Cytokinins are N6 substituted adenine derivatives that affect many aspects of plant growth and development. A multistep phosphorelay systém, including hybrid sensor kinases, histidinecontaining phosphotransfer proteins and two sets of response regulators, is the key part of cytokinin signaling. However, a recent evidence indicates a crucial role for the proteasomeubiquitin systém (UPS) in the cytokinin response. Here, in this thesis entitled 'Protein metabolism disruption and its effect on cytokinin signaling' the major protein degradation mechanisms are outlined and the present-day model of cytokinin metabolism and signaling is discussed. In the experimental part, the UPS-cytokinin interaction is probed in a growth response experiment, an LC-MS proteome analysis and by the datamining of previously published proteomics data. The results indicate an interesting dosage-dependent balance between cytokinin- and proteasome-mediated signaling, and a huge impact of proteasome inhibition on cytokinin response proteins. Key words: proteasome, ubiquitin, growth response, protein degradation, LC-MS, proteome

The HPLC and LC/MS analyses of the CNS from isopod crustacean the woodlouse, Porcellio scaber revealed a presence of the red pigment-concentrating hormone (Panbo-RPCH) in this species. It has been shown that this neuropeptide plays a role in mobilization of energy stores: topical treatments of P. scaber individuals by Panbo-RPCH in a concentration 20 pmol/{$\mu$}l increased the level of glucose in haemolymph about 4 times. Glucose was the main carbohydrate mobilized by the Panbo-RPCH treatment. Despite the demonstration of hyperglycaemic activity of Panbo-RPCH, no stimulatory effect of this hormone on the locomotory activity of P. scaber was observed. The present study is the first discovery of an occurrence of Panbo-RPCH and its hyperglycaemic activity in the representative of the isopod crustacean.

Orientador: Susanne Rath / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-10T13:13:12Z (GMT). No. of bitstreams: 1 Paschoal_JonasAugustoRizzato_D.pdf: 2545620 bytes, checksum: 047e1d72fd2de11da7411d954b23d23f (MD5) Previous issue date: 2007 / Resumo: Os antimicrobianos são largamente empregados na medicina veterinária, e resíduos destes podem permanecer nos alimentos de origem animal, acima de valores considerados seguros, quando não são respeitadas as boas práticas veterinárias. Este trabalho teve por objetivos: (i) desenvolver e validar métodos analíticos para a determinação de multi-resíduos de quinolonas (enrofloxacina, ciprofloxacina, danofloxacina, sarafloxacina, ácido oxolínico e flumequina) em carne de peixe, usando a cromatografia líquida de alta eficiência associada a detecção por fluorescência (HPLC-FL) e cromatografia líquida associada a espectrometria de massas em tandem por interface de ionização por electrospray e (LC-ESI-MS/MS Q-ToF); (ii) desenvolver e validar métodos analíticos para a determinação de oxitetraciclina (OTC) em ração e carne de tilápias por HPLC-DAD e HPLC-FL, respectivamente e (iii) realizar um ensaio com tilápias (Oreochromis niloticus) para avaliar a depleção da OTC na carne desses peixes. De modo geral, a extração das quinolonas e da OTC da carne de peixes foi conduzida por extração sólido líquido seguida da limpeza do extrato em cartuchos de extração em fase sólida. A separação cromatográfica dos antimicrobianos foi realizada em coluna de fase reversa octadecil híbrida. Os métodos foram validados mediante avaliação dos seguintes parâmetros: faixa linear, linearidade, sensibilidade, seletividade, limites de detecção e quantificação, precisão intra e inter-ensaios e exatidão. Para o método LC-MS/MS foi também avaliado o efeito matriz. Todos os métodos foram considerados adequados aos objetivos propostos neste trabalho. Para avaliar a depleção de OTC na carne de tilápias, os peixes (peso médio de 93 a 115 g) receberam o fármaco via ração na dose de 80 mg OTC/kg peso vivo/dia, por cinco dias consecutivos. A temperatura da água durante o ensaio variou de 16,5 a 24,5 °C. A curva de depleção se ajustou a um modelo exponencial de primeira ordem. O tempo de meia vida de eliminação foi de 2,5 dias e o período de carência estimado foi de 5 dias / Abstract: Antimicrobials are widely employed in veterinary medicine, and their residues could remain in food of animal origin above values considered safe if good veterinary practices are not followed. The aim of this work is to address (i) the development and validation of analytical methods using high performance liquid chromatography with fluorescence detection (HPLC-FL) and liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS Q-Tof) for the determination of multi residues of quinolones (enrofloxacin, ciprofloxacin, danofloxacin, sarafloxacin, oxolinic acid and flumequine) in fish; (ii) the development and validation of analytical methods for the determination of oxytetracycline (OTC) in fish feed and tilapia fish fillets using HPLC-DAD and HPLC-FL, respectively, and (iii) the study of tilapias (Oreochromis niloticus) in order to evaluate the depletion of OTC from the fish fillet. In general, the extraction of the quinolones and OTC from the fish matrix was conducted by solid-liquid extraction followed by clean-up on solid phase extraction cartridges. The antimicrobials chromatographic separation was performed on a reverse phase octadecyl hybrid column. The methods were validated through the following parameters: linear range, linearity, sensitivity, selectivity, detection limit, quantitation limit, intra- and inter-assay precision and accuracy. For the LC-MS/MS method, the matrix effect was also evaluated. All methods were adequate to the proposed objectives. In order to evaluate the depletion of OTC in the tilapia fillets, the fish (weight range 93 ¿ 115 g) were given medicated feed in a concentration of 80 mg OTC/kg body weight/day for five consecutive days. The water temperature was between 16.5 to 24.5 °C during the treatment. The depletion curve was fitted to an exponential first order model. The elimination half-life was 2.5 days and the withdrawal period was estimated as five days / Doutorado / Quimica Analitica / Doutor em Ciências

Abstract Sensitive chromatographic methods were developed for the quantitative analysis of secondary metabolites in Hypericum perforatum (St. John's wort) and Rhodiola rosea (Golden root, rose root) extracts. Sample preparation methods were developed for plant, cell culture and biotransformation suspension matrixes. High performance liquid chromatography (HPLC) was used for the separation of analytes, and chromatographic data was acquired using photodiode array (PDA) detection or atmospheric pressure ionization mass spectrometry (API-MS). Ionization efficiencies with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) were compared under different conditions. Specific mass spectrometric detection methods such as multiple reaction monitoring (MRM) and selective ion monitoring (SIM) were utilized. For identification of known and new secondary metabolites in plant tissues, mass spectrometric methods with triple quadrupole and time-of-flight mass spectrometers were used together with one- and two-dimensional nuclear magnetic resonance spectroscopy (NMR).

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22487号 / 農博第2391号 / 新制||農||1076(附属図書館) / 学位論文||R2||N5267(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 梅澤 俊明, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22732号 / 医博第4650号 / 新制||医||1046(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙橋 良輔, 教授 中山 健夫, 教授 川上 浩司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Drug-facilitated sexual assaults are a public health and safety concern. Liquid chromatography paired with tandem mass spectrometry is theoretically capable of detecting the scope of drugs commonly encountered in these types of cases. An analytical method was developed for the quantitative analysis of 40 drugs designated by Academy Standards Board 121 “Standard for the Analytical Scope and Sensitivity for Forensic Toxicological Testing of Urine in Drug Facilitated Crime” (ASB 121). The targeted analytes spanned a range of drug classes including antidepressants, antihistamines, barbiturates, benzodiazepines, cannabinoids, stimulants, and opioids. The final method utilized supported liquid extraction, followed by liquid chromatography tandem mass spectrometry with electrospray ionization in simultaneous positive and negative mode. Multiple reaction monitoring allowed quantification of analytes along with stable isotope internal standards. Validation parameters assessed included linearity, bias, precision, limit of detection, lower limit of quantitation, interference, and ion suppression or enhancement. The utilized sample preparation method was able to extract 36 of the 40 target analytes and the developed analytical method was able to detect and quantify all analytes to the sensitivities required by ASB 121.

No description available.

Genus Phytophthora represents a world-wide spread pathogen with more than hundred recognized species and its devastating effect on plants has a serious economic and ecological impact. This diploma thesis entitled „Proteome and metabolome of genus Phytophthora” briefly summarizes knowledge about this pathogen, including its life cycle and interactions with its host. Twelve species representing six Phytophthora clades that were selected for experimental work are then discussed in details. Phytophthora isolates were characterized on proteome and metabolome level employing an LC-MS untargeted proteome profiling and a GC-MS analysis of volatiles. The results were then processed to identify candidate molecules for a targeted identification of Phytophthora and these results were validated in an independent experiment with P. palmivora and Hordeum vulgare. We found that a proteome profiling can be employed as a tool to differentiate individual Phytophthora species and that the marker peptides can be employed for a targeted monitoring of Phytophthora presence in plants.

Rapid and simple HPLC-UV and LC-MS methods were developed and validated for the quantification of ertapenem (Invanz™) in human plasma. Ertapenem is a unique drug in that current dosing recommendations call for a 1g dose for normal renal function patients, despite body weight. These assays, which involve a protein precipitation followed by liquid-liquid extraction, allow for fast therapeutic drug monitoring of ertapenem in patients, which is especially useful in special populations. Both methods were sufficient to baseline resolve meropenem (internal standard) and ertapenem, and were validated over 3days using a six-point calibration curve (0.5-50μg/mL). Validation was collected using four different points on the calibrations curve yielding acceptable precision (<15% inter-day and intra-day; <20% for lower limit of quantitation, LLOQ) as well as accuracy (<15% inter-day and intra-day; <20% for LLOQ). The lower limit of detection (LOD) was determined to be 0.1 and 0.05μg/mL for the HPLC-UV and LC-MS methods, respectively. The developed HPLC-UV and LC-MS methods for ertapenem quantification are fast, accurate and reproducible over the calibration range and can be used to determine ertapenem plasma concentrations for monitoring clinical efficacy.