Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-09-21T18:28:49Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) FABIO DE SOUZA BARBOSA.pdf: 1689958 bytes, checksum: 1fe429224460761440aa3dfeb3cbe066 (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-09-21T18:29:20Z (GMT) No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) FABIO DE SOUZA BARBOSA.pdf: 1689958 bytes, checksum: 1fe429224460761440aa3dfeb3cbe066 (MD5) / Made available in DSpace on 2016-09-21T18:29:20Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) FABIO DE SOUZA BARBOSA.pdf: 1689958 bytes, checksum: 1fe429224460761440aa3dfeb3cbe066 (MD5) Previous issue date: 2015-07-17 / O doripenem é um antibiótico β-lactâmico de amplo espectro de ação. Pertencente ao grupo das carbapenemas, caracteriza-se por apresentar elevada potência e atividade frente à cepas Gram-negativas, produtoras de β-lactamases de espectro estendido (ESBL) e β-lactamases ampC. Apesar de sua grande importância clínica, os antibióticos carbapenêmicos não apresentam boa estabilidade quando em solução, o que é demonstrado em diversos trabalhos descritos na literatura científica. Para o doripenem, há vários trabalhos descritos na literatura que ressaltam sua importância clínica e sua atividade antibiótica. Porém, nota-se a escassez de trabalhos que enfoquem sua estabilidade físico-química, seus produtos e suas rotas de decomposição. O presente trabalho tem como objetivo a validação de um método analítico indicativo de estabilidade por ultra fast liquid chromatography (UFLC), e a avaliação da estabilidade do doripenem em solução, quando submetido a estresse térmico, oxidativo, fotólise e hidrólise em meio ácido e meio alcalino, e determinação da cinética química de decompsição. Para proposição da estrutura química dos produtos de degradação, foram realizadas análises por cromatografia líquida com detecção por espectrometria de massas (LC-MS). O método cromatográfico descrito neste trabalho demonstrou-se adequado para determinação do doripenem na forma de pó para solução injetável, possuindo performance indicativa de estabilidade. A faixa linear do método foi de 5,0 a 40,0 μg/mL, sem desvios de linearidade, sendo estatisticamente comprovada por meio de ANOVA. Com o auxilio do desenho experimental de Plackett–Burman, o método demonstrou-se robusto frente a uma série de fatores. O estudo de degradação forçada demostrou a susceptibilidade do doripenem a diversos fatores de degradação, com acentuada instabilidade frente à hidrólise ácida e alcalina, observando-se degradação aproximada de 60% do seu teor em apenas 2 minutos sob condições alcalinas. A decomposição oxidativa do fármaco seguiu uma cinética de segunda ordem, com constante de velocidade de reação de 0,000086 e 0,00010%-1.min-1, quando submetido à degradação em H2O2 a 3 e 10%, respectivamente. A decomposição térmica apresentou uma energia de ativação de aproximadamente 15 Kcal/mol, valor característico de reações de hidrólise. E na análise cromatográfica com detecção por espectrometria de massas, observou-se que os principais produtos de degradação formados sob condições de termólise e oxidação, apresentam massas moleculares semelhantes, sendo possível a proposição da estrutura química dos mesmos. / Doripenem is a β-lactam antibiotic with a broad spectrum of antimicrobial activity, including gram-negative strains, and producers of extended spectrum β-lactamases (ESBL) and ampC β-lactamases ampC. Despite its great clinical importance, carbapenems do not show good stability when incorporated as solution, as reported in several studies. In reference to doripenem, several works have describing its clinical use, efficacy data and cases of resistance. However, few works mention the drug stability, in terms of degradation products and routes of decomposition. The present work aimed to develop and validate a stability-indicating method by ultra-fast liquid chomatograph (UFLC) for doripenem in powder for injection, purposing an evaluation of stability of reconstituted solution using stress conditions of heat, oxidation, acid hydrolysis, alkaline hydrolysis and photolysis. The chemical kinetic of decomposition was also assayed for thermal and oxidative degradation. For identification of degradation products, the degraded samples where submitted to analysis by LC-MS. The chromatographic method described here proved to be stability-indicating and suitable for the determination of doripenem in drug formulation. The method linearity was performed in the range of 5 to 40 g mL-1, whose correlation coefficient (r) was 0.9999. The robustness testing, assayed against a variety of factors, allowed verifying that the method accept small variations in routine analysis. The forced degradation demonstrated the susceptibility of doripenem to several decomposition factors, being intense the instability to acidic and alkaline hydrolysis. In basic media, the drug residual content was approximately 40% in 2 minutes. At oxidative decomposition, the drug follows second-order kinetics, with a rate reaction of 0.000086 and 0.00010 %-1 min-1, respectively for H2O2 at 3.0 and 10.0 %. The thermal decomposition showed an activation energy of 15 kcal mol-1, a characteristic value for hydrolysis reactions. The analysis by LC-MS revealed that the major degradation products formed under oxidizing conditions and thermolysis present molecular weight (411, 427, 437, 634, 650 and 664). The stability of doripenem must be carefully observed, mainly after reconstitution and storage in adverse conditions of temperature.

Dietary intake is one of the major routes of human exposure to perfluoroalkyl and/or polyfluoroalkyl substances (PFAS). The objective of this study was to measure perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA) and perfluorohexane sulfonic acid (PFHxS) in organic and conventional egg from Sweden (n=8, consisted of 4 pooled eggs and 4 individuals) and China (n=9, consisted of 4 pooled eggs and 5 individuals) and compare the concentrations of PFAS between the two categories (organic and conventional). Also, to evaluate if there was any difference in concentrations of PFAS between both countries. In the end, evaluation of tolerable weekly intake of PFOS and PFOA due to consumption of egg recommended by the European Food Safety Authority (EFSA) was conducted if consuming these eggs would cause any human health risk. Liquid chromatography-mass spectrometry (LC-MS/MS) was used to analyze PFOS, PFOA and PFHxS in the egg samples. In egg samples from China, PFOA was the predominant PFAS; in an organic egg sample from Shenzhen with concentration up to 2000 pg/g, making up to 86% of the 3 PFAS. In contrast, PFOS had the greatest concentration of all PFAS in egg samples from Sweden and was detected in organic egg sample with concentration up to 184 pg/g, making up to 78% of the 3 PFAS. PFOA in samples from China was 18 times higher compared to egg samples to Sweden; results showed no significant differences in PFAS concentrations in egg samples between Sweden and China. In samples from China, concentrations of PFAS had total mean of 50 pg/g for PFOS, 373 pg/g for PFOA and 13 pg/g for PFHxS. In Sweden, mean concentrations of PFOS, PFOA, and PFHxS were found to be 5, 2, and 1,5 times (respectively) higher in organic eggs when compared to conventional. However, significant difference was only observed for PFOS in Swedish organic eggs (p<0.05, t-7.96, df=6). The different concentrations of contamination between organic and conventional egg could be due to the fish powder in organic chicken feed and ingestion of soil through pecking. The result suggests that current concentrations of PFOS and PFOA in organic and conventional chicken eggs are unlikely to cause any immediate harm to Swedish populations. For Chinese population since the consumption of egg has a high risk of exceeding the TWI, the current concentration of PFOA in organic chicken eggs may cause harm to the population based on TWIs established by EFSA. Further investigation is needed with more samples to be analyzed to confirm this point.

Opioid use during pregnancy can result in the newborn being physically dependent on the substance, thus experiencing drug withdrawal, termed neonatal abstinence syndrome (NAS). Buprenorphine and methadone are two drugs used to treat opioid withdrawal and are approved for use in pregnancy. Quantification of these compounds in umbilical cord plasma would help assess in utero exposure of neonates in cases of buprenorphine or methadone use during pregnancy. An LC-MS/MS method using solid-phase extraction sample preparation was developed and validated for the simultaneous quantification of methadone, buprenorphine, norbuprenorphine, and glucuronide metabolites in umbilical cord plasma. The average accuracy (percentage error) and precision (relative standard deviation) were

Mit der vorliegenden Studie sollte zu dem wichtigen Forschungsfeld der Pharmakogenetik beigetragen werden, indem zum einen eine einfache und sichere kombinierte Phänotypisierung der drei zuvor erwähnten CYPs (CYP2D6, CYP2C9 und CYP2C19) entwickelt, und zum anderen die Vorhersagekraft des Genotyps für den gemessenen Phänotyp näher untersucht werden sollte. Es ist uns gelungen eine sichere, einfache, schnelle und kombinierte Phänotypisierung der beiden wichtigen Monooxygenasen CYP2D6 und CYP2C9 zu etablieren. Zunächst wurden dazu Wechselwirkungsstudien mit den ausgewählten Testsubstanzen Dextromethorphan (DEX, CYP2D6), Flurbiprofen (FLB, CYP2C9) und Omeprazole (OME, CYP2C19) durchgeführt. Es konnte gezeigt werden, dass DEX und FLB als Kombination verabreicht werden können. Die Gabe von OME gemeinsam mit FLB verändert jedoch das Ergebnis der CYP2C9 Phänotypisierung. Dies ist eine neue Erkenntnis, denn noch 2004 wurde ein Phänotypisierungscocktail veröffentlicht, der die Kombination von FLB und OME enthielt. Bei der genannten Studie wurden jedoch, unseres Wissens nach, keine Wechselwirkungsstudien zu den einzelnen Testsubstanz-Kombinationen durchgeführt. Die von uns entwickelte Phänotypisierungsmethode wurde durch Wechselwirkungsstudien verifiziert. Sie ist jedoch auch in anderen Bereichen den bisher veröffentlichten phänotypisierungscocktails überlegen. Zum einen wurden nur sehr kleine Dosen sicherer Testsubstanzen verwendet. Dies wurde durch Entwicklung neuer, sensitiver LC-MS/MS Methoden ermöglicht. Zum anderen ist diese neue Prozedur schnell und nicht-invasiv durchführbar. Nach Verabreichung der Testsubstanz muss der Urin nur für zwei Stunden gesammelt werden. Zudem weisen unsere Ergebnisse darauf hin, dass die normalerweise durchgeführte, aufwendige Glucuronidspaltung des CYP2D6 abhängigen DEX-Metaboliten, Dextrorphan, vermutlich vernachlässigt werden kann. Die wichtigsten Ergebnisse dieser Studie sind jedoch die Einblicke, die in die Vorhersagekraft der CYP2D6 und CYP2C9 Genotypen für die entsprechenden Phänotypen gewonnen werden konnten. Fast 300 phänotypisierte Kaukasier wurden auch in Hinsicht auf die wichtigsten varianten Allele von CYP2D6, CYP2C9 und CYP2C19 mithilfe bekannter und neu etablierter Methoden genotypisiert. Aufgrund der parallelen Phäno- und Genotypisierung konnten Geno- und Phänotyp direkt korreliert werden. Mit linearen Modellen war es möglich, allen detektierten varianten CYP2D6- und CYP2C9-Allelen Aktivitätskoeffizienten zuzuweisen. Diese können nun verwendet werden, um den Beitrag der einzelnen Allele zur resultierenden Enzymaktivität zu bestimmen, wodurch sich die Vorhersage dieser Aktivität ausgehend vom Genotyp verbessern lassen sollte. Besonders für CYP2D6 ermöglicht das neue Korrelationsmodel präzisere Vorhersagen des Phänotyps als bisher veröffentlichte Modelle. Zusammengefasst leistet diese Studie durch die Entwicklung eines sicheren und einfachen Phänotypisierungsprozesses für CYP2D6 und CYP2C9 und durch die Bestimmung von Aktivitätskoeffizienten für alle einbezogenen CYP2D6 und CYP2C9 Allele und der damit verbundenen präziseren Vorhersage des Phänotyps ausgehend vom Genotyp einen wesentlichen Beitrag zum Forschungsfeld der Pharmakogenetik. / This study should contribute to the important field of pharmacogenetics by: firstly, establishing an easy and safe phenotyping method that combines the activity determination of all three previously mentioned CYPs (CYP2D6, CYP2C9, and CYP2C19) into one phenotyping cocktail and secondly, improving the knowledge about the predictive power of the genotype for the measured phenotype. It was indeed possible to develop a save, easy-to-use, fast and simultaneous phenotyping procedure for the important genetic polymorphic enzymes CYP2D6 and CYP2C9. To accomplish that, interaction studies with the chosen probe drugs dextromethorphan (DEX, CYP2D6), flurbiprofen (FLB, CYP2C9) and omeprazole (OME, CYP2C19) were conducted. It could be proven that DEX and FLB can be administered in combination, whereas OME alters the phenotyping results of CYP2C9. This is a new finding as in 2004 a phenotyping cocktail was published that used FLB and OME in combination. However, to our knowledge, no interaction tests were carried in that study. The new phenotyping procedure is not only verified by prior probe drug interaction studies, it also has other advantages over phenotyping cocktails found in literature. Firstly, save probe drugs are used in very small doses. This is possible due to the new sensitive LC-MS/MS methods that were evaluated. Secondly, the new phenotyping procedure is very fast and on-invasive. Urine has to be collected only for 2 h and the results also suggest that the time consuming glucuronide cleavage of the CYP2D6 dependent metabolite dextrorphan, usually carried out before CYP2D6 phenotyping, may be unnecessary. Most importantly, however, new insights into the phenotype prediction from genotype for CYP2C9 and CYP2D6 could be gained within this study. Nearly 300 phenotyped Caucasian subjects were also genotyped for the most important known variant alleles for CYP2D6, CYP2C9 and CYP2C19 using several established and newly developed genoptyping methods. Therefore, a direct correlation between phenotype and genotype could be conducted for CYP2D6 and CYP2C9. Employing linear modeling, it was possible to assign activity coefficients to each of the detected CYP2D6 and CYP2C9 alleles, thereby estimating their contribution to the resulting enzyme activity. This might facilitate the prediction of the CYP2D6 and CYP2C9 metabolic status of a subject knowing only its respective genotypes. Especially the new CYP2D6 genotype phenotype correlation model might allow for more precise phenotype prediction for the included variant alleles than was possible until now. Taken together, this study substantially contributes to the important research field of pharmacogenetics by (i) developing a save and easy-to-use phenotyping combination for CYP2D6 and CYP2C9, and (ii) by establishing activity coefficients for each of the detected CYP2D6 and CYP2C9 alleles, thereby allowing for a more precise prediction of the phenotype from genotype.

Secreted proteins play important roles in many cellular functions and molecular processes. Because secreted proteins potentially enter the blood stream, they can serve as valuable measures of health and disease useful for disease diagnosis and prognosis, therapeutic target identification, and patient stratification in personalized medicine. Consequently, significant interest exists in secreted protein analysis within complex biospecimens, particularly blood but significant bioanalytical challenges including the wide protein dynamic range >10 orders of magnitude remain. The cellular secretome therefore represents a viable alternative to direct biomarker discovery in biofluids. Finally, cellular systems are amenable to labeling for the production of intact stable isotope labeled (SIL) proteins that can be used as global internal standards for quantitative proteomics. In this dissertation, two secretome-focused studies were undertaken. The first study involving candidate biomarker discovery in radiation-induced autophagy utilized the p53-null and inducible H1299 non-small cell lung cancer (NSCLC) secretome. The study identified 364 secreted proteins that were mainly associated with exosomes (N=224) and chaperone activity (N=21). CHGB and SCG2 were identified as potential population-based biomarkers (for patient stratification) due to their consistent overexpression in p53-null H1299 cell secretomes compared to p53-wt cells before and after radiation. FAM3C, CANX, EIF5A, GPI, and TXNRD1 were identified as candidate biomarkers for patient prognosis following radiotherapy due to their differential expression only in response to radiation treatment. In the second study, a comprehensive glycoproteomics characterization of the SILAC-labeled HepG2 secretome was undertaken. 1635 SIL proteins, 492 of which were major plasma proteins including 192 cancer biomarkers were identified with high sequence coverage spanning six orders of magnitude. EDTA plasma spiked with the SIL secretomes yielded 63 proteins that were quantified with H/L ratios in all samples out of 1405 total proteins identified. Additionally, LC-MS/MS analysis of the Con A and WGA enriched 72h secretome:plasma sample afforded an opportunity to clearly distinguish between glycoproteins in plasma and the HepG2 secretome that share/differ in N-glycan structures. Collectively, the two studies reveal the suitability of the H1299 cancer cell secretome as an experimental model for biomarker studies and support the HepG2 secretome as a viable platform for producing SIL glycoproteins.

In the present thesis the development and validation of bioanalytical LC-MS/MS methods for the quantification of erythromycin A, erythromycin ethylsuccinate, roxithromycin, clarithromycin, 14 hydroxy clarithromycin, flucloxacillin, piperacillin and moxifloxacin in human plasma and human urine (piperacillin) is introduced. All methods were applied to analyze human plasma and urine samples from clinical trials and therefore, have been validated according to international guidelines. The methods were reliable in these studies and fulfilled all regulatory requirements known at the time of the study conduct. Moreover, the validation data of the macrolides were compared on three different mass spectrometers (API III Plus, API 3000™, API 5000™). The new innovations in the ion source (horizontal versus vertical electrospray), the ionpath (skimmer, QJet) and the diameter of the orifice resulted in better sensitivity and a larger linearity range for the majority of the analytes. Sensitivity was improved up to a factor of 12 (for clarithromycin) between API III Plus to API 3000™ and up to a factor of 8 (for erythromycin and roxithromycin) between API 3000™ and API 5000™, keeping the accuracy and precision data at about the same level. The high sensitivity was a benefit for example for the flucloxacillin study, because concentrations from all subject samples were detectable up to approximately eight half-lives, i.e. no concentrations needed to be reported below the quantification limit. Also the linearity range were extended from two orders of magnitude to up to four orders of magnitude, which increases the likelihood to allow to analyze all samples from a pharmacokinetic study in the same run. This is especially useful if a large concentration range needs to be analysed, for example, if the method shall be applied in an ascending dose study. Then, all low concentrations from the beginning of the study can be determined, as well as all high concentrations, without the need to dilute and analyse single samples repeatedly. The pharmacokinetic data were compared to previously reported literature data and correlated graphically with MIC values of popular microorganisms which might be a starting point for further PK/PD investigations. The PK/PD theory is a very helpful tool for prediction of the efficacy of given drugs against certain micro-organisms. Depending on the pharmacodynamic processes, e. g. the mode of action, three classes of drugs have been identified. In the same way this applies to adverse effects, which need to be minimised by reducing plasma concentrations. These coherences are not well-investigated, yet, and are not discussed further in this thesis. Still, a lot of research has to be done in this interdisciplinary field to minimise uncertainty in single values, like an AUC/MIC. These include: Improve accuracy and precision of bioanalytical methods determining total and free concentration data in biological matrices for calculation of AUC and Cmax These parameters are related to the MIC in pharmacodynamic considerations. Since the determination of the MIC often underlies significant variations and also differences between microbiological laboratories, the determination of concentrations of anti-infectives is particular important, being achievable by scientific exact techniques. Finally, from the volume of distribution of antibiotics can be used to derive information about intracellular concentrations and effectivity of antiinfectives. / In der vorliegenden Arbeit wird die Entwicklung und Validierung von bioanalytischen LC-MS/MS-Methoden zur Quantifizierung von Erythromycin A, Erythromycin-ethylsuccinat, Roxithromycin, Clarithromycin, 14 Hydroxy-clarithromycin, Flucloxacillin, Piperacillin und Moxifloxacin in Humanplasma und Humanurin (Piperacillin) vorgestellt. Alle Methoden wurden für die Analyse von Plasma- und Urinproben aus klinischen Studien beim Menschen eingesetzt und deshalb nach international anerkannten Richtlinien validiert. In diesen Studien haben sich die Methoden bewährt und alle Anforderungen der zum Zeitpunkt der Studie bekannten behördlichen Ansprüche erfüllt. Die Validierungsdaten der Makrolid-Methoden konnten zudem an drei Massenspektrometern der selben Baureihe verglichen werden. Dabei zeigte sich, dass die Neuerungen an der Ionenquelle (horizontale versus vertikale ESI), dem Ionenpfad (Skimmer, QJet), sowie das immer größere Orifice in der Baureihe, nicht nur stetig verbesserte Sensitivität ermöglichen, sondern auch immer größere Linearitätsbereiche. So konnte ein Sensitivitätsgewinn bis zu Faktor 12 (Clarithromycin) von API III Plus auf API 3000™ und bis zu Faktor 8 (Erythromycin und Roxithromycin) von API 3000™ auf API 5000™ bei etwa gleichen Werten für die Präzision erreicht werden. Die hohe Sensitivität zeigte sich zum Beispiel bei der Flucloxacillin Studie von Vorteil, da alle Konzentrationen bis circa acht Halbwertszeiten nach Wirkstoffgabe bestimmt werden konnten, ohne dass einzelne Proben als „BLOQ“ (unter dem Quantifizierungslimit) berichtet werden mussten. Während früher noch Linearitätsbereiche um zwei Größenordnungen üblich waren, sind jetzt am API 5000™ Konzentrationsbereiche über bis zu vier Größenordnungen reproduzierbar linear. Das ist insbesondere von Nutzen, wenn bei einer Substanz ein größerer Konzentrationsbereich gemessen werden muss. Das ist beispielsweise dann der Fall, wenn die Methode für eine Dosissteigerungsstudie eingesetzt werden soll. Dann können sowohl alle kleinen Konzentrationen zu Beginn der Studie gemessen werden, als auch alle hohen Konzentrationen zum Ende der Studie, und zwar ohne, dass einzelne Proben gesondert verdünnt und wiederholt gemessen werden müssen. Die pharmakokinetischen Daten der Antibiotika wurden in den Kontext mit Literaturdaten gestellt und graphisch mit MHK-Werten für gängige Mikroorganismen verglichen, was den Ausgangspunkt für spätere PK/PD-Untersuchungen darstellt. Für die Vorhersage der Wirksamkeit einer verabreichten Substanz gegenüber bestimmten Mikroorganismen ist die PK/PD-Korrelation ein gutes Hilfsmittel. Anhand der pharmakodynamischen Parameter, wie der Wirkungsweise, können drei Antibiotikaklassen gebildet werden. In analoger Weise gilt für die Nebenwirkungen, dass die Plasma-Konzentrationen verringert werden müssen. Diese Zusammenhänge sind jedoch weniger gut untersucht und werden in dieser Dissertation nicht näher diskutiert. In diesem interdisziplinären Feld ist immer noch viel Forschung nötig um beispielsweise Unsicherheiten bei einzelnen Werten wie z.B. AUC/MIC zu minimieren. Diese beinhalten: Verbesserung der Genauigkeit und Präzision von bioanalytischen Methoden welche für die Bestimmung der Gesamtkonzentration und der freien Konzentration in biologischen Matrices verwendet werden um AUC und Cmax zu bestimmen. Diese Parameter werden in pharmakodynamischen Überlegungen in Beziehung zur MHK gesetzt. Da die MHK-Bestimmung oft erheblichen Schwankungen und auch Unterschieden zwischen verschiedenen mikrobiologischen Laboratorien unterliegt, kommt der Konzentrationsbestimmung der Antibiotika besondere Bedeutung zu, weil sie mit naturwissenschaftlich exakten Methoden möglich ist. Aus den Verteilungsvolumina von Antibiotika lassen sich schließlich auch noch Informationen über die intrazelluläre Konzentration und Wirksamkeit von Antibiotika ableiten.

The detection of oxidative stress markers has gained increasing importancy in the early investigation of diseases like diabetes, cancer or hypertension. 8 oxo 2' deoxyguanosine (8-oxodG) is the main marker, which is used for the intracellular detection of oxidative stress levels. However, the oxidative stress markers 8 oxoguanine (8-oxoGua), a product of the DNA base excision repair and 8 oxoguanosine (8-oxoGuo), a marker for oxidative damaged RNA have received less attention up to now. The renin-angiotensin-aldosterone system (RAAS) plays an important role in the regulation processes of the blood pressure system. During hypertension angiotensin II (Ang II) and aldosterone (Aldo) are released in high concentrations over a longer period leading to non-physiological effects of the RAAS hormones. Subsequently, an increase of the intracellular oxidative stress level in kidney cells can be measured. The aim of this thesis is the in vitro and in vivo detection of the oxidative damage in DNA and RNA by measuring oxidative stress markers, especially 8-oxodG which is triggered by Ang II and Aldo. In vitro experiments were carried out in LLC-PK1, a cell line originated from porcine kidney cells. It could been shown that Ang II and Aldo led to a dose-dependent increase of DNA damage in the cells. A time-dependent increase was detected for the first 30 minutes of the treatment. For the rest of the experimental set up (4 h) the level of detected DNA damage remained constant. The FPG comet assay and the immunocytochemical staining showed a significant increase of 8-oxodG in the cells, whereas the HPLC-MS/MS measurement only detected a small increase of 8-oxodG in the DNA. The FPG enzyme, which recognises also other oxidized purines besides 8-oxodG, which led to an overestimation of 8-oxodG in the comet assay. Also, the 8 oxodG antibody, which was used in the immunocytochemical analysis, detected higher amounts of 8-oxodG most likely due to its side reactions with other oxidized DNA structures. One of the main advantages of the last mentioned methods is the direct measurement in damaged cells, whereas the HPLC-MS/MS requires an isolation of the DNA. During this isolation process the oxidative stress markers can be oxidized and the detection can become imprecise. The main purpose of the in vivo experiments was the detection of the oxidative stress marker 8-oxoGua, 8-oxodG and 8-oxoGuo in the urine of test animals. The treatment of C57BL/6 mice and Sprague Dawley (SD) rats with the RAAS hormones led to an increase of the blood pressure, higher DNA damage due to oxidative stress as well as an increased excretion rate of oxidative stress markers. The inhibition of the angiotensin II type 1- or mineralocorticoid receptor and a mutation of the AT1a gene could show, that the DNA damage is independent from the hypertension. In addition, it was shown that the NOX4 is not alone responsible for the oxidative stress. Other NADPH oxidases must contribute to the induction of oxidative stress inside the cell. Moreover, the activation of the Nrf2 pathway has an influence on the effect of Aldo in SD rats. The excretion rate of the oxidative stress markers in the 20 h urine of the treated animals showed how the equilibrium between the DNA repair and the oxidative stress level was changing over time. The measurement of 8-oxoGuo became more and more popular, because up to the fact that 80 % of the DNA is translated into RNA. Overall, the detection of 8-oxodG and 8-oxoGuo is feasible for monitoring the disease or the healing process, because the measurement is non-invasive. The detection of 8-oxodG and 8-oxoGuo in nucleic acids is a first step into the field of basic research methods, because it reveals a snapshot of the nucleic acid damage in the cell at a specific time point. Usually, there will be an overestimation of the oxidative stress marker resulting from the analytical method. Although, it is possible to detect an underestimation of oxidative stress markers in tissue samples if not all cell types are damaged equally. Therefore, a primary goal should be the detection of a stable oxidation product of guanine to insure a reliable detection strategy and for a better understanding of the equilibrium of DNA oxidation and repair. / Der Nachweis von oxidativen Stressmarkern hat bei der Untersuchung von Krankheiten wie Diabetes, Krebs und Hypertonie an großer Bedeutung gewonnen. Vor allem 8-Oxo-2’-desoxyguanosin (8-oxodG) wird gezielt mit verschiedenen Methoden gemessen und als Marker für oxidativen Stress herangezogen. Daneben haben 8 Oxoguanin (8-oxoGua), als Produkt aus der Basenexzisionsreparatur der DNA, sowie 8-Oxoguanosin (8-oxoGuo), als Biomarker für oxidativ geschädigte RNA, bisher weniger Aufmerksamkeit bekommen. Das Renin-Angiotensin Aldosteron System (RAAS) spielt eine wichtige Rolle in der Regulierung des Blutdrucks. Im Falle einer Hypertonie werden Angiotensin II (Ang II) und Aldosteron (Aldo) über einen langen Zeitraum in erhöhter Konzentration ausgeschüttet. Dieser Umstand bewirkt eine nicht physiologische Wirkung der Hormone des RAAS, welche zu einer Induktion von oxidativem Stress führt. Die Zielsetzung dieser Arbeit ist es, die oxidative Schädigung, ausgelöst durch Ang II und Aldo, in der DNA und der RNA in vitro und in vivo nachzuweisen und dabei speziell den Biomarker 8-oxodG zu untersuchen. In-vitro-Experimente wurden mit LLC PK1-Zellen, einer Schweinenierenzelllinie, durchgeführt. Ang II und Aldo lösten einen dosisabhängigen Anstieg der DNA Schäden in LLC PK1 Zellen aus. Eine Zeitabhängigkeit wurde für die ersten 30 Minuten gezeigt. Für die restliche Zeit (4 h) blieb der nachgewiesene DNA Schaden konstant. Der FPG Comet-Assay und die immunzytochemische Färbung zeigten jeweils eine signifikante Zunahme von 8-oxodG in LLC-PK1-Zellen an, während die HPLC MS/MS Messung nur geringe Veränderungen nachwies. Das FPG Enzym erkennt neben 8-oxodG auch andere oxidierte Purine und sorgte so für eine Überbestimmung des DNA-Schadens. Bei der immunzytochemischen Färbung entsteht die Überbestimmung durch Kreuzreaktionen des 8 oxodG Antikörpers mit oxidierten Strukturen in der DNA. Der Vorteil beider Analysemethoden ist die direkte Messung von Schädigungen in der Zelle, während die HPLC-MS/MS eine Isolierung der Nukleinsäuren voraussetzt. Bei diesem Schritt kann es zur Oxidation der Marker für oxidativen Stress kommen, welche einen genauen Nachweis erschwert. In vivo-Versuche hatten zum Ziel, die oxidativen Stressmarker 8-oxoGua, 8-oxodG und 8-oxoGuo im Urin nachzuweisen. Die Behandlung der C57BL/6-Mäuse und Sprague Dawley-Ratten (SD-Ratten) mit den Hormonen des RAAS zeigten einen Anstieg des Blutdrucks, erhöhte DNA Schäden durch oxidativen Stress sowie erhöhte Exkretionsraten der oxidativen Stressmarker. Durch eine Inhibierung des Angiotensin II-Typ1- oder Mineralkortikoidrezeptors sowie die Mutation des Gens AT1a konnte gezeigt werden, dass die Schädigungen unabhängig vom Blutdruck sind. Zudem konnte gezeigt werden, dass neben NOX4 auch andere NADPH Oxidasen für den oxidativen Stress verantwortlich sein müssen. Eine Aktivierung des Nrf2 Signalweges in den SD-Ratten hat Einfluss auf die Wirkung von Aldo. Die Exkretionsrate der oxidativen Biomarker im 20-h-Urin der behandelten Tiere zeigen, wie sich das Gleichgewicht zwischen DNA-Reparatur und oxidativem Stress verändert. Da 80 % der DNA in RNA umgeschrieben werden, ist der Nachweis von 8 oxoGuo in den Fokus gerückt. In der praktischen Anwendung kann mit der Messung von 8 oxodG und 8-oxoGuo ein Krankheits- oder Heilungsprozess auf nicht invasive Weise verfolgt werden. Der Nachweis von 8-oxodG und 8-oxoGuo in den Nukleinsäuren stellt einen Einstieg für die Grundlagenforschung dar, da sie nur eine Momentaufnahme der Nukleinsäureschädigung in der Zelle zeigen. Meist findet eine Überbestimmung, ausgelöst durch die Messmethode, statt. In Gewebeproben kann eine Unterbestimmung vorliegen, falls nicht alle Zelltypen vom oxidativen Stress betroffen sind. Daher sollte es ein vorrangiges Ziel sein, ein stabileres Oxidationsprodukt des Guanins nachzuweisen, um das Gleichgewicht der DNA-Oxidation und Reparatur besser zu verstehen.

Resumo: Produtos cárneos que contenham propriedades organolépticas específicas e que atendam às exigências dos consumidores, são cada vez mais valorizados no mercado. Apesar da sua importância, as características de qualidade da carne em geral não são consideradas em programas de melhoramento genético animal, em razão do alto custo e dificuldade de mensuração. Marcadores moleculares identificados em análises genômicas, têm sido estudados em rebanhos Nelore. Trabalhos que visam o entendimento da expressão de genes e proteínas envolvidos na determinação de características relacionadas a gordura intramuscular da carne, são realizados com o intuito de auxiliar no entendimento da expressão dos genes relacionados a possíveis biomarcadores. Assim, inúmeros trabalhos que visam avaliar a expressão diferencial de genes podem ser encontrados na literatura, no entanto, devido a divergências entre o manejo e as raças estudadas, os resultados são discordantes, e a validação dos genes mais comumente encontrados, se faz necessário para que essas informações possam ser úteis em programas de melhoramento genético. Assim, este estudo teve como finalidade buscar na literatura genes diferencialmente expressos associados à característica de deposição de gordura intramuscular, avaliada por meio de duas técnicas, e validá-los em uma população de bovinos Nelore com fenótipo extremo para a característica gordura intramuscular por meio da técnica de PCR quantitativa em tempo real. Além disto, foi verificado se... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Meat products with specific organoleptic properties and that attend the requirements of consumers, are increasingly valued in the market. Despite its importance, meat quality traits have not been widely used in animal breeding programs because of the high cost and difficulty of measurement. Molecular markers identified in genomic analyzes have been studied in Nellore herds. Studies aimed at understanding the expression of genes and proteins involved in the determination of characteristics such as intramuscular fat from meat are performed with the aim of helping to understand the expression of the genes related to possible biomarkers. Several studies that aim the expression of differentially expressed genes can be found in the literature, however, because of differences between management and breeds studied the results are discordant, and the validation of the common genes found is necessary for this information to be useful in breeding programs. Thus, this study aimed to search the literature for differentially expressed genes related to the marbling trait, by means of the two techniques for the evaluation of this trait and to validate them in a commercial herd of Nellore cattle, by quantitative real time PCR techniques. Besides that, this study will verify if the differentially expressed genes are being translated into differentially expressed proteins, using advanced mass spectrometry technique (LC-MS/MS). For this, 24 divergent samples were selected for each intramuscular ... (Complete abstract click electronic access below) / Mestre

In the last few years, the prevalence of legal party pills in New Zealand has risen dramatically. These pills contain new piperazine designer drugs, two of the more common being 1-benzylpiperazine (BZP) and m-trifluoromethylphenylpiperazine (TFMPP). This thesis describes an optimised LC-MS/MS method for the detection of BZP and TFMPP in whole blood, using an automated solid phase extraction (SPE) for sample clean-up. The method was validated on three different days using five replicate samples each day. The standard curve was linear from 7 - 7000 ng/mL for BZP and 10 - 10,000 ng/mL for TFMPP, with coefficients of variation (CV) below 10%, and accuracy greater than 90% for both drugs. The method was used to quantitate samples provided by the Medical Research Institute of New Zealand. Blood levels were used to show concentrations in the blood over time, and relate these to performance of subjects on a driving simulator. The study was stopped after 41% of the participants who received BZP and TFMPP had adverse reactions to the pills, including vomiting and migraines. The LC-MS/MS method was also used to detect and quantitate methamphetamine, amphetamine, methylenedioxymethamphetamine, methylenedioxyamphetamine, morphine, codeine and 6-monoacetylmorphine in hair. The drugs were extracted from 20 mg of hair using hydrochloric acid in a water bath overnight, then purified using SPE. Validation on three days with five replicate samples gave coefficients of variation (CV) below 12% and acceptable accuracy for all drugs. The method was tested on three samples, previously reported by Environmental Science and Research (ESR) using gas chromatography mass spectrometry (GC-MS) giving results in good agreement. This thesis describes a sensitive, accurate, reproducible LC-MS/MS method easily adapted to analyse drugs of abuse in different biological matrices. It demonstrates the versatility of LC-MS/MS and its applications in forensic work.

L'étude de protéines ciblées comme des biomarqueurs dans des matrices biologiques est un réel défi pour le diagnostic médical. De nos jours, la technique de choix est la chromatographie en phase liquide couplée à la spectrométrie de masse. Cependant, l'analyse d'une protéine induit une étape de traitement d'échantillon afin de purifier au mieux la matrice biologique. En vue d'une automatisation totale de l'analyse, une étape d'extraction sélective a été couplée à un réacteur enzymatique suivie de l'analyse par CPL-SM. Tout d'abord, différents réacteurs enzymatiques ont été préparés et évalués. Puis, une étape extraction a été développée via un immunoadsorbant (IS). L'IS a ensuite été couplé à l'analyse pour une application à un échantillon de plasma dopé. Enfin, un des défis analytiques actuels est la miniaturisation du système analytique afin d'augmenter la sensibilité. Ainsi, un réacteur enzymatique a été préparé dans en format capillaire. L'efficacité de digestion a été démontrée sur un substrat de faible poids moléculaire. La synthèse d'un IS miniaturisé sera envisagée dans le but d'un couplage avec le réacteur enzymatique et l'analyse par nano-CPL/SM.