Because it can expand the range of a protein’s interactions or modulate its activity, post-translational methylation of arginine residues in proteins must be duly coordinated and ‘decoded’ to ensure appropriate cellular interpretation of this biological cue. This can be achieved through modulation of the enzymatic activity/specificity of the protein arginine methyltransferases (PRMTs) and proper recognition of the methylation ‘mark’ by a subset of proteins containing ‘methyl-sensing’ protein modules known as ‘Tudor’ domains. In order to gain a better understanding of these regulatory mechanisms, we undertook a detailed biochemical characterization of the predominant member of the PRMT family, PRMT1, and of the novel Tudor domain-containing protein 3 (TDRD3). First, we found that PRMT1 function can be modulated by 1) the expression of up to seven PRMT1 isoforms (v1-7), each with a unique N-terminal region that confers distinct substrate specificity, and by 2) differential subcellular localization, as revealed by the presence of a nuclear export sequence unique to PRMT1v2. Second, our findings suggest that TDRD3 is recruited to cytoplasmic stress granules (SGs) in response to environmental stress potentially by engaging in methyl-dependent protein-protein interactions with proteins involved in the control of gene expression. We also found that arginine methylation may serve as a general regulator of overall SG dynamics. Finally, we uncovered that alteration of PRMT1, TDRD3, and global arginine methylation levels in breast cancer cells may be closely associated with disease progression and poor prognosis. Therefore, further studies into the pathophysiological consequences ensuing from misregulation of arginine methylation will likely lead to the development of novel strategies for the prevention and treatment of breast cancer.

.L’objectif de cette étude était, en premier lieu, de mettre en place en Thaïlande un testd’adhésion par traction-écaillage sur une machine de traction classique (test« macroscopique »), de le comparer au test « microscopique » Grenoblois fonctionnant dansla chambre du MEB et de l’utiliser pour évaluer l’adhérence des calamines de process sur desaciers industriels. Deux paramètres ont été étudiés, la vitesse de déformation et la teneur desaciers en silicium. Il apparaît que l’écaillage des calamines au cours du test augmente quandaugmente la vitesse de déformation. Une vitesse de déformation élevée entraîne unedéformation au premier écaillage plus faible, donc une adhérence mesurée plus faible. Ceteffet est lié aux phénomènes de relaxation. On a pu alors montrer que la présence d’oxyde(s)contenant Si, situé(s) à l’interface avec le métal, augmentait l’adhérence. Les étudesd’oxydation dans la vapeur d’eau qui ont aussi été réalisées ont révélé que la présence desilicium réduisait la vitesse d’oxydation. En augmentant la teneur en Si, les couches defayalite et de wüstite s’épaississent ; par contre, les couches externes s’amincissent. Pour lesaciers contenant du cuivre, la vitesse d’oxydation est réduite quand la teneur en Cu estaugmentée. De la même façon, les couches internes sont plus épaisses et on observe uneaugmentation du nombre de précipités de Cu quand la teneur en cet élément augmente. / The purpose of this study was first to develop in Thailand a “macroscopic” adhesion testusing a conventional tensile machine, to compare it to the micro-tensile test used in Grenobleand sitting in the SEM chamber, and to use it for measuring adhesion of scales grown duringprocessing on industrial steels. Parameters affecting the test, i.e. strain rate and Si content ofsteels were investigated. The results showed that spallation of scales during strainingincreased with increasing tensile strain rate. A higher strain rate resulted in lower straininitiating the first spallation and lower mechanical adhesion of scales, which could beexplained by a relaxation effect. Oxide containing Si existed at the steel-scale interface andpromoted adhesion of scales. Oxidation studies were also performed, and the behaviour inwater vapour of steels with different contents of Si and Cu was investigated. Increasing Sicontent tended to decrease oxidation rate. It also resulted in the thickening of the wüstite andfayalite layers which formed by internal oxidation. When Si in steel increased, theintermediate (FeO + Fe3O4) and outermost (Fe2O3 sitting on Fe3O4) layers formed by externaloxidation were thinner. For Cu containing steel, increasing Cu content tended to decrease theoxidation rate. It also decreased the innermost and intermediate layers and resulted in moreCu precipitates along steel-scale interface.

Abstract Hematopoietic cells, i.e., erythrocytes, platelets and white blood cells, differentiate from hematopoietic stem cells in a process that is similar in vertebrates. Hematopoiesis is regulated by molecules expressed by both the hematopoietic stem and progenitor cells and the surrounding microenvironments. Knowledge of these molecules is important since many of the genes involved in normal hematopoiesis are mutated in leukemia. Furthermore, this information can be utilized in more efficient isolation and expansion of hematopoietic cells in vitro. However, these molecules are not yet sufficiently characterized. Transmembrane immunoglobulin and mucin domain (TIM) genes form a known family of immunoregulators. In mammals, TIM-4 is expressed by antigen presenting cells, while TIM-1, TIM-2 and TIM-3 are expressed by T cells, in which they regulate differentiation of TH cells. The role of TIM molecules in hematopoiesis has not yet been investigated. The aim of this thesis work was to identify and analyze novel molecules involved in embryonic hematopoiesis using chicken and mouse as model organisms. This was carried out by generating a cDNA library of hematopoietic stem and progenitor cells from embryonic chicken para-aortic region. Both previously known and novel candidate genes were identified from the library. Among them, we found homologs to tim genes. Their expression and role in hematopoiesis was studied further. TIM-2 expression was shown to be tightly governed during B cell development. It is expressed by common lymphoid progenitors and highly proliferative large-pro and large pre-B cells during both fetal liver and adult bone marrow hematopoiesis. In mouse, tim-4 expression was restricted to fetal liver CD45+F4/80+ cells. Furthermore, two distinct populations were identified: F4/80hiTIM-4hi and F4/80loTIM-4lo. The results suggest that the F4/80hiTIM-4hi cells are yolk sac-derived macrophages and the F4/80loTIM-4lo cells myeloid progenitors. This work shows for the first time that TIM family molecules are expressed during hematopoiesis. TIM-2- and TIM-4 are expressed by specific cell types during hematopoietic cell development, and in the future they may be utilized as markers in isolation of hematopoietic progenitor cells. / Tiivistelmä Verisolut eli punasolut, verihiutaleet ja immuunipuolustuksessa tärkeät valkosolut kehittyvät alkion veren kantasoluista prosessissa, joka on kaikissa selkärankaisissa samankaltainen. Veren kanta- ja esisolujen sekä ympäröivän mikroympäristön tuottamat molekyylit säätelevät hematopoieesia eli verisolujen kehitystä. Näiden molekyylien tunteminen on tärkeää, sillä useat normaalia verisolujen kehitystä säätelevät geenit ovat osallisena myös verisyöpien synnyssä. Lisäksi tätä tietoa on mahdollista hyödyntää verisolujen tehokkaammassa eristämisessä ja kasvattamisessa hoitoja varten. Immuunipuolustuksen solut, kuten syöjäsolut eli makrofagit ja T-solut, ilmentävät TIM-molekyylejä (Transmembrane Immunoglobulin and Mucin). Ne toimivat immunologisen vasteen säätelyssä sekä solusyönnissä, mutta niiden roolia verisolujen kehittymisessä ei ole selvitetty aikaisemmin. Tässä väitöstutkimuksessa etsittiin uusia hematopoieesiin vaikuttavia geenejä käyttäen mallieläiminä sekä kanaa että hiirtä. Tutkimuksessa luotiin geenikirjasto kanan alkion para-aortaalisen alueen veren kanta- ja esisoluista. Kirjastosta tunnistettiin useita ennalta tiedettyjä sekä uusia verisolujen kehitykseen vaikuttavia geenejä. Tutkimuksessa analysoitiin tarkemmin kirjastosta löytyneiden TIM-geeniperheen jäsenten ilmentymistä ja roolia verisolujen kehityksessä. Tutkimuksessa osoitettiin, että TIM-2 proteiinin ilmentymistä säädellään tarkasti B-solujen kehityksen aikana. Lymfosyyttien yhteiset esisolut sekä suuret pro-B- ja pre-B-solut ilmentävät TIM-2 proteiinia B-solukehityksen aikana sekä alkion maksassa että aikuisen luuytimessä. Hiiren alkiossa tim-4 geenin ilmentyminen oli rajoittunut maksaan, jossa erottui kaksi erillistä solupopulaatiota: F4/80hiTIM-4hi ja F4/80loTIM-4lo. Tutkimuksen tulokset viittaavat siihen, että maksan F4/80hiTIM-4hi solut ovat ruskuaispussista lähtöisin olevia syöjäsoluja ja F4/80loTIM-4lo solut myeloidisen linjan esisoluja. Tämä tutkimus on ensimmäinen osoitus TIM-molekyylien ilmentymisestä kehittyvissä verisoluissa. Havaitsimme, että TIM-2 ja TIM-4-molekyylejä ekspressoidaan tietyissä soluissa verisolujen erilaistumisen aikana, joten tulevaisuudessa niitä on mahdollista käyttää merkkiproteiineina hematopoieettisten solujen esiasteita eristettäessä.

Because it can expand the range of a protein’s interactions or modulate its activity, post-translational methylation of arginine residues in proteins must be duly coordinated and ‘decoded’ to ensure appropriate cellular interpretation of this biological cue. This can be achieved through modulation of the enzymatic activity/specificity of the protein arginine methyltransferases (PRMTs) and proper recognition of the methylation ‘mark’ by a subset of proteins containing ‘methyl-sensing’ protein modules known as ‘Tudor’ domains. In order to gain a better understanding of these regulatory mechanisms, we undertook a detailed biochemical characterization of the predominant member of the PRMT family, PRMT1, and of the novel Tudor domain-containing protein 3 (TDRD3). First, we found that PRMT1 function can be modulated by 1) the expression of up to seven PRMT1 isoforms (v1-7), each with a unique N-terminal region that confers distinct substrate specificity, and by 2) differential subcellular localization, as revealed by the presence of a nuclear export sequence unique to PRMT1v2. Second, our findings suggest that TDRD3 is recruited to cytoplasmic stress granules (SGs) in response to environmental stress potentially by engaging in methyl-dependent protein-protein interactions with proteins involved in the control of gene expression. We also found that arginine methylation may serve as a general regulator of overall SG dynamics. Finally, we uncovered that alteration of PRMT1, TDRD3, and global arginine methylation levels in breast cancer cells may be closely associated with disease progression and poor prognosis. Therefore, further studies into the pathophysiological consequences ensuing from misregulation of arginine methylation will likely lead to the development of novel strategies for the prevention and treatment of breast cancer.

Les relais de phosphorylation de type histidine/aspartate constituent des voies de signalisation impliquées dans la perception et la transduction des signaux jusqu’à la mise en place de réponses spécifiques. Ils mettent en jeu un récepteur ou Histidine aspartate Kinase (HK), des protéines navettes en charge de la transmission du phosphate (HPt) et des Régulateurs de Réponse (RR). L’implication d’un tel système dans la transduction du signal de la contrainte osmotique est avérée chez la levure et fortement suspectée chez Arabidopsis. Ce travail de thèse visait d’une part à caractériser l’implication de cette voie de transduction de la contrainte osmotique chez le peuplier, avec l’identification de partenaires HPt et RR en aval du récepteur HK1 et d’autre part à caractériser le mode de fonctionnement d’un RR de type-B. HK1, un osmosenseur membranaire détecterait le signal et le transmettrait à trois HPt préférentielles. De plus, un partenariat d’interaction se dégagerait entre ces trois HPt et certains RR-B. La régulation transcriptionnelle observée lors d’une contrainte osmotique pour deux des représentants des RR-B témoigne d’une possible implication de ces RR dans cette voie. Ces protéines sont des facteurs de transcription dont la fonction a été confirmée in planta pour l’un d’entre eux. La dimérisation du domaine receveur du RR et son interaction avec le domaine de fixation à l’ADN ou domaine GARP apparaissent comme des points de contrôle clés dans la régulation de l’activité effectrice des RR-B. De plus, la capacité d’un RR-B à se fixer sur ses motifs de reconnaissance (boîtes AGAT) a pu être vérifiée in vitro et la présence de ces séquences a d’ailleurs été retrouvée dans des gènes régulés par la contrainte osmotique. Ce travail prospectif ouvre des perspectives concernant l’implication des RR-B dans la voie de transduction du signal de la contrainte osmotique, et propose notamment des mécanismes fins pour l’élaboration d’une réponse hautement spécifique. / Multistep His-to-Asp phosphorelay systems are signaling pathways devoted to signal perception and transduction for establishment of specific responses. These systems are composed of three successive partners: Histidine-aspartate Kinases (HKs), Histidine-containing Phosphotransfer proteins (HPts), and Response Regulators (RRs). One of the best characterized corresponding systems is the osmo-responsive pathway in yeast. Such systems are also suspected in Arabidopsis. This work aimed to characterize the involvement of an osmosensing pathway in Populus by identifying HPt and RR elements downstream of HK1 and to reveal the underlying mechanisms for the activity of a RR-B. HK1, membrane osmosensor, is expected to be responsible for signal detection and propagation by triggering the activation of three preferential HPt. Furthermore, an interacting partnership between those HPts and particular B-type RRs was observed. Two of them appear to be regulated by an osmotic stress, suggesting their possible involvement in this pathway. The B-type RR members, the final output elements of the pathway, act as transcription factors, as shown for at least for one of them in planta. Taken together, the dimerization of the RR receiver domain and its interaction with its DNA binding domain (GARP), are likely key checkpoints in the regulation of RR-B activity. Besides, the ability of one RR-B to bind its cognate specific DNA sequences (AGAT boxes) was confirmed in vitro and those were found in promoters of osmotic response genes. This work opens up prospects for the involvement of RR-B in the osmotic stress signaling pathway and suggests mechanisms tuning induction of specific responses.

Bile is a biofluid synthesized by liver and concentrated in the gallbladder. Interference with the bile flow may cause cholestasis. Primary sclerosing cholangitis (PSC) is an inflammatory cholestatic disorder which eventually may result in liver cirrhosis and failure. The management of PSC is controversial. The only effective treatment for end stage disease is orthotopic liver transplantation (OLT). However, cholangiocarcinoma (CC), which is the major complication of this long-lasting disease, is an absolute contraindication for the surgery. Therefore, early diagnosis of the disease can not only improve the outcome of PSC, but also facilitate the allocation of donated livers to those who can benefit from transplantation. Unfortunately, the diagnosis of CC is challenging. Endoscopic retrograde cholangiopancreatography (ERCP), the gold standard technique, is highly invasive. Non-invasive alternatives such as magnetic resonance cholangiopancreatography (MRCP) have lower accuracy. Therefore, it is essential to develop more accurate and less invasive diagnostic techniques. Magnetic resonance spectroscopy (MRS) is an evolving technique with potential to detect disease-related metabolic changes. In vitro studies have proven the capacity of MRS in the early detection of hepatopancreatobiliary (HPB) disorders based on the metabolic analysis of bile obtained invasively. An in vivo alternative has been attempted by others on human bile within the gallbladder. However, due to the poor quality of the acquired spectra, quantification of most major bile metabolites was not possible, except for choline-containing phospholipids (chol-PLs). In the current study, the quality of the in vivo 1D spectra has been greatly improved, and we have obtained the first 2D L-COSY spectra from bile within the gallbladder. Spectral data from healthy controls and PSC patients were compared. Statistically significant differences in the concentrations of chol-PLs, and glycine- and taurine-conjugated bile acids were revealed in the 1D analysis. Our 2D spectra also demonstrated potential for the detection of metabolic differences between the two groups. The success of these studies indicates a strong potential of in vivo bile MRS techniques to characterize and diagnose a wide variety of HPB disorders. / May 2014

<p>Coeliac disease is a chronic inflammatory disease treated with a gluten-free diet, excluding barley, rye and wheat. Hence, there is a demand for methods able to detect gluten in foods in order to ensure correct labeling of products. According to the Codex Alimentarius Commission, 20ppm gluten is the maximum amount allowed in food labeled gluten-free.</p><p>PCR can detect DNA from cereals in food. Four real-time PCR-systems,</p><p>using TaqMan®-probes for detection of barley, oat, rye and wheat were optimized and evaluated. Evaluations were carried out using seeds. Primers were targeted to genes coding for prolamines, seed storage proteins. PCR-systems targeted to barley, oat and wheat were shown to be specific for the cereals corresponding to each system. The system targeted to rye showed cross-reactions with durum wheat and spelt wheat. Detection limits were 50pg, corresponding to <10 haploid genome copies for each cereal. All systems were able to detect 250ppm amounts of DNA, most likely even smaller amounts are detectable. All systems showed an amplification efficiency of ≥95%.</p><p>Systems for detection of barley, oat and wheat are ready for further evaluation, using food products as samples. The rye system however, needs to be re-designed before further evaluation can take place.</p>

This thesis presents an experimental study of non-thermal atmospheric pressure gas plasmas in presence of liquid as an efficient source of transient and reactive species to initiate chemical reactions necessary for many important applications. Two types of liquid-containing plasmas are considered: discharges formed between a needle electrode and a liquid electrode, and plasma jets formed in a water vapour flow mixed in helium or argon gas. Two plasma modes (the pulsed and the continuous mode) are observed in the needle-to-liquid plasma. A comparative study of the needle-to-liquid plasma in the continuous mode with DC and AC excitations reveals that the plasmas are glow discharges, and AC excited plasmas have the highest energy efficiency. A study of helium/water vapour plasma jet shows that “plasma bullets” are formed even with water vapour in the gas mixture, but become quenched when the moist helium flow rate is above 300sccm (~1800ppm water concentration). Moderate amount of water vapour (~250ppm water concentration) is beneficial for active species production mainly due to the high electron density. Hydrogen peroxide production in saline solution with three different plasma sources is investigated due to the importance of H2O2 in several important applications. Long lifetime of H2O2 in the liquid after plasma treatment indicates an exciting possibility of plasma pharmacy.

This study is divided into three parts. Part I deals with the mechanism of the self-initiated polymerization (or thermal polymerization) of styrene in the gas phase. In this work, we present the first direct evidence for the thermally self-initiated polymerization of styrene in the gas phase. Our approach is based on on-line analysis of the gas phase Oligomers by mass-selected ion mobility. The mobility measurements provide structural information on the ionized oligomers based on their collision cross-sections (Ω) which depend on the geometric shapes of the ions. Theoretical calculations of possible structural candidates of the Oligomers ions are then used to compute angle averaged Ω for comparison with the measured ones. The agreement between the measured and calculated Ω of the candidate structures provides reliable assignments to the structures of the oligomers. Furthermore, collisional-induced dissociations of the mass-selected oligomer ions provide further support for the structures obtained from the mobility measurements. Our results indicate that the gas phase polymerization of styrene proceeds via essentially the same initiation mechanism (the Mayo mechanism) as in condensed phase polymerization. The structural evidence, the mechanism of formation and the observed fragmentation pathway of the growing dimers and trimers in the gas phase are presentedIn Part II the solvation of a variety of metal cations by benzene clusters have been studied using laser vaporization, cluster beam and time-of-flight mass spectrometry techniques. In this work strong magic numbers were observed for clusters containing 10, 13 and 14 benzene molecules depending on the nature of the metal cation involved. The metal cations exhibiting preference solvation by 14 benzene molecules show a strong tendency to form sandwich structures with two benzene molecules. The interpretation of these results in view of the proposed structures and the growth patterns of the clusters are presented. In Part III, the work is focused on the investigation of the intracluster ion molecule reactions following the generation of Mg+ within the polar clusters (water, methanol, ether and acetonitrile).

In recent years, tyrosine kinase inhibitors have been widely used for the treatment of certain tumors as so-called targeted therapy. Many studies are concerned with their metabolism and the role of enzymes in the biotransformation process, but very little is known about the impact of tyrosine kinase inhibitors on the expression and activity of biotransformation enzymes. Nevertheless modification of the expression and activity of enzymes may cause adverse interactions of co-administered drugs and their negative impact on the human body. This diploma thesis studies the effect of tyrosine kinase inhibitors vandetanib and lenvatinib and cytotoxic alkaloid ellipticine on biotransformation enzymes in a rat model organism in vivo. The aim was to characterize the effect of the investigated compounds on gene expression, protein expression and activity of cytochromes P450 (CYP) 1A1, 1A2 and 1B1 and flavin-containing monooxygenases FMO1 and FMO3 in renal and hepatic microsomes. Microsomes and RNA were isolated from kidneys of control rats and the pretreated rats. Western blot and immunodetection was used to compare the protein expression levels of studied enzymes in kidney and liver. By reverse transcription, cDNA was prepared from isolated RNA and used as a template for quantitative PCR to compare the...