This thesis concentrates on the methodology of semiconductor samples preparation for low voltage scanning electron microscopy. In the first part a detailed theory of sample imaging using electron beam and difference between classical scanning electron microscopy (SEM) and low voltage scanning electron microscopy (LVSEM) is described. It is given a description of a contrast formation in SEM and LVSEM and theories describing a contrast formation of differently doped semiconductors. The second part contains experimental data. The advantages and disadvantages of cleavage and focused ion beam (FIB) milling as sample preparation techniques are discussed. FIB was found as the best method for sample preparation for the analysis of precisely defined location on the sample. It is necessary to use the lowest possible FIB accelerating voltage for final polishing, ideally 1 kV.

Recently much attention is given to lanthanides and their complexes as excellent catalysts for cleavage of nucleic acids. The thesis has been focused on the cleavage of plasmid and bacterial DNA by ions Nd3+ and Y3+ and by different carriers containing the lanthanide compounds. The creation of single-stranded nicks and double-stranded ones in the plasmid DNA molecules was studied by agarose gel electrophoresis. Verification of the cleavage of bacterial DNA was made by polymerase chain reaction using primers specific for the domain Bacteria and genus and species-specific primers. The results will be used in the development of the method that will allow perfect carriers`s coverage verification with the magnetic perovskit nucleus and other carriers with the lanthanide compounds.

2013 DIPLOMOVÁ PRÁCE Lukáš Novák Abstract Free-living microaerophilic protist Trimastix pyriformis is closely related to oxymonads which are the largest eukaryotic group without any known mitochondrion. In contrast to oxymonads, an enigmatic reduced mitochondrion has been found in the cell of T. pyriformis. In EST data of T. pyriformis, a number of genes has been identified whose products are putatively localized in the mitochondrion. Among these are genes for all the components of the glycine cleavage system, [FeFe]hydrogenases and the mitochondrial marker Cpn60. We performed experiments in order to determine the cellular localization of these proteins. Our results show that the glycine cleavage system is localized in the mitochondrion. Results of the experiments carried out in order to localize two hydrogenases suggest also the mitochondrial localization but are not fully convincing. The attempt to localize Cpn60 has failed. We have also identified a set of new genes in transcripts of T. pyriformis and Monocercomonoides sp. (Oxymonadida). These genes code for some components of the SUF system of FeS cluster synthesis and a peroxidase rubrerythrin. Key words: Trimastix, Monocercomonoides, mitochondrion, hydrogenosome, mitosome, hydrogenase, glycine cleavage system, SUF system.

This work introduces a database of experimentally verified structures of nucleic acids which were collected from published scientific literature. The database is annotated and the structures are analysed from the perspective of quality and it was found that the experimentally obtained data are not always sufficient - their supporting evidence is often limited and their quality is not convincing. This work also discusses some of the problems, that can be encountered when the structures are experimentally probed. Contents of the database were compared to the RFAM database and despite of its small range it contains 80 new structures. The complete database of 166 structures can be possibly used to optimise software used to predict derived structures of nucleic acids. Furthermore, the work presents several possible ways of improvement of the quality of contained structures.

The current discussion about party-based Euroscepticism forms in the Federal Republic of Germany is usually limited to the Alternative für Deutschland issue and neglect inside-parties forms before its formation. This master thesis examines this type of Euroscepticism in Germany in relevant political parties between 1990 and 2013. This period is defined by the time between German reunification and the formation of the AfD. The term 'Euroscepticism' is conceptualized in the theoretical section, and theories defined by selected scholars are reflected. Subsequently, the theoretical section reflects the perception of Euroscepticism as a thin- centered ideology, its applications in the left-right dimension, and focus on 'new cleavages' GAL/TAN theory. This section emphasizes a deficiency of 'classic' hard-soft Euroscepticism theory and reveals empirical limits of any typologies. The author uses the methodology of content analysis of program papers (PDS/Die Linke, Die Grünen, SPD, and CSU) in the empirical section. Its corpus is defined by the program thesis for Bundestag and European Parliament elections. The coding divides analyzing statements into logical topic groups, which correspond to a subject of research. The synthesis of this coding introduces a clear image of EU attitudes by every single party,...

Every year millions of tons of lignin, a complex biopolymer present in plants that naturally contains aromatic subunits, are produced as a by-product of industries like the food sector and paper sector. Until now it has been considered as a waste but proper valorisation through optimized depolymerization techniques would allow to recover of high-added value fine chemicals and bulk commodities. Vanadium(V) triphenolamine complexes (VO-TPA), developed and studied in the group of Giulia Licini (University of Padova, IT), are of interest in this context as they are efficient catalysts for the oxidative cleavage of carbon-carbon bonds typical of those present in lignin. However, the mechanism of this reaction had not yet been elucidated and the catalysts have exclusively been used in organic solvents. This Thesis is a contribution to the further development of these catalysts and takes different Green Chemistry principles into account such as waste prevention, atom economy, renewable feedstocks, catalysis, energetic efficiency and the use of benign solvents. A first part of this thesis is a contribution to the elucidation of the reaction mechanism. The work was undertaken with vicinal diols, which are the simplest model compounds of lignin. Based on a review of the literature, radical trapping experiments, kinetics, 13C KIE experiments, Hammett plots and multi-parametric correlations, the C-C cleavage of a non-oxo or oxo chelate has been identified as the rate determining step. Attempts were made to synthesise the chelate and identify it via MS. Moreover, three hypothetic pathways were posited to reach this intermediate. Isotopic labelling experiments, determination of reaction activation parameters via an Eyring plot and computations of the different pathways along EPR elucidation of reduced species, have been used to discriminate the operative mechanism. The second part of this thesis is devoted to the transfer of the reaction into water, which can be considered a benign solvent. The VO-TPA complexes were successfully transferred to water using both non-ionic TPGS-750-M and zwitterionic DPC micelles. The latter maintained the catalyst stability over a broad pH range and temperature range which was not the case with the non-ionic micelles. The substrate scope was evaluated with the DPC micelles and a strong selectivity was observed for hydrophobic substrates. After scaling up the process to reach standards used in the literature, mixed DPC/TPGS micelles were characterized and used as a simple solution to combine the pH stability of the former and extractive properties of the latter. This allowed to completely recycle the micellar phase while maintaining good yields. / Doctorat en Sciences de l'ingénieur et technologie / info:eu-repo/semantics/nonPublished

Le facteur neurotrophique dérivé du cerveau (BDNF, Brain-Derived Neurotrophic Factor) est une protéine qui joue un rôle essentiel dans la survie et la différenciation des neurones, ainsi que dans l'induction et l'expression de la plasticité synaptique (Deinhardt and Chao, 2014; Lu et al., 2005). Le BDNF est très exprimé à l'âge adulte et la réduction de son expression est impliquée dans de nombreuses maladies neurodégénératives et troubles psychologiques (Anastasia and Hempstead, 2014). Son action sur la plasticité synaptique est majeure pour la mise en place de fonctions cognitives et pour la mise en place de la mémoire (Bekinschtein et al., 2008; Egan et al., 2003).Le BDNF existant sous deux formes ayant des fonctions opposées, on peut parler d'effet Yin et Yang du BDNF (Lu et al., 2005). En effet, le BDNF est synthétisé en tant que molécule précurseur, le proBDNF, qui a des effets négatifs " Yin ". Par sa liaison au récepteur p75NTR, le proBDNF favorise l'apoptose, la rétraction dendritique et la dépression à long-terme. Au contraire, la forme clivée, le BDNF mature (mBDNF), se lie préférentiellement au récepteur TrkB qui active les voies de signalisation de survie cellulaire, de différentiation et de potentialisation à long terme. On parle alors d'effets positifs ou " Yang ". Le clivage de BDNF joue donc un rôle capital dans la régulation de sa balance fonctionnelle vers l'une ou l'autre voie. Il est généralement admis que le proBDNF est clivé en mBDNF par la furine dans le Golgi ou par la pro-protéine convertase 1/3 dans les vésicules de sécrétion (Mowla et al., 2001; Seidah et al., 1996). Le proBDNF restant n'ayant pas été clivé dans la cellule peut être sécrété et être rapidement clivé par la plasmine ou les métalloprotéases de matrice (MMP7) dans l'espace extracellulaire (Lee et al., 2001). Cependant, si les enzymes de conversion du BDNF sont bien connues, les mécanismes de régulation du clivage ne sont pas encore compris.Nous montrons ici un nouveau mécanisme de régulation de la maturation de BDNF via une phosphorylation qui impacte directement la balance fonctionnelle. En effet, nous montrons que la phosphorylation du résidu S130, localisé à l'interface entre le pro-domaine et le domaine mature, diminue l'efficacité du clivage du BDNF par la furine, régulant ainsi l'équilibre entre les formes immature et mature. Cette phosphorylation au site S130 est catalysée par les ectokinases FJX1 et FAM69B qui sont localisées dans l'appareil de Golgi avec BDNF. De plus, grâce à l'utilisation de souris transgéniques knock-in phospho-mutantes, nous montrons que la phosphorylation de BDNF, en favorisant la forme proBDNF, inhibe la potentialisation à long-terme et diminue la dynamique de plasticité des épines dendritiques après stimulation neuronale. Ces résultats suggèrent ainsi une nouvelle voie de régulation de la balance fonctionnelle de BDNF et suggèrent un rôle critique de la phosphorylation S130 dans les processus d'apprentissage et de la mémoire.En parallèle, la recherche de kinases potentielles de BDNF nous a amenés à identifier une exoPKA, localisée dans le Golgi et qui interagit directement avec BDNF. Cette exoPKA atypique phosphoryle BDNF au niveau de la sérine S130 et régule sa maturation et donc l'équilibre entre les formes pro et matures de BDNF. Cette exoPKA est différente de la PKA cytosolique ce qui suggère l'existence d'un nouveau mécanisme de régulation de la plasticité par PKA via BDNF. Enfin, nous sommes en train de tester le rôle de l'exoPKA sur la régulation négative de la plasticité synaptique via la régulation du clivage de BDNF. Ces résultats permettront de déterminer l'existence d'une dichotomie d'action de PKA sur la plasticité en fonction de la forme activée, cytosolique ou golgienne/sécrétée. / Brain-Derived Neurotrophic Factor (BDNF) is a protein that plays an essential role in the survival and differentiation of neurons, as well as in the induction and expression of synaptic plasticity (Deinhardt and Chao, 2014; Lu et al., 2005). BDNF is highly expressed during adulthood and the reduction of its expression is implicated in many neurodegenerative diseases and psychological disorders (Anastasia and Hempstead, 2014). Its action on synaptic plasticity is critical for the establishment of cognitive functions and for the establishment of memory (Bekinschtein et al., 2008; Egan et al., 2003).BDNF exists as two forms with opposite functions, or Yin and Yang effects (Lu et al., 2005). Indeed, BDNF is synthesized as a precursor molecule, proBDNF, which has negative effects "Yin". By binding to the p75NTR receptor, proBDNF promotes apoptosis, dendritic retraction and long-term depression. In contrast, the cleaved form, mature BDNF (mBDNF), binds preferentially to the TrkB receptor that activates the signaling pathways promoting cell survival, differentiation, and long-term potentiation. Thus, mBDNF has positive effects or "Yang". The cleavage of BDNF therefore plays a key role in regulating its functional balance towards one or the other pathway. It is believed that proBDNF is cleaved into mBDNF by furin in the Golgi network or by pro-protein convertase 1/3 in secretory vesicles (Mowla et al., 2001; Seidah et al., 1996). The remaining proBDNF that has not been cleaved in the cell can be secreted and rapidly cleaved by plasmin or matrix metalloproteases (MMP7) into the extracellular space (Lee et al., 2001).Having diametrically opposite roles, together, mature BDNF and proBDNF allow fine regulation of neuronal survival and differentiation and of their activity-dependent synaptic plasticity (Lu et al., 2014; Yang et al., 2014). Since BDNF has critical roles in neuronal functions, it is not surprising that its expression and action are extremely regulated in time and space. However, even if the enzymes processing BDNF are well known, the mechanisms regulating BDNF cleavage are not yet understood.Here we show a new mechanism for the regulation of BDNF maturation via phosphorylation which directly impacts the functional balance. Indeed, we show that phosphorylation of the S130 residue, located at the interface between the pro- and the mature domain, decreases the efficiency of BDNF cleavage by furin, thus regulating the balance between immature and mature forms. This phosphorylation at the S130 site is catalyzed by ectokinases FJX1 and FAM69B which are localized in the Golgi apparatus with BDNF. Furthermore, by using phospho-mutant knock-in transgenic mice, we show that the phosphorylation of BDNF, by promoting the proBDNF form, inhibits the long-term potentiation and decreases the plasticity dynamics of the dendritic spines after neuronal stimulation. These results suggest a novel regulatory pathway for BDNF functional balance and suggest a critical role for S130 phosphorylation in learning and memory processes.In parallel, the search for potential kinases of BDNF led us to identify an exoPKA, located in the Golgi and interacting directly with BDNF. This atypical exoPKA phosphorylates BDNF at serine S130 and regulates its maturation and therefore the balance between the pro- and mature forms of BDNF. This exoPKA is different from the cytosolic PKA which suggests the existence of a new mechanism of regulation of plasticity by PKA via BDNF. Finally, we are testing the role of exoPKA on the downregulation of synaptic plasticity via the regulation of BDNF cleavage. These results will allow to determine the existence of a dichotomous action of PKA on plasticity whether cytosolic or Golgi/secreted PKA is activated.

EPHB2, an ephrin receptor (EPH) from receptor tyrosine kinase (RTK) family, is one of the substrates for tissue factor (TF) - coagulation factor VIIa (FVIIa) complex and it is cleaved in its ectodomain. EPHB2 cleavage is important for ephrin receptor (EPH) - ephrin ligand (EFN) signaling and cell repulsion. TF has been reported to be overexpressed in different cancer types such as breast and colorectal cancer (CRC). Furthermore, EPHB2 R155C mutation, at the TF/FVIIa-mediated cleavage site, has been identified as one of the somatic mutation sites in human metastatic CRC. Therefore, the aim of the present work was to characterize the EPHB2 R155C mutation and its effect on the cleavage by TF/FVIIa on EPHB2 in context to CRC. We generated overexpression cell models for EPHB2 wild type (wt) and R155C mutant in human CRC DLD-1 cell line for in vitro compartmentalization assay analysis to demonstrate repulsion event in EPH-EFN signaling. Whereas low endogenous TF expression led to incomplete cleavage of EPHB2 wt protein, stable overexpression of TF resulted in complete cleavage. Moreover, overexpression of TF resulted in reduced compartmentalization in EPHB2 wt cells after FVIIa treatment. Transient expression of TF in EPHB2 wt and R155C cells showed no clear difference in EPHB2 cleavage. Interestingly, it was difficult to obtain similar stable overexpression level of TF in EPHB2 R155C cells compared to EPHB2 wt cells. This may lead to further research in context to the role of TF/FVIIa-mediated EPHB2 cleavage in CRC by the generation of TF overexpression cell lines using lentiviral transduction.

No description available.

Mass spectrometers have proven to be vital to understanding the Solar System and the planets within it. Spacecraft containing mass spectrometers have been sent to numerous remote places and have determined important information about the atmospheric composition of Venus, Earth, Mars, Jupiter, and Saturn, along with other celestial bodies. Such results have shown a variety of small neutral molecules, such as CH4 NH3, H2O, CO2, and CO, neutral radicals such as atomic O, H, and N, and a host of small ions, such as H+, N+, and NH4+. Closed ion source inlets, which allow for the detection of these small neutral molecules, contain a spherical antechamber that allows the neutrals to thermalize with the walls of the chamber through many successive collisions before they are introduced into the ionization region of the spacecraft mass spectrometer. These collisions, however, energetically excite neutral molecules and lead to many chemical changes, such as racemization, ionization, or even dissociation. When these changes occur, smaller neutrals can be produced, even if they were not in the original sample from the atmosphere or surface. As a result, the determination of the true composition of an atmosphere or a surface is cast into doubt. Herein is given a brief description of mass spectrometry in space research and how the closed ion source has greatly assisted this process. Dissociation and other chemical changes caused by the high velocity impacts that occur in closed source antechambers is also addressed. A theoretical approach to understanding such dissociative processes that occur after high energy collisions in closed source antechambers is described and undertaken. Chapter 2 describes a proof-of-concept study using hexane as a representative molecule and determines the velocity at which widespread dissociation of hexane molecules is likely to occur in closed source antechambers. This same theoretical process is then utilized in Chapter 3 with many more members of the n-alkane family to probe what effect molecular weight has on the amount of dissociation. Alkanes of both higher and lower molecular weight than hexane (C6H14) are used to show the effect as a function of molecular weight. In all cases, it was found that the velocity at which half of the incoming neutral n-alkane molecules dissociate is roughly the same for all molecular weights studied. This result is then applied to current and future space research through a proposed hardware solution, which will reduce the amount of dissociation and a discussion of how this effect may be seen in the results obtained from future mission instruments. Lastly, future work with different molecular weights and with successive collisions (the second, third, fourth, etc.) is described. This future work will further expand the present study to show how different functional groups, which may be partly responsible for higher-than-expected levels of NH3 and CO2, are affected after a high velocity, high energy impact.