abstract: Dealloying induced stress corrosion cracking is particularly relevant in energy conversion systems (both nuclear and fossil fuel) as many failures in alloys such as austenitic stainless steel and nickel-based systems result directly from dealloying. This study provides evidence of the role of unstable dynamic fracture processes in dealloying induced stress-corrosion cracking of face-centered cubic alloys. Corrosion of such alloys often results in the formation of a brittle nanoporous layer which we hypothesize serves to nucleate a crack that owing to dynamic effects penetrates into the un-dealloyed parent phase alloy. Thus, since there is essentially a purely mechanical component of cracking, stress corrosion crack propagation rates can be significantly larger than that predicted from electrochemical parameters. The main objective of this work is to examine and test this hypothesis under conditions relevant to stress corrosion cracking. Silver-gold alloys serve as a model system for this study since hydrogen effects can be neglected on a thermodynamic basis, which allows us to focus on a single cracking mechanism. In order to study various aspects of this problem, the dynamic fracture properties of monolithic nanoporous gold (NPG) were examined in air and under electrochemical conditions relevant to stress corrosion cracking. The detailed processes associated with the crack injection phenomenon were also examined by forming dealloyed nanoporous layers of prescribed properties on un-dealloyed parent phase structures and measuring crack penetration distances. Dynamic fracture in monolithic NPG and in crack injection experiments was examined using high-speed (106 frames s-1) digital photography. The tunable set of experimental parameters included the NPG length scale (20-40 nm), thickness of the dealloyed layer (10-3000 nm) and the electrochemical potential (0.5-1.5 V). The results of crack injection experiments were characterized using the dual-beam focused ion beam/scanning electron microscopy. Together these tools allow us to very accurately examine the detailed structure and composition of dealloyed grain boundaries and compare crack injection distances to the depth of dealloying. The results of this work should provide a basis for new mathematical modeling of dealloying induced stress corrosion cracking while providing a sound physical basis for the design of new alloys that may not be susceptible to this form of cracking. Additionally, the obtained results should be of broad interest to researchers interested in the fracture properties of nano-structured materials. The findings will open up new avenues of research apart from any implications the study may have for stress corrosion cracking. / Dissertation/Thesis / Ph.D. Materials Science and Engineering 2012

Magister Scientiae - MSc / The action of Mung Bean Nuclease (MBN) on DNA makes it possible to clone intact gene fragments from genes of the malaria parasite, Plasmodium. This “genease” activity has provided a foundation for further investigation of the coding elements of the Plasmodium genome. MBN has been reported to cleave genomic DNA of Plasmodium preferentially at positions before and after genes, but not within gene coding regions. This mechanism has overcome the difficulty encountered in obtaining genes with low expression levels because the cleavage mechanism of the enzyme yields sequences of genes from genomic DNA rather than mRNA. However, as potentially useful as MBN may be, evidence to support its genease activity comes from analysis of a limited number of genes. It is not clear whether this mechanism is specific to certain genes or species of Plasmodia or whether it is a general cleavage mechanism for Plasmodium DNA .There have also been some projects (Nomura et al., 2001;van Lin, Janse, and Waters, 2000) which have identified MBN generated fragments which contain fragments of genes with both introns and exons, rather than the intact genes expected from MBN-digestion of genomic DNA, which raises concerns about the efficiency of the MBN mechanism in generating complete genes.Using a large-scale, whole genome mapping approach, 7242 MBN generated genome survey sequences (GSSs) have been mapped to determine their position relative to coding sequences within the complete genome sequences of the human malaria parasite Plasmodium falciparum and the incomplete genome of a rodent malaria parasite Plasmodium berghei. The location of MBN cleavage sites was determined with respect to coding regions in orthologous genes, non-coding intergenic regions and exon-intron boundaries in these two species of Plasmodium. The survey illustrates that for P. falciparum 79% of GSSs had at least one terminal mapping within an ortholog coding sequence and 85% of GSSs which overlapped coding sequence boundaries mapped within 50 bp of the start or end of the gene. Similarly, despite the partial nature of P.berghei genome sequence information, 73% of P.berghei GSSs had at least one terminal mapping within an ortholog coding sequence and 37% of these mapped between 0-50 bp of the start or end of the gene. This indicates that a larger percentage of cleavage sites in both P.falciparum and P.berghei were found proximal to coding regions. Furthermore, 86% of P.falciparum GSSs had at least one terminal mapping within a coding exon and 85% of GSSs which overlapped exon-intron boundaries mapped within 50bp of the exon start and end site. The fact that 11% of GSSs mapped completely to intronic regions, suggests that some introns contain specific cleavage sites sensitive to cleavage and this also indicates that MBN cleavage of Plasmodium DNA does not always yield complete exons. Finally, the results presented herein were obtained from analysis of several thousand Plasmodium genes which have different coding sequences, in different locations on individual chromosomes/contigs in two different species of Plasmodium. Therefore it appears that the MBN mechanism is neither species specific nor is it limited to specific genes. / South Africa

The main objective of the thesis was to gain better understanding of key parameters associated with Cold Work (CW) and their possible effects on Stress Corrosion Cracking (SCC) susceptibility of Austenitic Stainless Steels (ASS) cold rolled to different degrees. The microstructural characterisation of the cold rolled ASS was carried out using optical microscopy to determine and correlate the average grain size with the level of CW. The assessment of martensite development during the CW was carried out using the neutron diffraction technique. The effects of CW levels and strain paths on the lattice strain evolution during the in-situ loading and on the mechanical failure of cold worked ASS were studied. The electrochemical behaviour of cold rolled ASS was also studied. Finally, The SCC susceptibility of ASS was investigated using the Slow Strain Rate Testing (SSRT) techniques. The post-mortem analyses of the failed samples were carried using the optical and Scanning Electron Microscopy (SEM). The study showed that the average grain size decreases with CW and reaches minimum at 20%CW. The smallest and the largest grain size occurred consistently on the Longitudinal (L) and Short-transverse (S) plane respectively. Evidence of martensite development was only found during the plastic deformation at cryogenic temperature and none was observed at ambient temperature. The study showed that the strength of material increases with the level of CW. The Bauschinger effect occurred when the strain path is reversed and its magnitude is independent on whether the tensile or compressive prestraining comes first or last but rather dependent on the amount of CW. The correlation between the CW levels and the lattice strain evolution during the in-situ loading showed that, the lattice strain increases with prestrain and reaches saturation in the material prestrained to 20%CW.The result of the mechanical failure test showed that, 20% cold rolled material loaded along the L and Transverse (T) directions showed a gradual failure, whilst the material loaded along the S direction exhibited a rapid failure. The SEM micrographs suggest that materials loaded along the L and T direction failed with the characteristic features of pure ductile failure while the specimen loaded along S direction showed mixed features of the ductile and brittle failure. The electrochemical properties of the cold rolled materials are more affected by sample orientation than the levels of CW. The short-transverse plane was observed to be most noble whilst the longitudinal plane was the least noble. The results of the SSRT in the chloride environment showed that the plastic elongation, the ultimate tensile strength and the time to failure decrease as the applied potential increases. The post-mortem analysis of the failed samples with the SEM showed that, the fracture surface contained region of ductile failure characterised by dimples, and region of SCC with secondary cracks along the loading axis. Whilst the cross sectional analysis, showed evidence of predominant transgranular stress corrosion cracks. The study found that SCC susceptibility of the ASS is directly linked to strain heterogeneity and directional anisotropy caused by cold working.

Alarmins and damage associated molecular pattern (DAMP) are endogenous proteins with distinct and various intracellular roles that when released extracellularly act as startingsignals for inflammatory immune responses. The endogenous protein High mobility group box 1 (HMGB1) acts as a DAMP and has been shown to drive progression of multiple inflammatory and autoimmune diseases. During homeostasis HMGB1 is localized in the nucleus of almost any cell, where its main function is organization of the DNA and regulation of transcription. Upon cell death or immune cell activation HMGB1 can be translocated into the cytoplasm for subsequent release into the extracellular space. Extracellular HMGB1 can act as a DAMP by activating several receptors of the immune system. Recent studies focus on HMGB1 release and functional regulation due to prost-translational modifications (PTMs) on cysteine residues. However, little is known about enzymatic regulation of HMGB1. The aim of this thesis was to investigate the possibility of proteolytic processing of HMGB1 by enzymes, which play a crucial role in inflammatory diseases and their progression. We utilized an in vitro model that mimics natural conditions of the autoimmune disease arthritis. Enzymatic digestion of HMGB1 was performed in kinetics studies using the neutrophilic enzymes cathepsin G, neutrophil Elastase as well as matrix metalloproteinase-3, which is released from tissues at the site of inflammation. We defined that HMGB1 is a novel substrate of all of the tested enzymes. All enzymes induced different cleavage pattern. In conclusion, my findings open up the possibility for future studies involving the observed fragments of HMGB1 and their functional features. It also demonstrated that HMGB1 is affected by protease modifications in a disease relevant environment.

Chez Arabidopsis thaliana, les gènes d’ARNr 5S sont transcrits par l’ARN polymérase III qui fait intervenir plusieurs facteurs de transcription dont TFIIIA qui reconnaît spécifiquement le promoteur interne de ces gènes et permet ainsi le recrutement de l’ensemble du complexe de transcription. Le gène TFIIIA est composé de sept exons dont le troisième, résultant de l’exonisation d’une molécule d’ARNr 5S, est épissé de façon alternative produisant ainsi deux transcrits. Le transcrit ES, qui ne contient pas cet exon, code pour la protéine TFIIIA pleine longueur et fonctionnelle. Le transcrit EI, dans lequel l’exon « 5S-like » est maintenu, est reconnu et dégradé par la voie NMD (Non-Mediated Decay). L’exon « 5S-like » a, en effet, la particularité de contenir un codon stop prémature qui en fait une cible de la voie NMD. Lors de l’étude de l’expression des transcrits ES et EI ainsi que celle de l’accumulation de la protéine TFIIIA au cours du développement, nous avons montré que le taux de la protéine TFIIIA fonctionnelle est soumis à plusieurs niveaux de régulation. En effet, la production de la protéine TFIIIA pleine longueur est le résultat de la synthèse du transcrit ES et de sa traduction mais également de l’efficacité du clivage protéolytique de la protéine. Lors de la maturation de la graine, l’accumulation croissante de protéine TFIIIA résulte d’une augmentation des quantités de transcrits ES couplée à une diminution du clivage protéolytique. Dans les premiers jours du développement, la protéine TFIIIA n’est détectée qu’après le 4ème jour, suite à la diminution du clivage. Sa présence est corrélée au remodelage de la chromatine de l’ADNr 5S. La combinaison de ces deux mécanismes permet ainsi la production de TFIIIA et de son produit de transcription l’ARNr 5S en fonction des besoins de la plante. / In Arabidopsis thaliana, 5S rRNA genes are transcribed by RNA polymerase III. TFIIIA, specifically required for transcription of these genes, binds to the internal control region of the 5S rRNA genes and allows the assembly of the full transcription complex pol III. The TFIIIA gene consists of seven exons, the third of which results in the exonisation of one 5S rRNA molecule. This exon “5S-like” is alternatively skipped or included to produce either of two transcript isoforms. The ES transcript encodes the fully functional TFIIIA protein. The EI transcript, which contains the exon “5S-like”, is a target of the NMD pathway (Non-Mediated Decay). Indeed, the exon “5S-like” contains a premature stop codon, which is recognized by this RNA decay pathway. During the study of the ES and EI transcripts expression and the TFIIIA protein accumulation throughout the plant development, we show that TFIIIA functional protein levels are under control of many regulation steps. Actually the production of the full-length TFIIIA protein results from production and translation of the ES transcript but also from the proteolytic cleavage efficiency of TFIIIA protein. During the seed maturation, the strong accumulation of TFIIIA results from an increase in ES amounts and a proteolytic cleavage decrease. After the fourth day post-germination, TFIIIA protein is detected because the proteolytic cleavage decreases. TFIIIA presence is correlated with 5S rDNA chromatin reorganization. The combination of these two mechanisms allows TFIIIA production and its transcription product 5S rRNA according to the plants needs.

京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第22085号 / エネ博第393号 / 新制||エネ||76(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー社会・環境科学専攻 / (主査)教授 河本 晴雄, 教授 石原 慶一, 教授 川那辺 洋 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DFAM

In the food industry, it is very important to take care of the quality, safety and organoleptic properties of the products supplied. For this reason, food must be checked. However, not all information can be found using conventional techniques such as immunoassays, chromatographic techniques, etc. DNA-based techniques can be used for these cases where traditional procedures are insufficient. Among them, the best known technique is PCR. The aim of the thesis was to isolate DNA from vegetable samples (broccoli, beetroot, carrot and pepper). DNA was isolated using the magnetic particle method and the traditional CTAB method. Both methods were able to isolate the DNA from the vegetable samples in quality and at a concentration suitable for PCR, where the 35S rDNA gene region was amplified (more precisely about 700 bp of the 18S-ITS1-5,8S region). After amplification, the PCR products were subjected to restriction reactions and the results compared to bioinformatic analysis. These steps have succeeded in finding suitable enzymes for diferentiation of PCR products from the tested vegetable species.

Les microARN (miARN) constituent une classe de petits ARN non-codants qui jouent un rôle clé dans la régulation de l’expression des gènes au niveau post-transcriptionnel. De nombreuses études ont démontré que la surexpression de certains miARN est liée au développement de plusieurs types de cancer. C’est pour cette raison les miARN représentent une nouvelle classe de cibles thérapeutiques à haute potentielle. Dans ce contexte, l’objectif de mon travail de thèse était la découverte de nouveaux inhibiteurs de la production de miARN oncogène. Dans ce but, j’ai suivi deux approches, différentes mais complémentaires : (i) le criblage d’une petite librairie de composés et (ii) la conception et la synthèse de nouveaux conjugués en tant que ligands de précurseurs de miARN (pré-miARN). En particulier, nous avons ciblé les miARN-372 et -373 qui sont oncogènes dans plusieurs cancers, tels que le cancer gastrique. Nous avons ainsi démontré que certains des composés criblés ou synthétisés sont capables de se lier efficacement à la structure secondaire en tige-boucle de pré-miARN avec une haute affinité, conduisant à l’inhibition de la production des miARNs correspondants. Par ailleurs, nous avons montré que certains composés possèdent une activité anti-proliférative spécifique pour les cellules de cancer gastrique et que cette activité est directement liée à une diminution de la production de miARN ciblés et au rétablissement de la traduction de ARN messenger / MicroRNAs (miRs) are a class of small non-coding RNAs that act as regulators of gene expression at the post-transcriptional level. Increasing evidence has indicated that the deregulation of miR expression is linked to various human cancers and therefore, miRs represent a new class of potential drug targets. In this context, my PhD project focused on the discovery of new inhibitors of oncogenic miRs production. Toward this aim, two different but complementary approaches were followed: (i) the screening of small libraries of compounds and (ii) the design and synthesis of new classes of conjugates as binders of miRNA precursors (pre-miRs). In particular, we focused our attention on miR-372 and miR-373, two oncogenic miRs overexpressed in various cancers, such as gastric cancer. We showed that some of the screened or of the newly synthesized compounds are able to efficiently bind to stem-loop structured precursors of the targeted miRs with high affinity, thus inhibiting the production of their corresponding mature miRs at the level of Dicer cleavage. Moreover, we found compounds bearing a specific anti-proliferative activity in gastric cancer cells overexpressing targeted miRs and this activity is directly linked to a decrease in the production of oncogenic miR-372 and -373 and to the restoration of normal mRNA translation.

Parasitic protists with modified mitochondria represent important and exciting group of organisms, not only from the view of eukaryotic cell evolution but also because these parasites are causative agents of serious and widespread diseases. The study and understanding of their biology is thus necessary for the development of new antiparasitic drugs. These organisms reside in host body cavities with low concentrations of oxygen and while they lack typical mitochondria, they possess mitochondrion-related organelles which still integrate many physiologically important processes. Trichomonas vaginalis is an anaerobic flagellate inhabiting mucosal surface of vagina. Instead of canonical mitochondria, T. vaginalis possesses organelles termed hydrogenosomes. These organelles harbor pathways of ATP-generating metabolism via substrate-level phosphorylation, dependent on enzymes prone to oxidative damage, such as pyruvate:ferredoxin oxidoreductase and Fe-Fe hydrogenase. Because the environment of trichomonads is not fully anaerobic, the parasite had to develop complex strategies to cope with both oxygen and reactive oxygen species (ROS) generated by host immune system cells. Recent data from T. vaginalis proteomic and genomic analyses revealed the presence of bacterial-type proteins potentially participating...

Theory of Political Spectrum Proposed New Model of Political Spectrum in the Context of Political Changes Caused by Globalisation Major political changes are happening across Europe and the world during the second decade of the 21 century, represented by the collapse of traditional party systems or Brexit. Current models of political spectrum cannot convincingly explain these changes. In my thesis, I propose a new model of political spectrum which reflects contemporary political changes and contributes to their explanation. Main innovation of the model is the inclusion of political conflict based on globalisation. In the first part of the thesis, I describe historic and modern models of political spectrum including the well-known political compass. I analyse them, critically assess them and form general methodological requirements, which should be met by functional models of political spectrum. In the second part of the thesis, I integrate cleavage theory into the theory of political spectrum. Cleavage theory addresses the most salient political conflicts in political systems. Furthermore, I describe the decline of traditional cleavages and changes in party systems. I dedicate most of my attention to a new cleavage caused by globalisation, described as transnational cleavage. In the third part of the...