Les Rétinal déshydrogénases (RALDHs) catalysent irréversiblement la déshydrogénation du Rétinal en Acide Rétinoïque (AR) qui est impliqué dans l’embryogenèse et la différenciation tissulaire. Pour comprendre le rôle dans la biosynthèse de l’AR des RALDHs type 3 et 4 de souris, nous avons déterminé leurs propriétés cinétiques ainsi que leur comportement en présence de différents inhibiteurs. Les tests enzymatiques sont effectués avec une préparation d’enzyme recombinante, tagguée avec 6 histidines, purifiée sur colonne Ni-NTA (Qiagen). L’activité enzymatique est évaluée en quantifiant la production d’AR par chromatographie liquide à haute performance (HPLC) en phase inversée. Les constantes cinétiques ont été déterminées pour les isomères du rétinal tout-trans, 9-cis et 13-cis. La RALDH4 catalyse les isomères 9-cis et 13-cis de rétinal, elle présente un faible KM (3μM) pour les deux isomères et a une efficacité catalytique élevée pour le 9-cis rétinal 3.4 fois supérieure au 13-cis rétinal. La RALDH3 est spécifique au tout-trans rétinal avec un KM de 4 μM et une efficacité élevée. β-Ionone, inhibiteur possible pour la RALDH4, inhibe l’activité avec le rétinal 9-cis et 13-cis, mais n’influence pas l’activité de la RALDH3. Le para-hydroxymercuribenzoïque (p-HMB) inhibe l’activité de deux isoenzymes. Le cation MgCl2 augmente par 3 fois l’oxydation du rétinal 13-cis par la RALDH4, diminue l’oxydation du 9-cis rétinal et influence faiblement la RALDH3. Ces données enrichissent les connaissances sur les caractéristiques cinétiques des RALDHs recombinantes de souris de types 3 et 4 et fournissent des éclaircissements sur la biogenèse de l’acide rétinoïque in vivo. / SUMMARY Retinal dehydrogenases (RALDHs) catalyze the dehydrogenation of retinal into retinoic acids (RA) that are required for embryogenesis and tissue differentiation. This study sought to determine the detailed kinetic properties of 2 mouse RALDHs, namely RALDH3 and 4, for retinal isomer substrates, to better define their specificities in RA isomer synthesis. RALDH3 and 4 were expressed as His-tagged proteins and affinity-purified. RALDH3 oxidized all-trans retinal with high catalytic efficiency but did not show activity for either 9-cis or 13-cis retinal substrates. RALDH4 was inactive for all-trans retinal substrate, exhibited high activity for 9-cis retinal oxidation, and oxidized 13-cis retinal with lower catalytic efficiency. β-ionone, a potent inhibitor of RALDH4 activity, suppressed 9-cis and 13-cis retinal oxidation competitively, but had no effect on RALDH3 activity. The p-HMB inhibited the activity for both RALDH3 and RALDH4. The divalent cation MgCl2 activated 13-cis retinal oxidation by RALDH4 by 3-fold, slightly decreased 9-cis retinal oxidation, and did not significantly influence RALDH3 activity. These data extend the kinetic characterization of RALDH3 and 4, providing their specificities for retinal isomer substrates, which should help in determining their functions in the synthesis of RAs in specific tissues.

Exosomes are small secreted vesicles of endocytic origin with a size range of 50-150 nm. They are secreted by many cell types and display multiple biological functions including immune-activation, immune-suppression, antigen presentation, and the shuttling of mRNA and miRNA, as well as other cargo. We have characterised the exosomes secreted from two breast cancer cell lines, MDA-MB-231 and MCF7. Exosomes secreted from both cell lines display typical markers including ALIX, Tsg101, CD9 and CD63, and were capable of inducing apoptosis of the Jurkat T cell line, indicating the potential immune-suppressive function of such tumour-derived exosomes. To further investigate the biological potential of exosomes, we loaded purified exosomes with gene specific siRNAs using electroporation, and observed the targeted inhibition of both a known component of the exosome pathway, Rab27a, and also the arthritis associated gene ERAP1, demonstrating the potential novel use of exosomes as therapeutic gene delivery vectors. We have also shown that exosomes derived from MDA-MB-231 cells and the parental cells have different lipid composition, as analysed by lipidomics study. Nanoparticle tracking analysis (NTA), which allows the rapid detection of size and concentration of nanoparticles within the size range 10 nm-1000 nm was tested for its ability to accurately measure size and concentration of exosomes and microvesicles under different conditions. NTA was capable of detecting apoptotic vesicles induced by Taxol and Curcumin treatment. Immunodepletion was used to determine the percentage of CD9 and CD63 positive vesicles. Our data suggest that NTA is a useful technique for measuring size and concentration of exosomes and microvesicles. We hypothesized that NTA could assist in the screening of agents that interfere or promote exosome release. NTA was therefore used to detect increases in exosomes secretion induced by Tamoxifen and Thimerosal treatment, and to monitor the inhibition of exosome secretion from MDA-MB-231 breast cancer cells expressing inhibitory RNA targeted for Rab27a, a component of the exosome pathway. Increases in exosome release induced by Tamoxifen and Thimerosal was detected by NTA and a significant reduction in the release of exosomes by inhibition of Rab27a expression was also observed. Treatment with the known exosomal pathway inhibitor DMA also reduced exosome release, establishing the principle of NTA as a screening technique. We further compared the siRNA targeted cells for their ability to migrate, invade and form anchorage-independent colonies, which were all significantly reduced. Supplementation with MDA-MB-231 derived exosomes restored the ability to form colonies, suggesting exosomes may contribute to metastatic lesion formation. These data suggest that the exosomal pathway is a valid target to disrupt the behaviour of tumour cells and NTA can be used to monitor its activity.

<p>Several plant defense systems consist of enzymes that act on glucosides and produce a toxic compound. In the intact plant tissue the substrate and enzyme are kept apart. The system studied here consists of the substrate 2-O-β-D-glucopyranosyl-4-dihydroxy-1,4-benzoxazin-3-one and the enzyme glucan 1,3-β-glucosidase in rye. The aim was to determine the properties of a cell wall β-glucosidase. Two different systems for expression and purification of β-glucosidase fused to a tag were used: a 6xHistidine tag system and a thioredoxin tag system. The sequence of the β-glucosidase had previously been determined so now the gene was subcloned into E.coli. A direct PCR on colonies, a test expression, a restriction digestion of plasmids and sequencing was made to analyze the transformation, which all turned out successful. Then the β-glucosidase solubility was determined. Finally a purification of the β-glucosidase from E.coli under native conditions and a pNPG assay was carried out. For the (His)6-tagged protein, the recombinant β-glucosidase tended to end up in the insoluble pelleted fraction which indicated formation of inclusion bodies. The cell wall 1,3-β-glucosidase was soluble with the thioredoxin system, but the percentage of soluble protein fraction was around 5% only of the total protein. In eluates from a nickel-nitrilotriacetic acid column the presence of recombinant protein was confirmed with Western blot, but contaminating bands were also present. Purified elauted fractions did not exhibit detectable β-glucosidase activity. It was not possible to purify active enzyme. From a BLAST search it was clear that the most similar enzymes all had putative glycosylation sites and lack of glycosylation could be a reason for the protein not to fold properly.</p>

Several plant defense systems consist of enzymes that act on glucosides and produce a toxic compound. In the intact plant tissue the substrate and enzyme are kept apart. The system studied here consists of the substrate 2-O-β-D-glucopyranosyl-4-dihydroxy-1,4-benzoxazin-3-one and the enzyme glucan 1,3-β-glucosidase in rye. The aim was to determine the properties of a cell wall β-glucosidase. Two different systems for expression and purification of β-glucosidase fused to a tag were used: a 6xHistidine tag system and a thioredoxin tag system. The sequence of the β-glucosidase had previously been determined so now the gene was subcloned into E.coli. A direct PCR on colonies, a test expression, a restriction digestion of plasmids and sequencing was made to analyze the transformation, which all turned out successful. Then the β-glucosidase solubility was determined. Finally a purification of the β-glucosidase from E.coli under native conditions and a pNPG assay was carried out. For the (His)6-tagged protein, the recombinant β-glucosidase tended to end up in the insoluble pelleted fraction which indicated formation of inclusion bodies. The cell wall 1,3-β-glucosidase was soluble with the thioredoxin system, but the percentage of soluble protein fraction was around 5% only of the total protein. In eluates from a nickel-nitrilotriacetic acid column the presence of recombinant protein was confirmed with Western blot, but contaminating bands were also present. Purified elauted fractions did not exhibit detectable β-glucosidase activity. It was not possible to purify active enzyme. From a BLAST search it was clear that the most similar enzymes all had putative glycosylation sites and lack of glycosylation could be a reason for the protein not to fold properly.

Jag har valt att med hjälp av en kvalitativ metod undersöka hur lärare arbetar eller har arbetat med Natur och Teknik för Alla (NTA), som är ett verktyg till för att stödja lärares kompetensutveckling och undervisning i naturvetenskap och teknik (Anderhag &amp; Wickman 2006). NTA inkluderar fortbildning, ett nätverk av pedagoger, samt tillgång till temalådor att ta med in i klassrummet som bygger på ett undersökande och praktiskt arbetssätt. Syftet med undersökningen är att få ta del av hur lärare väljer att arbeta med NTA i NO-undervisningen. Vidare undersöks om lärarna upplever att NTA underlättar genomförandet av praktiska inslag i undervisningen, om NTA synliggör kopplingen mellan teori och praktik, samt om de anser att NTA höjer kvaliteten på deras planering och undervisning. Undersökningen baseras på sex genomförda intervjuer med lärare som undervisar i årskurserna 1-6.I Bakgrund tydliggörs vad NTA är för något; var det har sitt ursprung, vad NTA-konceptet innebär, hur man undervisar på klassrumsnivå samt hur relationen mellan NTA och de aktuella styrdokumenten ser ut. Förutom detta finns ett avsnitt som förklarar experiment och praktiskt arbete i NO-undervisningen.I Tidigare forskning presenteras tidigare analyser av NTA, Skolinspektionens kvalitetsgranskning av NO-undervisningen och Skolverkets kunskapsöversikt över NO-undervisningen. I detta kapitel redogörs likaså för det teoretiska perspektivet, konstruktivism, som ligger till grund för denna undersökning. Konstruktivism innebär att det finns en bestämd syn på individers lärande och på den organiserade kunskap som överförs från en generation till en annan (Sjøberg, 2010)Mina huvudsakliga resultat från de genomförda intervjuerna är att samtliga lärare tycker att NTA-undervisningen stödjer deras utförande av praktiska moment. De tycker även att NTA förenklar arbetet med att sträva mot det som står i kursplanerna, men fem av sex lärare anser att NTA inte är heltäckande i det avseende att det täcker vad som står i styrdokumenten. Detta medför att man måste vara noggrann i sin planering och undervisning. De sex lärarna berättar likaså att de inte väljer att fullfölja den lärarhandledning som finns att tillgå fullt ut i sin undervisning.

Uppsatsens syfte är att undersöka om NTA, ett undervisningsmaterial som går ut på att tillhandahålla planeringar och material för praktisk NO-undervisning, skapar ett undersökande arbetssätt utifrån naturvetenskapens karaktär. Tidigare forskning är positiv till NTA-materialet men visar att ett undersökande arbetssätt inte blir en självklarhet. Genom en kvalitativ enkätundersökning i fyra skolklasser och intervjuer med två lärare, som undersöker elever och lärares syn på NTA-materialet, kommer vi fram till att ett undersökande arbetssätt är möjligt men att undervisande lärare aktivt måste se till att det sker.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18303号 / 工博第3895号 / 新制||工||1598(附属図書館) / 31161 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 濵地 格, 教授 森 泰生, 教授 跡見 晴幸 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Computer security is to protect the data inside the computer, relay the information, expose the information, or reduce the level of security to some extent. The communication contents are the main target of any malicious intent to interrupt one or more of the three aspects of the information security triad (confidentiality, integrity, and availability). This thesis aims to provide a comprehensive idea of network traffic analysis, various anomaly or intrusion detection systems, the tools used for it, and finally, a comparison of two Network Traffic Analysis (NTA) tools available in the market: Splunk and Security Onion and comparing their finding to analyse their feasibility and efficiency on Anomaly detection. Splunk and Security Onion were found to be different in the method of monitoring, User Interface (UI), and the observations noted. Further scope for future works is also suggested from the conclusions made.

Characterising the geochemistry and speciation of major contaminant radionuclides is crucial in order to understand their behaviour and migration in complex environmental systems. Organic complexing agents used in nuclear decontamination have been found to enhance migration of radionuclides at contaminated sites; however, the mechanisms of the interactions in complex environments are poorly understood. In this work, radionuclide speciation and sorption behaviour were investigated in order to identify interactions between four key radionuclides with different oxidation states (Cs(I) and Sr(II) as important fission products; Th(IV) and U(VI) as representative actinides), three anthropogenic organic complexing agents with different denticities (EDTA, NTA and picolinic acid as common co-contaminants), and natural sand (as simple environmental solid phase). A UV spectrophotometric and an IC method were developed to monitor the behaviour of EDTA, NTA and picolinic acid in the later experiments. The optimised methods were simple, applied widely-available instrumentation and achieved the necessary analytical figures of merit to allow a compound specific determination over variable background levels of DOC and in the presence of natural cations, anions and radionuclides. The effect of the ligands on the solubility of the radionuclides was studied using a natural sand matrix and pure silica for comparison of anions, cations and organic carbon. In the silica system, the presence of EDTA, NTA and, to a lesser extent, picolinic acid, showed a clear net effect of increasing Th and U solubility. Conversely, in the sand system, the sorption of Th and U was kinetically controlled and radionuclide complexation by the ligands enhanced the rate of sorption, by a mechanism identified as metal exchange with matrix metals. Experiments in which excess EDTA, NTA and picolinic acid (40 – 100 fold excess) were pre-equilibrated with Th and U prior to contact with the sand, to allow a greater degree of radionuclide complex formation, resulted in enhanced rates of sorption. This confirmed that the radionuclide complexes interacted with the sand surface more readily than uncomplexed Th or U. Overall this shows that Th and U mobility would be lowered in this natural sand by the presence of organic co-contaminants. In contrast, the complexation of Sr with the complexing agents was rapid and the effect of the ligands was observed as a net increase on Sr solubility (EDTA, picolinic acid) or sorption (NTA). As expected, Cs did not interact with the ligands, and showed rapid sorption kinetics. Finally, ESI-MS was used to study competitive interactions in the aqueous Th-Mn-ligand ternary system. Quantification presented a challenge, however, the careful approach taken to determine the signal correction allowed the competitive interactions between Mn and Th for EDTA to be studied semi-quantitatively. In an EDTA limited system, Th displaced Mn from the EDTA complex, even in the presence of a higher Mn concentration, which was consistent with the higher stability constant of the Th-EDTA complex.

Yaku'amide A shows a unique inhibitory profile against a series of 39 human cancer cell lines (JFCR39). In our efforts to synthesize yaku'amide A, we have optimized our regioselective base-free aminohydroxylation method with a series of nitrogen sources, developed a chiral reagent-mediated aminohydroxylation strategy and chemoselective deprotections of the resulting aminohydroxylation product, and explored a stereospecific E2 dehydration and O-N acyl transfer sequence. In addition, we have prepared the right-hand tetrapeptide and the NTA subunit. For our bulky α,β-dehydroamino acids project, we have developed strategies to incorporate α,β-dehydroamino acids such as ΔVal and ΔEnv into small synthetic peptides via Solid Phase Peptide Synthesis (SPPS). We have also prepared two analogues of a monomeric helical peptide with 13 residues.