases still fail to show clonality.

<p>This thesis deals with the development of analytical methods for the determination of new antimalarial drugs in biological fluids. The goal was to develop methods that facilitate clinical studies performed in the field, such as capillary blood sampling onto sampling paper.</p><p>Methods for the determination of atovaquone (ATQ) in plasma, whole blood and capillary blood applied onto sampling paper were developed and validated. </p><p>Automated solid-phase extraction (SPE) and liquid chromatography (LC) with UV absorbance detection was used to quantify ATQ. Venous blood contained higher levels of ATQ than capillary blood after a single dose of Malarone (ATQ + proguanil).</p><p>Ion-pairing LC was used to separate amodiaquine (AQ), chloroquine (CQ) and their metabolites on a CN-column. A method for quantification of AQ, CQ and their metabolites in capillary blood applied onto sampling paper was developed and validated. Perchloric acid and acetonitrile were used to facilitate the extraction of the analytes from the sampling paper. The liquid extract was further cleaned by SPE.</p><p>Methods for the determination of piperaquine (PQ) in plasma and whole blood using SPE and LC were developed and validated. Addition of trichloroacetic acid (TCA) to the samples prior to injection into the LC-system significantly enhanced the efficiency for the PQ peak. Serum and whole blood contained higher levels (about 300 nM) of PQ than plasma (about 200 nM) after a single oral dose of 340 mg PQ. This indicates that PQ may be taken up in the leucocytes and thrombocytes.</p>

<p>Liquid chromatography – mass spectrometry (LC-MS) methods were developed for the analysis of positron emission tomography (PET) radiotracers in biological matrices. Additionally, radiotracer chemistry was explored as an analytical tool for supporting LC-MS method development and imaging molecular interactions in miniaturised chemical analysis systems.</p><p>Conventional radiodetection methods can offer high sensitivity in the analysis of radiotracers in biological matrices, although with the short half-life of PET tracers, this mass sensitivity decreases rapidly with time. This limits the time frame for analysis, and may compromise the precision and accuracy of the later measurements. Performing LC-MS analysis of the dominant stable isotope form of the tracer removes such time restrictions.</p><p>An LC-MS/MS method was developed for determination of the tracer flumazenil in human plasma, with high inter-assay precision (RSD < 7%) and accuracy (95 – 104%). The method was applied in a multiple scan PET study where the plasma concentration spanned from 0.07 to 0.21 nM. The method removed the time restrictions associated with radiodetection methods and thus provided the opportunity of analysing a greater number of samples than would have been possible with radioanalysis.</p><p>Furthermore, an LC-MS/MS method was developed that provided an efficient metabolic screening tool of potential PET tracers, whereby the substrates could be collected directly from 11C-labelling batches. This permitted repeated incubation experiments without the need of repeated labelling syntheses. A para-methoxy-benzamide analogue of the radiotracer WAY-100635 was thus identified as a potential tracer with improved metabolic stability. Additionally, a capillary LC-MS method was developed with rapid (0.75 min) and efficient (> 99%) on-line high flow-rate extraction for determination of metabolic stability of PET radiotracers.</p><p>Finally, the concept of radionuclide imaging of miniaturised chemical analysis systems was demonstrated with the direct study of interactions within capillary extraction columns and microchannels moulded in a plastic CD and poly(dimethylsiloxane).</p>

This thesis deals with the development of analytical methods for the determination of new antimalarial drugs in biological fluids. The goal was to develop methods that facilitate clinical studies performed in the field, such as capillary blood sampling onto sampling paper. Methods for the determination of atovaquone (ATQ) in plasma, whole blood and capillary blood applied onto sampling paper were developed and validated. Automated solid-phase extraction (SPE) and liquid chromatography (LC) with UV absorbance detection was used to quantify ATQ. Venous blood contained higher levels of ATQ than capillary blood after a single dose of Malarone (ATQ + proguanil). Ion-pairing LC was used to separate amodiaquine (AQ), chloroquine (CQ) and their metabolites on a CN-column. A method for quantification of AQ, CQ and their metabolites in capillary blood applied onto sampling paper was developed and validated. Perchloric acid and acetonitrile were used to facilitate the extraction of the analytes from the sampling paper. The liquid extract was further cleaned by SPE. Methods for the determination of piperaquine (PQ) in plasma and whole blood using SPE and LC were developed and validated. Addition of trichloroacetic acid (TCA) to the samples prior to injection into the LC-system significantly enhanced the efficiency for the PQ peak. Serum and whole blood contained higher levels (about 300 nM) of PQ than plasma (about 200 nM) after a single oral dose of 340 mg PQ. This indicates that PQ may be taken up in the leucocytes and thrombocytes.

Liquid chromatography – mass spectrometry (LC-MS) methods were developed for the analysis of positron emission tomography (PET) radiotracers in biological matrices. Additionally, radiotracer chemistry was explored as an analytical tool for supporting LC-MS method development and imaging molecular interactions in miniaturised chemical analysis systems. Conventional radiodetection methods can offer high sensitivity in the analysis of radiotracers in biological matrices, although with the short half-life of PET tracers, this mass sensitivity decreases rapidly with time. This limits the time frame for analysis, and may compromise the precision and accuracy of the later measurements. Performing LC-MS analysis of the dominant stable isotope form of the tracer removes such time restrictions. An LC-MS/MS method was developed for determination of the tracer flumazenil in human plasma, with high inter-assay precision (RSD &lt; 7%) and accuracy (95 – 104%). The method was applied in a multiple scan PET study where the plasma concentration spanned from 0.07 to 0.21 nM. The method removed the time restrictions associated with radiodetection methods and thus provided the opportunity of analysing a greater number of samples than would have been possible with radioanalysis. Furthermore, an LC-MS/MS method was developed that provided an efficient metabolic screening tool of potential PET tracers, whereby the substrates could be collected directly from 11C-labelling batches. This permitted repeated incubation experiments without the need of repeated labelling syntheses. A para-methoxy-benzamide analogue of the radiotracer WAY-100635 was thus identified as a potential tracer with improved metabolic stability. Additionally, a capillary LC-MS method was developed with rapid (0.75 min) and efficient (&gt; 99%) on-line high flow-rate extraction for determination of metabolic stability of PET radiotracers. Finally, the concept of radionuclide imaging of miniaturised chemical analysis systems was demonstrated with the direct study of interactions within capillary extraction columns and microchannels moulded in a plastic CD and poly(dimethylsiloxane).

Deep subsurface rock samples from Upper Ordovician strata in the Michigan Basin were analyzed for the presence of microbial communities. High concentrations of biogenic methane were observed in the Upper and Middle Ordovician formations. Total porosity values for the shale, shale hard bed and limestone samples were 7.4%, 2.5% and 1.9%, respectively. Hydrocarbon presence ranged from petroliferous shale, to bituminous layering in shale hard beds, to hydrocarbon odour in limestone. Organic carbon content ranged from 0.5 to 2.5%, highest amount being present in the shale. Environmental DNA was extracted from core samples and PCR amplified using 16S rDNA bacterial primers. PCR performed with archaeal 16S rDNA and methanogen-specific (mcrA) primers did not yield DNA amplification. Gene analysis indicated that bacterial sequences similar to Proteobacteria, Cyanobacteria, Firmicutes, and Actinobacteria were present. Most sequences were not related to known cultivated species. Proteobacteria was the most dominant phyla at all depths and included heterotrophic, lithotrophic, acidophilic, radiotolerant, and sulphate-reducing species of bacteria. This study concludes that the observed biogenic methane is a product of ancient methanogenesis.

Deep subsurface rock samples from Upper Ordovician strata in the Michigan Basin were analyzed for the presence of microbial communities. High concentrations of biogenic methane were observed in the Upper and Middle Ordovician formations. Total porosity values for the shale, shale hard bed and limestone samples were 7.4%, 2.5% and 1.9%, respectively. Hydrocarbon presence ranged from petroliferous shale, to bituminous layering in shale hard beds, to hydrocarbon odour in limestone. Organic carbon content ranged from 0.5 to 2.5%, highest amount being present in the shale. Environmental DNA was extracted from core samples and PCR amplified using 16S rDNA bacterial primers. PCR performed with archaeal 16S rDNA and methanogen-specific (mcrA) primers did not yield DNA amplification. Gene analysis indicated that bacterial sequences similar to Proteobacteria, Cyanobacteria, Firmicutes, and Actinobacteria were present. Most sequences were not related to known cultivated species. Proteobacteria was the most dominant phyla at all depths and included heterotrophic, lithotrophic, acidophilic, radiotolerant, and sulphate-reducing species of bacteria. This study concludes that the observed biogenic methane is a product of ancient methanogenesis.