Humans are exposed to electrophilically reactive compounds, both formed endogenously and from exogenous exposure. Such compounds could react and form stable reaction products, adducts, at nucleophilic sites in proteins and DNA. The formation of adducts constitutes a risk for effects, such as cancer and contact allergy, and plays a role in ageing processes. Adducts to proteins offer a possibility to measure electrophilic compounds in vivo. Adductomic approaches aim to study the totality of adducts, to specific biomolecules, by mass spectrometric screening. This thesis describes the development and application of an adductomic approach for the screening of unknown adducts to N-terminal valine (Val) in hemoglobin (Hb) by liquid chromatography tandem mass spectrometry (LC/MS/MS). The adductomic approach is based on the FIRE procedure, a modified Edman procedure for the analysis of adducts to N-terminal Val in Hb by LC/MS/MS. The adduct screening was performed by stepwise scanning of precursor ions in small mass increments and monitoring four fragments common for derivatives of detached Val adducts, in the multiple reaction monitoring mode. Samples from 12 smokers/nonsmokers were screened with the adductomic approach, and seven previously identified adducts and 19 unknown adducts were detected. A semiquantitative approach was applied for approximate quantification of adduct levels. A strategy for identifying unknown Hb adducts using adductome LC/MS/MS data was formulated and applied for the identification of unknown adducts. Identifications were based on the observed m/z of precursor ions and retention times combined with databases and Log P calculations. Hypothesized adducts were generated in vitro for comparison and matching with the corresponding unknown adducts. Five identified adducts correspond to the precursor electrophiles ethyl vinyl ketone (EVK), glyoxal, methylglyoxal, acrylic acid, and 1-octen-3-one. These adducts, except the adducts corresponding to glyoxal and methylglyoxal, have not been observed as protein adducts before.  Probable exposure sources to these electrophiles are diet and/or endogenous formation. The observation of these adducts motivate further studies to evaluate possible contributions to health risks, as well as their potential as biomarkers of exposure. The adduct from EVK was quantitatively assessed through different experiments to estimate the daily internal dose (area under the concentration-time-curve, AUC). EVK is about 2 × 103 more reactive than the reference compound acrylamide. The EVK adduct was shown to be unstable, with a relatively short half-life. The daily AUC in humans of EVK was estimated to be about 20 times lower than the corresponding AUC of acrylamide from intake via food. To confirm the observation of the detected unknown adducts and obtain a statistical foundation, analysis of unknown adducts were performed in large sets of blood samples (n = 50–120) from human cohorts. The majority of the previously detected unknown adducts were found in all analyzed samples, and the levels of many adducts showed large variations between individuals. The cause and significance of these observed variations are not yet clarified, but are of importance for the directions of future studies. In conclusion, a new approach for identification of unknown human exposure to electrophiles was developed and successfully applied. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Submitted.</p>

Alors que le protéome de l'ovocyte isolé, aux stades VG et MII a déjà été étudié, celui du follicule en croissance n'a jamais été décrit.Dans cette étude, nous avons cherché à identifier, comparer et caractériser les profils protéiques de follicules ovariens de souris à trois stades de leur développement in vitro distincts morphologiquement : follicules secondaires en début de culture - stade initial (IS), follicules avec une rupture complète de la membrane de Slavjanski (RMS) et follicules avec une cavité similaire à l'antrum (FA).Après un préfractionnement par IEF et une analyse LC-MS/MS en deux configurations (1D et 2D), 1403 protéines ont pu être identifiées dans le follicule ovarien de souris. 43,4 % (609) des protéines identifiées étaient communes aux trois stades et d'autres ont été identifiées uniquement à un stade : 71 au stade IS, 182 au stade RMS et 193 au stade FA. De plus, on a identifié 365 protéines qui n'avaient pas été décrites antérieurement dans le protéome de l'ovocyte ce qui pourrais indiquer qu'elles sont exprimées dans les cellules somatiques du follicule. Des analyses qualitatives et quantitatives complémentaires ont démontré une surreprésentation pour 44 fonctions biologiques par rapport aux fonctions biologiques des gènes constituant le génome de Mus musculus et mis en évidence des différences d'expression et d'abondance des protéines liées au cycle cellulaire, à la fixation des ions de calcium et à la glycolyse selon le stade de développement. Ces résultats représentent un point de départ pour beaucoup d'autres études de caractérisation moléculaire du développement folliculaire. / Until now only the proteome of isolated oocyte from fully grown follicle were described with the aim of oocyte maturation characterization. However the ability of oocyte to mature is acquired during its development within the follicle. Thus in this study we proposed a protein identification and characterization of whole mice ovarian follicle at three morphological stages during in vitro development: early secondary stage, described as initial stage (IS), follicles with a complete Slavjanski membrane rupture (RMS) and follicles with an antrum like cavity formation (FA). Using an IEF pre fractionation before protein digestion and two configurations of LC-MS/MS analysis (1D and 2D), 1403 proteins were successfully identified in the murine ovarian follicle. From those, 43.4 % (609) were commonly identified in the three stages and some were identified only at one single stage: 71 at IS stage, 182 at RMS stage and 193 at FA stage. Compared to the proteomes of isolated oocyte previously described, 365 proteins were only identified in our samples indicating that those ones were probably expressed in the somatic cells of the follicle. Additional qualitative and quantitative analysis highlighted 44 biological processes over represented in our samples when compared to Mus musculus gene database and different expressions and protein abundance implicated in cell cycle, calcium ion binding and glycolysis, throughout in vitro follicle development. This report represents so far the most complete catalogue of follicle proteins and could be an important milestone in the proteomic study of the follicle metabolism throughout in vitro development.

HIV antiretroviral therapy spans several different drug classes, meant to combat various aspects of viral infection and replication. Many authors have argued the benefits of therapeutic drug monitoring (TDM) for the HIV patient including compliance assurance and assessment of appropriate drug concentrations; however, the array of drug chemistries and combinations makes TDM an arduous task. HPLC-UV and LC-MS/MS are both frequent instruments for the quantification of HIV drugs in biological matrices with investigators striving to balance sensitivity and affordability. Plasma, the dominant matrix for these analyses, is prepared using protein precipitation, liquid-liquid extraction or solid-phase extraction depending on the specific complement of analytes. Despite the range of polarities found in drug classes relevant to HIV therapeutics, most chromatographic separations utilize a hydrophobic column (C18 ). Additionally, as the clinically relevant samples for these assays are infected with HIV, along with possible co-infections, another important aspect of sample preparation concerns viral inactivation. Although not routine in clinical practice, many published analytical methods from the previous two decades have demonstrated the ability to conduct TDM in HIV patients receiving various medicinal combinations. This review summarizes the analytical methods relevant to TDM of HIV drugs, while highlighting respective challenges.

Primary marine aerosol particles containing biogenic polymer microgels play a potential role for cloud formation in the pristine high Arctic summer. One of the major sources of the polymer gels in Arctic aerosol was suggested to be the surface water and more specifically, the surface microlayer (SML) of the open leads within the perennial sea ice as a result of bubble bursting at the air-sea interface.  Phytoplankton and/or ice algae are believed to be the main origins of the polymer gels. In this thesis, we examine the chemical composition of biogenic polymers, with focus on polysaccharides, in seawater and airborne aerosol particles collected during the Arctic Summer Cloud Ocean Study (ASCOS) in the summer of 2008. The main results and findings include: A novel method using liquid chromatography coupling with tandem mass spectrometry was developed and applied for identification and quantification of polysaccharides. The enrichment of polysaccharides in the SML was shown to be a common feature of the Arctic open leads. Rising bubbles and surface coagulation of polymers are the likely mechanism for the accumulation of polysaccharides at the SML. The size dependencies of airborne polysaccharides on the travel-time since the last contact with the open sea are indicative of a submicron microgel source within the pack ice.  The similarity of polysaccharides composition observed between the ambient aerosol particles and those generated by in situ bubbling experiments confines the microgel source to the open leads. The demonstrated occurrence of polysaccharides in surface sea waters and in air, with surface-active and hygroscopic properties, has shown their potential to serve as cloud condensation nuclei and subsequently promote cloud-drop activation in the pristine high Arctic. Presumably this possibility may renew interest in the complex but fascinating interactions between marine biology, aerosol, clouds and climate. / At the time of doctoral defence, the following paper was unpublished and had a status as follows: Paper 4: Manuscript

<p>Proteomics is the analysis of the total complement of proteins expressed by a cell or organism grown under a specified condition. The obtained protein profile would provide a better understanding of phenotypic characteristics of a cell grown under pre-determined conditions. Mass spectrometric-based protein analysis is currently the standard method in proteomic studies; however, there are many limitations associated with its application. The major objectives of this study included the development of a strategy to analyze the confidence of identified proteins and the development of an algorithm to interpret the experimentally obtained mass spectral data. </p> <p>A two-step strategy was developed to analyze the confidence of identified proteins. In the first step, the proteins identified by a single protein identification tool were classified into two groups: high confidence proteins that were identified by unique peptides, and low confidence proteins that were identified by non-unique peptides. In the second step, the proteins identified by different tools (e.g., SEQUEST and Mascot in our work) were cross-compared. After integrating the two-step analysis, the identified proteins were classified into four levels of confidence. The proteins that were identified by the presence of unique peptides and that were commonly identified by different tools were grouped into the highest confidence level - Level 4. Even though the number of proteins in Level 4 was reduced significantly, the conclusions drawn from the proteins were more reliable.</p> <p>According to the operation of tandem mass spectrometry and the characteristics of the peptides generated by site-specific protease digestion, a two-pass approach for identifying the species-specific proteins was developed. The approach can find all possible peptides corresponding to a precursor ion and gives detailed matching information of each peptide candidate to the experimental product ion series. According to the total number of matched product ions, the total number of matched b- and y- ions, and the contiguity characteristic of identified product ions, the peptide candidates were ranked decreasingly from the most probable to the least. Combined with the concept of unique peptide, the obtained most probable peptide can then be used to predict proteins existing in the original sample.</p> <p>The developed two-pass approach and two-step strategy were then used to study the protein profiling of <i>Saccharomyces cerevisiae</i> cultivated in various gravity conditions (10 and 300 g glucose/l) in order to investigate the changes in central metabolic pathways of <i>S. cerevisiae</i>. Our fermentation data indicated that the higher glucose contents would result in lower cell growth and higher ethanol production (e.g., high ethanol concentration in fermentation broth). However, the relative ethanol yield as related to the glucose consumption was lower under higher glucose concentrations. The protein profile showed that a higher flux of nutrient was channelled into the pentose phosphate pathway when <i>S. cerevisiae</i> was grown under a high glucose concentration. The reason for this phenomenon might be that the cell needs more reducing power (e.g., NADPH) for the synthesis of macromolecules such as proteins, nucleic acids, and lipids. These materials are essential to the cell in order to modify its structure (e.g., cell wall), to survive osmotic stress and to replicate.</p>

A rapid and accurate quantitative method was developed and validated for the analysis of four urinary organic acids with nitrogen containing functional groups, formiminoglutamic acid (FIGLU), pyroglutamic acid (PYRGLU), 5-hydroxyindoleacetic acid (5-HIAA), and 2-methylhippuric acid (2-METHIP) by liquid chromatography tandem mass spectrometry (LC/MS/MS). The chromatography was developed using a weak anion-exchange amino column that provided mixed-mode retention of the analytes. The elution gradient relied on changes in mobile phase pH over a concave gradient, without the use of counter-ions or concentrated salt buffers. A simple sample preparation was used, only requiring the dilution of urine prior to instrumental analysis. The method was validated based on linearity (r2 ¡Ý 0.995), accuracy (85¨C115%), precision (C.V. < 12%), sample preparation stability (¡Ü 5%, 72h), and established patient ranges. The method was found to be both efficient and accurate for the analysis of urinary zwitterionic organic acids.

<p>Proteomics is the analysis of the total complement of proteins expressed by a cell or organism grown under a specified condition. The obtained protein profile would provide a better understanding of phenotypic characteristics of a cell grown under pre-determined conditions. Mass spectrometric-based protein analysis is currently the standard method in proteomic studies; however, there are many limitations associated with its application. The major objectives of this study included the development of a strategy to analyze the confidence of identified proteins and the development of an algorithm to interpret the experimentally obtained mass spectral data. </p> <p>A two-step strategy was developed to analyze the confidence of identified proteins. In the first step, the proteins identified by a single protein identification tool were classified into two groups: high confidence proteins that were identified by unique peptides, and low confidence proteins that were identified by non-unique peptides. In the second step, the proteins identified by different tools (e.g., SEQUEST and Mascot in our work) were cross-compared. After integrating the two-step analysis, the identified proteins were classified into four levels of confidence. The proteins that were identified by the presence of unique peptides and that were commonly identified by different tools were grouped into the highest confidence level - Level 4. Even though the number of proteins in Level 4 was reduced significantly, the conclusions drawn from the proteins were more reliable.</p> <p>According to the operation of tandem mass spectrometry and the characteristics of the peptides generated by site-specific protease digestion, a two-pass approach for identifying the species-specific proteins was developed. The approach can find all possible peptides corresponding to a precursor ion and gives detailed matching information of each peptide candidate to the experimental product ion series. According to the total number of matched product ions, the total number of matched b- and y- ions, and the contiguity characteristic of identified product ions, the peptide candidates were ranked decreasingly from the most probable to the least. Combined with the concept of unique peptide, the obtained most probable peptide can then be used to predict proteins existing in the original sample.</p> <p>The developed two-pass approach and two-step strategy were then used to study the protein profiling of <i>Saccharomyces cerevisiae</i> cultivated in various gravity conditions (10 and 300 g glucose/l) in order to investigate the changes in central metabolic pathways of <i>S. cerevisiae</i>. Our fermentation data indicated that the higher glucose contents would result in lower cell growth and higher ethanol production (e.g., high ethanol concentration in fermentation broth). However, the relative ethanol yield as related to the glucose consumption was lower under higher glucose concentrations. The protein profile showed that a higher flux of nutrient was channelled into the pentose phosphate pathway when <i>S. cerevisiae</i> was grown under a high glucose concentration. The reason for this phenomenon might be that the cell needs more reducing power (e.g., NADPH) for the synthesis of macromolecules such as proteins, nucleic acids, and lipids. These materials are essential to the cell in order to modify its structure (e.g., cell wall), to survive osmotic stress and to replicate.</p>

novel ozone-assisted aerobic sludge digestion process to stabilize and decrease the amount of excess sludge produced during biological treatment is presented in this study. Excess sludge production is a well known burden for the treatment plants both legally and financially. Moreover, with the arise in the knowledge in recalcitrant compounds it is understood that it can act as a significant secondary pollutant. With the developed pulse ozonation method, waste activated sludge samples from Ankara Tatlar and other Wastewater Treatment Plants (WWTP) were ozonated for different periods in Erlenmeyer flasks once a day on each of four consecutive days. Flasks were continuously aerated between ozone applications on an orbital shaker. The MLVSS, MLSS, COD and OUR parameters were measured routinely during the course of four days of digestion in order to optimize the process. Also pH, CST(capillary suction time) and SVI (sludge volume index) were followed. As a result MLVSS reductions of up to 95% were achieved with an ozone dose of only 0.0056 kg O3/kg-initial MLSS, at the end of the fourth day. In another experimental set, ozone dose was increased on the last day in order to destroy the selected endocrine disrupting compounds, namely diltiazem, carbamazepine, butyl benzyl phthalate and acetaminophen and two natural hormones estrone and progesterone, which accumulated onto the sludge. Over 99% removal of these contaminants were achieved on the fourth day. The analyses were conducted by using LC(ESI) MS/MS after solid phase extraction (SPE). By this process it became possible to save on contact time, as well as achieving a bio-solids digestion far exceeding the standard aerobic process at the expense of a minimum of ozone dose with the additional micropollutants removal. The developed process is deemed superior over side-stream ozonation of activated sludge in that it does not cause any reduction in active biomass amount that should be maintained in the aeration tank.

La protéine kinase TOR (Target Of Rapamycin) a été identifiée chez la levure et les mammifères comme participant à deux complexes protéiques qui servent de carrefour entre la perception des facteurs endogènes et exogènes et la stimulation de la croissance cellulaire. Depuis la découverte de la kinase AtTOR chez Arabidopsis thaliana, des études ont été menées afin de mieux caractériser son rôle chez les plantes et l'influence de son niveau d'expression sur la régulation du métabolisme et du développement.Au cours de ce travail, j'ai contribué à l'étude de cette kinase en étudiant l'influence de l'inactivation de TOR sur la composition du ribosome au niveau protéique et sur le niveau de phosphorylation de ces protéines, ainsi que sur l'organisation du méristème au niveau moléculaire et cytologique Au cours de cette étude, j'ai montré que certaines protéines constitutives du ribosome pourraient être des cibles de l'activité TOR au niveau de leur abondance et/ou de leur état de phosphorylation. Ainsi, l'inactivation de TOR entraine une diminution du niveau de phosphorylation des protéines RPS6 et pourrait influencer l'abondance des protéines acides constitutives du stalk ribosomal, une structure importante dans la régulation de la traduction. Les résultats obtenus suggèrent également que l'activité TOR est nécessaire au maintien du méristème à l'état fonctionnel en régulant les voies importantes contrôlant la division et la différentiation au sein de cette structure.

The use of nanocarriers is an intriguing approach in the development of efficacious treatment for brain disorders. The aim of the conducted research was to evaluate and quantify the impact of a liposomal nanocarrier formulation on the brain drug delivery. A novel approach for investigating the blood-brain barrier transport of liposomal DAMGO is presented, including in vivo microdialysis in rat, a high quality LC-MS/MS bioanalytical method and pharmacokinetic model analysis of the data. Factors limiting the brain distribution of the free peptide DAMGO were also investigated. Microdialysis, in combination with plasma sampling, made it possible to separate the released drug from the encapsulated and to quantify the active substance in both blood and brain interstitial fluid over time. The opioid peptide DAMGO entered the brain to a limited extent, with a clearance out of the brain 13 times higher than the clearance into the brain. The brain to blood ratio of unbound drug was not affected when the efflux transporter inhibitors cyclosporine A and elacridar were co-administered with DAMGO. Nor was the transport affected in the in vitro Caco-2 assay using the same inhibitors. This indicates that DAMGO is not transported by P-glycoprotein (Pgp) or breast cancer resistant protein (Bcrp). The blood-brain barrier transport was significantly increased for DAMGO when formulated in liposomes, resulting in 2-3 fold higher brain to blood ratio of unbound DAMGO. The increased brain delivery was seen both for glutathione tagged PEGylated liposomes, as well as for PEGyalted liposomes without specific brain targeting. The improvement in brain delivery was observed only when DAMGO was encapsulated into the liposomes, thus excluding any effect of the liposomes themselves on the integrity of the blood-brain barrier. Modeling of the data provided additional mechanistic understanding of the brain uptake, showing that endocytosis or transcytosis of intact liposomes across the endothelial cell membranes were unlikely. A model describing fusion of the liposomes with the luminal membrane described the experimental data the best. In conclusion, the studies presented in this thesis all contribute to an increased understanding of how to evaluate and improve brain delivery of CNS active drugs and contribute with important insights to the nanocarrier field.