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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
201

Evaluation de l'effet mélange sur la clairance hépatique humaine de pesticides appartenant à deux groupes de matières actives communément retrouvées dans l'alimentation française : développement analytique et études cinétiques / Assessment of the mixture effect on human hepatic clearance of pesticides from two mixtures usually found in the French diet : analytical development and kinetic studies

Kadar, Ali 17 December 2018 (has links)
La population générale est exposée à un grand nombre de pesticides, principalement via son alimentation. Alors que ces matières actives ont reçu une autorisation de mise sur le marché individuelle, l’effet sur l’organisme humain d’un mélange de tels composés est en revanche très peu connu. Afin de contribuer à mieux appréhender cet « effet mélange », notre étude s’est attachée à étudier le métabolisme hépatique humain in vitro de deux mélanges de pesticides communément retrouvés dans l’alimentation française / General population is exposed to several pesticides, mainly via the diet. Whereas these active ingredients obtained their marketing authorization individually, the available data regarding their impact as a mixture on the human body are scarce. In order to help better understanding this “mixture effect”, we aimed at studying the human hepatic in vitro metabolism of two pesticides mixtures commonly found in the diet
202

MULTI-DIMENSIONAL MASS SPECTROMETRY, MICROBES, AND THE DEMONS AMONGST THEM: RAPID UNTARGETED PROFILING OF MICROORGANISMS

L. Edwin Gonzalez (7289045) 30 November 2023 (has links)
<p dir="ltr">Mass spectrometry has been at the forefront of complex mixture analysis and, as a result, has greatly advanced the understanding of biological systems with its application in the biological sciences. One area in which mass spectrometry has succeeded is the area of microbiology and the identification of pathogens and has gained much attention from the biothreat detection community. Although this technology has matured in the past decade, very few systems have been developed for point-of-need analysis in cases such as the detection of biothreats. Current MS systems for the analysis of microbes utilizing MALDI-TOF-MS require large instruments to accommodate a drift tube long enough for high resolution mass analysis and high vacuum which is not amenable to the miniaturization requirements of point-of-need analysis. The previously mentioned methods also require extensive manipulation of the sample which takes time and can pose a risk to instrument operators in the biothreat detection space. Additionally, most mass spectrochemical instruments provide only one-dimension of data which can limits classification accuracy when using classification algorithms to provide an identity on a microbiological sample which could consist of any of the numerous common bacterial pathogens or biothreats.</p><p dir="ltr">A possible solution to this problem is the implementation of two-dimensional tandem mass spectrometry (2D MS/MS) which allows the analysis of the product ions of all precursor ions representing the result in the 2D MS/MS data domain. This methodology is possible with a linear quadrupolar ion trap mass analyzer and can be applied to miniature ion trap technology for portability. In this dissertation, a progression of mass spectrochemical analysis of biological systems from conventional methods to the implementation of 2D MS/MS is demonstrated: by (i) the development of a rapid biomolecule extraction method to analyze bacterial spores, using a (ii) modified linear quadrupolar ion trap mass spectrometer, (iii) then a miniature ion trap mass spectrometer, and (iv) finally adding numerical methods to discriminate between biological systems using data acquired on each 2D MS/MS instrument. This work is then taken a step further by developing a high throughput experimentation method in which DESI is coupled to 2D MS/MS to analyze a moderate number of samples rapidly, automatically, and with high reproducibility.</p>
203

[pt] DERRAME DE ÓLEO E SAÚDE HUMANA-METABÓLITOS DE HPAS EM URINA COMO TRAÇADORES DE EXPOSIÇÃO UTILIZANDO ESPECTROMETRIA DE MASSAS TANDEM / [en] OIL SPILLS AND HUMAN HEALTH - PAH METABOLITES IN URINE AS EXPOSURE TRACERS USING TANDEM MASS SPECTROMETRY

LIVIA ARAUJO LOREDO 29 August 2023 (has links)
[pt] Um grande vazamento de óleo bruto ocorreu na costa brasileira em 2019 afetando uma extensão de 4.334 km de faixa litorânea, 11 estados e várias localidades. Com isso, moradores locais entraram em contato direto com óleo cru ficando expostos à contaminação por hidrocarbonetos policíclicos aromáticos (HPA). Os HPA são compostos orgânicos carcinogênicos de origem petrogênica e pirogênica, que são ubíquos e persistentes. Quando seres humanos são expostos a esses contaminantes, o organismo pode metabolizar e gerar metabólitos desses HPA com prováveis efeitos negativos à saúde humana. Com isso, este trabalho visou analisar a exposição de uma comunidade de pescadores (localizada em Canavierias, BA) aos HPA provindos de derrames de petróleo através da análise de metabólitos de HPA em urina. Para isso, as 44 amostras foram extraídas por extração em fase sólida (SPE) e analisadas por cromatografia líquida acoplada a espectrômetro de massas triplo quadrupolo (HPLC-MS/MS). Este trabalho também contempla a comparação de diferentes métodos de extrações em 18 amostras de urina selecionadas. No Centro de Pesquisas, Desenvolvimento e Inovação Leopoldo Américo Miguez de Mello (CENPES) realizou-se extração em fase sólida e a extração líquido-líquido, enquanto na PUC-Rio foi realizada a extração por SPE. Os resultados referentes às 44 amostras analisada na PUC-Rio, demonstraram que 4 dos 11 metabólitos de interesse foram identificados e quantificados. Para o 1-OH-Nap a faixa de concentração foi (menor que LQ – 44 n mL(-1) ), (menor que LQ-21 ng mL(-1) ) para o 2-OHNap, (menor que LQ -5,4 ng mL(-1) ) para o 3,9-OH-Phe e (menor que LQ- 0,76 ng mL(-1) ) para o 6-OH-Chr. Ao comparar os resultados obtidos com trabalhos da literatura, observou-se que os moradores da comunidade de Canavieiras apresentaram valores baixos de concentração dos metabólitos que foram quantificados. Mesmo assim, é importante a existência de trabalhos voltados para o monitoramento destes compostos já que não somente o ambiente como também apresentam-se como um risco à saúde humana. / [en] A major crude oil spill occurred off the coast of Brazil in 2019 affectinga 4,334 km stretch of coastline, 11 states and several localities. As a result,residents came into direct contact with crude oil and were exposed tocontamination by polycyclic aromatic hydrocarbons (PAHs). PAHs arecarcinogenic organic compounds of petrogenic and pyrogenic origin, whichare ubiquitous and persistent. When humans are exposed to thesecontaminants, the organism can metabolize and generate metabolites ofthese PAHs with probable negative effects on human health. Therefore,this work aimed to analyze the exposure of a fishing community (located inCa-navierias, BA) to PAHs from oil spills through the analysis of PAHmetabolites in urine. For this, the 44 samples were extracted by solid phaseextraction (SPE) and analyzed by liquid chromatography coupled to triplequadrupole mass spectrometer (HPLC-MS/MS). This work also includes thecomparison of different extraction methods in 18 selected urine samples.Solid phase extraction and liquid-liquid extraction were performed at theLeopoldo Américo Miguez de Mello Research, Development and InnovationCenter (CENPES), while SPE extraction was performed at PUC-Rio. Theresults for the 44 samples analyzed at PUC-Rio showed that 4 of the 11metabolites of interest were identified and quantified. For 1-OH-Nap theconcentration range was (less than LQ - 44 n mL(-1)), (less than LQ-21 ng mL(-1)) for 2-OH-Nap,(less than LQ -5.4 ng mL(-1)) for 3,9-OH-Phe and (less than LQ- 0.76 ng mL(-1)) for 6-OH-Chr.When comparing the results obtained with literature works, it was observedthat the residents of the community of Canavieiras had low concentrationvalues of the metabolites that were quantified. Even so, it is important theexistence of works focused on the monitoring of these compounds since notonly the environment but also present themselves as a risk to human health.
204

Development and validation of an ultrafiltration-UHPLC-MS/MS method for the quantification of unbound Beta-Lactam antibiotics cefotaxime, piperacillin, cloxacillin and flucloxacillin in plasma / Utveckling och validering av en UHPLC-MS/MS-metod med ultrafiltrering för kvantifiering av icke-proteinbunden beta-lactam-antibiotika cefotaxim, piperacillin, kloxacillin och flukloxacillin i plasma

Clarin, Leona January 2020 (has links)
Infections in critically ill patients are a problem for the healthcare system and at any one time, 70 % of all intensive care unit (ICU) patients are treated with antibiotics. Antibiotics bind toproteins in the blood, but only unbound drug can diffuse over capillary membranes and bindto the targeted receptor. Standard protein binding percentages for antibiotics have been developed from studies on healthy volunteers and dosing regimens for patients are adapted accordingly. The determination of the total concentration of antibiotics in patients’ bloodsamples is, based on the standard percentages, ordinarily representative for the pharmacological effect of the antibiotic. However, certain conditions that are common incritically ill patients can alter protein binding percentages, resulting in a larger or smaller unbound fraction. This in turn can result in toxicity or therapeutic failure. The aim of this project was to develop an analytical method for the determination of the unbound concentration of the Beta-Lactam antibiotics cefotaxime, flucloxacillin, cloxacillin and piperacillin in plasma. A method was successfully developed using ultrafiltration for the extraction of unbound analytes and ultra high performance liquid chromatography tandem mass spectrometry, UHPLC-MS/MS, for their quantification. The method was partly validated according to the European Medicines Agency’s guidelines on bioanalytical method validation. / Kritiskt sjuka patienter med infektioner är en börda för sjukvården och 70 % av alla patienter på intensivvårdsavdelningar är ordinerade antibiotika. Antibiotika binder till proteiner i blodet, men enbart den icke-proteinbundna (fria) fraktionen kan diffundera över kapillära membran och binda till receptorer. Standardproteinbindningsgrad för olika antibiotika har utvecklats från studier på friska frivilliga och doseringen av läkemedlen är anpassade därefter. Den totala koncentrationen av antibiotika i patienters blod är vanligen representativ för den farmakologiska effekten. Dock kan vissa sjukdomar påverka proteinbindningsgraden vilket resulterar i en större eller mindre mängd fria antibiotika i blodcirkulationen. Det här kan i sintur resultera i toxicitet eller otillräcklig effekt av läkemedlet. Syftet med det här projektet var att utveckla en analytisk metod för att bestämma den fria koncentrationen av Beta-Lactam antibiotikan cefotaxim, flukloxacillin, kloxacillin och piperacillin i plasma. En metod utvecklades med ultrafiltrering för extraktion av den fria fraktionen och högupplösande vätskekromatografi och tandem masspektrometri, UHPLCMS/MS, för kvantifiering av analyterna. Metoden validerades delvis enligt den Europeiska Läkemedelsmyndighetens riktlinjer för bioanalytisk metodvalidering.
205

DEVELOPMENT OF COCAINE HYDROLASE FOR THERAPEUTIC TREATMENT OF COCAINE ABUSE

Chen, Xiabin 01 January 2016 (has links)
Cocaine abuse is a world-wide public health and social problem without a U.S. Food and Drug Administration (FDA)-approved medication. An ideal anti-cocaine medication would accelerate cocaine metabolism producing biologically inactive metabolites by administration of an efficient cocaine-specific exogenous enzyme. Recent studies in our lab have led to discovery of the desirable, highly efficient human cocaine hydrolases (hCocHs) that can efficiently detoxify and inactivate cocaine without affecting normal functions of central nervous system (CNS). Preclinical and clinical data have demonstrated that these hCocHs are safe for use in humans and effective for accelerating cocaine metabolism. However, the actual therapeutic use of a hCocH in cocaine addiction treatment is limited by the short biological half-life (e.g. 8 hours or shorter in rats) of the hCocH. In the investigation described in this thesis, we have demonstrated that mCocH and hCocH have improved the catalytic efficiency of mBChE and hBChE against cocaine by ~8- and ~2000-fold, respectively, although the catalytic efficiencies of mCocH and hCocH against other substrates, including acetylcholine (ACh) and butyrylthiocholine (BTC), are close to those of the corresponding wild-type enzymes mBChE and hBChE. In addition, we have identified the first benzoylecgonine-metabolizing enzymes that can hydrolyze benzoylecgonine and accelerate its clearance in rats. The developed LC-MS/MS method has enabled us to simultaneously determine cocaine and nine cocaine-related metabolites in whole blood samples. In development of the long-acting hCocHs, we have designed and discovered a novel hCocH form, catalytic antibody analog, which is an Fc-fused hCocH dimer (hCocH-Fc). The hCocH-Fc has not only a high catalytic efficiency against cocaine, but also a considerably longer biological half-life. A single dose of hCocH-Fc was able to accelerate cocaine metabolism in rats even after 20 days and, thus, block cocaine-induced hyperactivity for a long period of time. In consideration of the general observation that the biological half-life of a protein drug in humans is significantly longer than that in rodents, the hCocH-Fc could allow dosing once every 2-4 weeks, or longer for cocaine addiction treatment in humans.
206

HIGH DOSE SIMVASTATIN AS A POTENTIAL ANTICANCER THERAPY IN LEUKEMIA PATIENTS

Ahmed, Tamer 01 January 2013 (has links)
Simvastatin is a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor that is used for the treatment of hyperlipidemia. Simvastatin has recently been studied for its potential use in cancer therapy. In-vitro studies have shown that simvastatin displays anticancer activity, but at concentrations unlikely to be achieved in patients being receiving typical antihyperlipidemic treatment doses. Thus, several clinical trials were conducted to study the tolerability of high dose statins in cancer patients. The maximum tolerated dose of simvastatin was determined to be 15 mg/kg/day, 25-fold higher than a typical dose. However, it is not known if simvastatin plasma concentrations can reach those found to be effective in-vitro. In this context, we initiated a clinical study to determine the pharmacokinetics of high dose simvastatin in patients with chronic lymphocytic leukemia. For this purpose, an LC-MS/MS method was developed and validated for the quantitation of simvastatin and its acid form in plasma and peripheral blood mononuclear cells obtained from CLL patients. Results show that simvastatin concentrations were dose proportional relative to the antihyperlipidemic doses, but lower than those required for in-vitro cytotoxicity against cancer cells. These findings demonstrate that the in-vitro effective concentrations of simvastatin are not achievable clinically, which might explain the limited effectiveness of high dose simvastatin in this study and in previous clinical trials. In view of these data, the use of simvastatin as a sole therapy in cancer treatment was not encouraging and led us to examine the use in combination with other anticancer drugs. After screening several chemotherapeutic agents in combination with simvastatin, we showed that tipifarnib (a farnesyltransferase inhibitor) interacts synergistically in several leukemia cell lines. Mechanistically we showed that simvastatin augments the cytotoxicity of tipifarnib by disrupting the localization of RAS in the cell membrane and by subsequent deactivation of the ERK pathway. Consistent with this observation, drug treatment led to the induction of apoptosis through the caspase cascade activation and the cleaved PARP upregulation. Notably, this synergistic effect was observed at clinically achievable concentrations of simvastatin and tipifarnib. Thus, the effectiveness of this combination should be explored further in future clinical studies.
207

DATA MINING OF PEPTIDE MS/MS SPECTRA TO ELUCIDATE GAS-PHASE PEPTIDE DISSOCIATION MECHANISMS AND IMPROVE PROTEIN IDENTIFICATION

Huang, Yingying January 2005 (has links)
Mining of datasets obtained from proteomics experiments was performed to investigate the dissociation pathways of protonated peptides activated in the gas phase under low energy collision-induced dissociation (CID). Intensity patterns in ion trap tandem mass spectra were exploited and different statistical approaches were employed to elucidate the dissociation mechanisms.Chapter 2 describes a study of 506 doubly-protonated tryptic peptides that shows the presence of an internal basic residue can increase the preferential fragmentation C-terminal to aspartic acid (Asp-Xxx) significantly. The degree of enhancement varies with the identity of the basic residues. The result corroborates a previously published mechanism based on studies from model peptides, and was incorporated into an existing peptide sequencing algorithm. A preliminary test on a separate dataset of 119 spectra shows that implementing rules to predict enhanced cleavages at Asp-Xxx improves the ability of the algorithm to identify the correct sequence from a list of candidates.Chapters 3-4 describe much more elaborate analyses on 28,330 peptides of different sequences and charge states. Extensive sorting based on prior knowledge was first performed to probe the correlation of fragmentation patterns with structural features. Pair-wise fragmentation maps reveal that the difference in basicity between Arg and Lys results in different dissociation patterns among singly-protonated tryptic peptides. While one dominant protonation form (proton localized) exists for Arg-ending peptides, a heterogeneous population of two or more protonated forms (proton partially-mobile) exist for Lys-ending peptides. Asp/Glu-Xxx dominates spectra from peptides that have a localized proton(s) and Xxx-Pro dominates those that have a mobile or partially mobile proton(s). When Pro is absent from peptides that have a mobile or partially mobile proton(s), cleavage at each peptide bond becomes more prominent. A fundamental dependence of gas phase peptide fragmentation on conformational constraints was found.A knowledge mining scheme was proposed in Chapter 5 to bypass the prior knowledge constraints and cluster the dissociation behaviors of 28,330 peptides into four distinct categories. The most influential factors in the fragmentation process are: the mobility of the proton(s), the presence and the location of Pro and Arg. Structural motifs responsible for each dissociation behavior are also elucidated.
208

Comprehensive Glycoproteomics and Glycomics Study of N-Linked Glycans and N-Glycoproteins

Li, Xu 06 January 2017 (has links)
N-linked glycosylation is the most common post-translational modification (PTM) of proteins that exist in nature. N-glycosylation and change in cells serve as a criterion to monitor the activity of developmental stages and diseases severity. Currently, there is an increasing application of mass spectrometry on glycoprotein for malicious, chronic or acute diseases, such as cancers, rheumatoid arthritis (RA) or influenza. In this dissertation, several mass spectrometric assays have been utilized to, quantitatively and qualitatively, characterize protein N-glycosylation at the glycan, glycopeptide and peptide levels. The goals are to identify serum-based RA biomarker (Chapter 2), or to determine possible glycan structures from monoclonal antibody (Chapter 3), or comprehensively to study one influenza glycoprotein, hemagglutinin (Chapter 4). In Chapter 2, LC-MS/MS with CID as MS 2 is the primary technique that is applied to collect raw data for RA biomarker screening; western blot is the verification method for newfound biomarkers. This mass spectrometry based comparative analysis of N-glycoprotein in RA and healthy patients’ sera reveal 41 potential biomarkers for RA that can be applied in clinical research. Chapter 3 describes another LC-MS/MS based method developed for the structural analysis of N-glycan released from the monoclonal antibody, immunoglobin G. Higher-energy collision dissociation (HCD) was the surprior technique utilized to identify glycopeptide fragments. The results show that 19 and 23 N-glycan structures were determined from standard and modified mAb samples respectively by using SimGlycan software, while 38 and 35 glycan structures were recognized by manually mapping respectively. 13 N-glycoforms, out of 26 overlapped glycan structures, were identified with significant alterations by comparing standard sample (sample A) and modified mAb (sample B) utilizing our method. In Chapter 4, we comprehensively studied hemagglutinin by using LC-MS/MS and MALDI from both proteomic perspective and glycomics prospective. After confirmed and verified protein sequence and glycosylation sites, galactose-specific quantitation was performed with exoglycosidase digestion combined HPLC with fluorescence detection. The MALDI-MS/MS based method was utilized to confirm glycan structures. The results in this dissertation provide insights into the significance of protein glycosylation alterations as RA biomarkers, and these quantitative methods can be reapplied to any other disease biomarkers screening for clinical researchers.
209

Characterization of Lysophosphatidic Acid Subspecies Using a Novel HPLC ESI-MS/MS Method

Mayton, Eric 14 July 2011 (has links)
Lysophosphatidic acid (LPA) is a bioactive lipid with a plethora of biological functions, including roles in cell survival, proliferation, and migration. Although high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC ESI-MS/MS) technology has been used to measure the levels of LPA in human blood, serum and plasma, current methods cannot readily detect the minute levels of LPA from cell culture. In this study, a novel HPLC ESI-MS/MS method with enhanced sensitivity was developed which allows accurate measurements of LPA levels with a limit of quantitation at approximately 10 femtomoles. The method was validated by quantitation of LPA levels in the media of previously characterized cell lines ectopically expressing autotaxin. Autotaxin overexpression induced an increase in several subspecies of LPA while others remained unchanged. Lastly, this HPLC ESI-MS/MS method was validated via biological assays previously utilized to assay LPA production. Hence, this new HPLC ESI-MS/MS will allow researchers to measure in vitro LPA levels and also distinguish between specific LPA subspecies for the delineation of individual biological mechanisms.
210

Quantitative Analysis of Multiple Charged Large Molecules in Human or Rat Plasma Using Liquid Chromatography Tandem Mass Spectrometry

Halquist, Matthew 27 April 2012 (has links)
Immunoassays have traditionally been employed for the determination of plasma concentration-time profiles for pharmacokinetic studies of therapeutic proteins and peptides. These ligand binding assays have high sensitivity but require significant time for antibody generation (1 to 2 years) for assay development. Despite high sensitivity, these assays suffer from cross-reactivity that can lead to inaccurate results. As an alternative to immunoassays, this dissertation was focused on the development and validation of assays that can be used for quantitative analysis of peptides or proteins in plasma using liquid chromatography tandem mass spectrometry (LC-MS/MS). Two approaches were considered for measurement of proteins and peptides fortified in plasma. The first approach involved employing signature peptides as quantitative surrogates of a target protein. This approach is a multistep process that includes: computer simulated (in silico) peptide predictions, protein purification, proteolytic digestion, peptide purification, and ultimately mass spectrometry. Signature peptides were determined through in silico peptide predictions and iterative tuning processes to represent Amevive® (Alefacept), a therapeutic for psoriasis, for quantification in human plasma. Horse heart myoglobin was chosen as a protein analogue internal standard to compensate for errors associated with matrix effects and to track recovery throughout the entire sample pretreatment process. Samples were prepared for analysis by selective precipitation of the target proteins with optimized pH and heat conditions followed by enzymatic digestion, dilution, and filtration. Combining selective precipitation and protein analogue internal standard lead to a method validated according to current FDA guidelines and achieved a linear range (250-10,000 ng/mL) suitable for monitoring the therapeutic levels of Alefacept (500 -6000 ng/mL) without the use of antibodies. A second approach exploited the mass spectrometric behavior of intact polypeptides. A polypeptide can exist in multiple charge states separated by mass to charge ratio (m/z). Herein, the charge state distribution and the formation of product ions to form selected reaction monitoring (SRM) transitions for intact polypeptide quantitative analysis was evaluated in plasma. Oxyntomodulin, a 37 amino acid anorectic peptide (4449 Da), was employed as a model for analysis in rat plasma. The +7 charge state form of OXM was used to form an SRM for quantitative analysis. Two-dimensional reversed phase ion pair chromatography, a modified solid phase extraction, and a multiply charged SRM of oxyntomodulin enabled a lower limit of quantification of 1 ng/mL. Following development of the LC-MS/MS method, a validation of this approach was performed according to FDA guidelines. Finally, to show further utility of LC-MS/MS, the validated oxyntomodulin method was used in a pharmacokinetic study with sprague-dawley rats. Rats were dosed with oxyntomodulin through intravenous or intratracheal instillation routes of administration. Plasma concentration-time profiles were determined. Using these profiles, noncompartmental parameters were determined for each dose and routes of administration.

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