La particule de reconnaissance du signal (signal recognition particle-SRP) et son récepteur (FtsY chez Escherichia coli) médiatise le processus simultané de traduction-ciblage de la protéine en dirigeant le complexe ribosome-nascent chain (RNCs) vers la membrane de destination. La reconnaissance par la SRP d'une charge RNC à transporter dépend de la présence de la partie N-terminale. L'assemblage de Ftsy au complexe RNC-PRS entraine plusieurs changements de configuration de SRP et de FtsY durant le cycle de direction. D'abord un stade « précoce » sans GTP est adopté. Celui-ci est stabilisé par le RNC. Ensuite une configuration « fermée » avec GTP est formée. Cette dernière peut s'activer pour hydrolyser GTP, elle entre alors dans sa configuration « active ». La succession de ces trois étapes conduit à la libération du complexe SRP-récepteur d'avec le ribosome et de sa protéine en cours de traduction, et leur mise à disposition au pore de la membrane. Dans ce projet, notre intérêt se limite à la traduction par le ribosome de la séquence signale EspP (RNCEspP). In vivo, EspP est une protéine dont le ciblage vers le récepteur membranaire se réalise après la traduction. Cependant il arrive que RNCEspP se lie au complexe SRP-FtsY, faisant échouer le ciblage. Nous avons étudié les bases structurales du rejet de RNCEspP par SRP et FtsY. Pour cela nous avons effectué la comparaison de la structure RNCEspP-SRP-FtsY obtenue par observation au cryo-microscope électronique avec d'autres complexes ribosome-SRP-récepteurs traduisant la charge FtsQ, qui est elle normalement ciblé par SRP. Nous avons cherché à observer la différence de structure entre les complexes SRP-FtsY dans les deux cas. Deux différences majeurs entre les complexes de ciblages contenants les séquences RNCFtsQ et RNCEspP ont été observés. Premièrement, dans le cas de la structure de RNCEspP le domaine M -Ffh est attaché à l'hélice 59 du ribosome, alors que celui-ci est détaché dans le cas de la structure de RNCFtsQ. Nous pensons que le domaine M empêche la libération de la séquence de signal, étape nécessaire à la réalisation du ciblage. Deuxièmement, dans le cas de la structure du complexe avec RNCEspP l'arrangement Ffh-FtsY avec le domaine NG était flexible. Ceci indiquerait que le complexe “précoce” formé sur RNCEspP est moins stable que celui formé sur RNCFtsQ. Une étude biochimique utilisant le transfert d'énergie via résonance fluorescente a corroboré ce résultat, montrant que FTS Y est lié avec une affinité moindre dans le cas du complexe précoce formé sur RNCEspP et que la reconfiguration au stade de complexe fermé est moins efficace. Une analyse biochimique plus poussée des variantes de la séquence de EspP montre que la partie N-Terminale de la séquence est la principale cause de rejet du cycle de ciblage via SRP.Dans un second projet, nous avons étudié la configuration “fermée” de SRP et ftsY en complexe avec une charge RNC stabilisée par un analogue non-hydrolysable de GTP (GMP-PCP). Pour franchir la barrière cinétique qui permet de passer du complexe précoce au complexe fermé, nous avons utilisé une version tronquée de FtsY, à laquelle la séquence terminale avait été amputée de tout le domaine acide (A-) ainsi que de la première hélice alpha du domaine NG. De plus, pour la formation du complexe, nous avons utilisé une construction contenant les 50 premiers acides aminés du leader peptidase (RNCLep50). En l'absence de nucléotides, notre reconstruction au cryo-EM a montré une configuration similaire à celle du stade précoce, dans laquelle Ftsy et Ffh- domaine NG, sont proche du tetraloop de la 4.5 S ARN. Une incubation avec GMP-PCP induit un détachement du domaine NG d'avec la queue du tetraloop. Il semblerait que les domaines NG soient flexibles dans l'état clos, et non attaché à la terminaison ouverte de l'ARN. / The signal recognition particle (SRP) and its receptor (FtsY in Escherichia coli) mediate co-translational protein targeting by delivering ribosome nascent chain complexes (RNCs) to the target membrane. Recognition of an RNC cargo by SRP is dependent on an N-terminal signal sequence. Binding of FtsY to the RNC-SRP complex leads to several conformational changes of SRP and FtsY during the targeting cycle: first, an “early” GTP-independent state is adopted which is stabilized by the RNC, subsequently a “closed” GTP- dependent conformation is formed which can activate itself to hydrolyze GTP (the “activated” state). Faithful completion of all three steps leads to release of the cargo from SRP-FtsY and hand over of the RNC to the translocation pore.It has been shown for E. coli that cargos can be rejected from the SRP pathway during all targeting steps. In the first project, our interest concentrates on ribosomes translating the EspP signal sequence (RNCEspP). In vivo, EspP is a post-translationally targeted protein, but RNCEspP has been shown to be bound by SRP and FtsY leading to a non-productive “early”-like RNCEspP-SRP-FtsY complex. Using single particle cryo-electron microscopy (EM), we analysed the structural basis for the rejection of RNCEspP by SRP and FtsY. Comparison of our RNCEspP-SRP-FtsY cryo-EM structure to other available cryo-EM structures of co-translational targeting complexes containing the correct cargo RNCFtsQ unravelled differences in the SRP-FtsY structure between a correct cargo and an incorrect cargo. Two major differences between the targeting complexes containing the cargos RNCFtsQ and RNCEspP were observed: first, the Ffh M-domain was attached to ribosomal RNA helix 59 of RNCEspP, while it was detached from this site in the case of RNCFtsQ. It could be that such an ordered M-domain is hampering the release of the signal sequence which is required for successful completion of targeting. Second, the Ffh-FtsY NG-domain arrangement was flexible in the complex with RNCEspP in comparison to RNCFtsQ indicating that the "early"-like complex formed on RNCEspP is less stable. Biochemical data using fluorescence resonance energy transfer corroborated these results, showing that FtsY is bound with lower affinity in the RNCEspP “early” complex and that the rearrangement to the “closed” conformation is less efficient. Further biochemical analysis of EspP signal sequence variants showed that mainly the N-terminal extension of the EspP signal sequence is responsible for its rejection from the SRP pathway.

The peroxisome is a subcellular organelle that carries out a diverse range of metabolic functions, including the b-oxidation of very long chain fatty acids, the breakdown of peroxide and the a-oxidation of fatty acids. Disruption of peroxisome metabolic functions leads to severe disease in humans. These diseases can be broadly grouped into two categories: those in which a single enzyme is defective, and those known as the peroxisome biogenesis disorders (PBDs), which result from a generalised failure to import peroxisomal matrix proteins (and consequently result in disruption of multiple metabolic pathways). The PBDs result from mutations in PEX genes, which encode protein products called peroxins, required for the normal biogenesis of the peroxisome. PEX1 encodes an AAA ATPase that is essential for peroxisome biogenesis, and mutations in PEX1 are the most common cause of PBDs worldwide. This study focused on the identification of mutations in PEX1 in an Australasian cohort of PBD patients, and the impact of these mutations on PEX1 function. As a result of the studies presented in this thesis, twelve mutations in PEX1 were identified in the Australasian cohort of patients. The identified mutations can be broadly grouped into three categories: missense mutations, mutations directly introducing a premature termination codon (PTC) and mutations that interrupt the reading frame of PEX1. The missense mutations that were identified were R798G, G843D, I989T and R998Q; all of these mutations affect amino acid residues located in the AAA domains of the PEX1 protein. Two mutations that directly introduce PTCs into the PEX1 transcript (R790X and R998X), and four frameshift mutations (A302fs, I370fs, I700fs and S797fs) were identified. There was also one mutation found in an intronic region (IVS22-19A>G) that is presumed to affect splicing of the PEX1 mRNA. Three of these mutations, G843D, I700fs and G973fs, were found at high frequency in this patient cohort. At the commencement of these studies, it was hypothesised that missense mutations would result in attenuation of PEX1 function, but mutations that introduced PTCs, either directly or indirectly, would have a deleterious effect on PEX1 function. Mutations introducing PTCs are thought to cause mRNA to be degraded by the nonsense-mediated decay of mRNA (NMD) pathway, and thus result in a decrease in PEX1 protein levels. The studies on the cellular impact of the identified PEX1 mutations were consistent with these hypotheses. Missense mutations were found to reduce peroxisomal protein import and PEX1 protein levels, but a residual level of function remained. PTC-generating mutations were found to have a major impact on PEX1 function, with PEX1 mRNA and protein levels being drastically reduced, and peroxisomal protein import capability abolished. Patients with two missense mutations showed the least impact on PEX1 function, patients with two PTC-generating mutations had a severe defect in PEX1 function, and patients carrying a combination of a missense mutation and a PTC-generating mutation showed levels of PEX1 function that were intermediate between these extremes. Thus, a correlation between PEX1 genotype and phenotype was defined for the Australasian cohort of patients investigated in these studies. For a number of patients, mutations in the coding sequence of one PEX1 allele could not be identified. Analysis of the 5' UTR of this gene was therefore pursued for potential novel mutations. The initial analyses demonstrated that the 5' end of PEX1 extended further than previously reported. Two co-segregating polymorphisms were also identified, termed –137 T>C and –53C>G. The -137T>C polymorphism resided in an upstream, in-frame ATG (termed ATG1), and the possibility that the additional sequence represented PEX1 coding sequence was examined. While both ATGs were found to be functional by virtue of in vitro and in vivo expression investigations, Western blot analysis of the PEX1 protein in patient and control cell extracts indicated that physiological translation of PEX1 was from the second ATG only. Using a luciferase reporter approach, the additional sequence was found to exhibit promoter activity. When examined alone the -137T>C polymorphism exerted a detrimental effect on PEX1 promoter activity, reducing activity to half that of wild-type levels, and the -53C>G polymorphism increased PEX1 promoter activity by 25%. When co-expressed (mimicking the physiological condition) these polymorphisms compensated for each other to bring PEX1 promoter activity to near wild-type levels. The PEX1 mutations identified in this study have been utilised by collaborators at the National Referral Laboratory for Lysosomal, Peroxisomal and Related Genetic Disorders (based at the Women's and Children's Hospital, Adelaide), in prenatal diagnosis of the PBDs. In addition, the identification of three common mutations in Australasian PBD patients has led to the implementation of screening for these mutations in newly referred patients, often enabling a precise diagnosis of a PBD to be made. Finally, the strong correlation between genotype and phenotype for the patient cohort investigated as part of these studies has generated a basis for the assessment of newly identified mutations in PEX1.

分散式系統具有資源共享及運算更有效率等優點，使得分散式系統的應用越來越廣泛，有關分散式系統的研究也越受重視。一些存在分散式系統中的不正常現象，如：死結(deadlock)，饑餓(stravation)，及臨界競賽(critical race)往往會抵消掉資源共享的好處並且容易產生通訊(communication)的錯誤，另外，保證一分散系統中行程(process)均能正確的執行終止(termination)也是一項值得研究的重要課題。 　　本篇論文將就分散系統中的死結、饑餓、臨界競賽及中止等問題之過去的文獻提出討論，並分別比較優、缺點，最後列出一些尚未解決及值得深入研究的問題。 / There are some addvantages of distributed systems, such as resources sharing and computing efficiency. Thus, the applications of distributed systems are more prevalent. The research of distributed systems had got great worth. Some undesired or abcdrmal behaviors such as deadlock, starvation, and critical race exist in distributed systems, which often offset the advantages of resources sharing and are prone to communication errors. It is very important to guarantee the processes of a distributed system which can terminate finitely. 　　In this thesis, we'll have thorough discussions and comparisons of those four properties through literature survey and then point out some unsolved problems at last.

<p>Advances in genetics, proteomics and chromatography techniques have enabled the successfully generation of a cell-free bacterial translation system composed of highly pure and active components. This system provided an ideal platform for better elucidating the mechanism of each individual step of the prokaryotic protein biosynthesis and the function of the translation factors involved in the process. </p><p>In doing so, we have discovered that the N-terminal domain or complete deletions of the ribosomal protein L11 reduced the termination efficiency of RF1 on cognate stop codons by four to six folds. The L11 deletions also conferred a two folds decrease in the missense error suggesting the increased nonsense termination accuracy of RF2 by two folds, which would clarified previous in vivo observations. </p><p>The versatility of the cell-free system has provided the additional possibility to study the effects of class II release factor RF3 mutations in mediating fast dissociation of class I release factors RF1 and RF2 from the post-termination ribosome complexes. The results show a series of mutations within RF3 conferring considerable reduction of the class I release factors recycling rate. These observations together with sequence alignment studies suggest the possible location on RF3 of the class I release factors interaction site. </p><p>In addition, the utilization of the cell-free system has made it possible to develop a new biotechnological approach for continuous production of polypeptides, based on gel filtration chromatography. The pilot trials have so far resulted in a six fold production increase of the MFTI test peptide compared to the conventional batch method.</p>

<p>This composite dissertation explores mediation in internal armed conflicts from a bargaining perspective. Four separate essays investigate why mediation occurs, why it is successful, and why peace guarantors’ commitments are credible. Essay 1 examines the conditions under which mediation takes place. The study argues that whereas it is costly for governments to accept international mediation, it is a less costly intervention tool for potential third parties. This argument implies that mediation will be more likely when and where negotiated settlements are least likely to be reached, a contention that is supported by empirical tests. Essay 2 reviews the contemporary debate on what types of mediators that can disseminate information in a credible manner, and formulates a set of testable hypotheses on mediation partiality. The analysis shows that negotiated settlements are more likely if biased or interested mediators intervene, while neutral mediators are not associated with mediation success. Essay 3 elaborates on the role of biased mediators. It proposes that rebels face a commitment problem when negotiated settlements are to be reached, which government-biased mediators can mitigate. The study finds that such types of mediators outperform rebel-biased mediators in terms of helping combatants to settle the armed conflict. Essay 4 deals with the commitment problem that comes to pass between, on the one hand the primary parties, and on the other, the potential peace guarantors. The study probes the requests and promises for third-party security guarantees and suggests that the reputation of the United Nations (UN) enhances its credibility as peace guarantor compared to non-UN actors. It finds that although the UN is more restrictive with its promises, it is more likely that peacekeeping forces will be provided if the UN is one of the guarantors. In sum, utilizing unique data from two time-periods (post World War II and post Cold War), this dissertation arrives at new insights on the role of mediators in bringing about negotiated settlements of internal armed conflicts.</p>

Advances in genetics, proteomics and chromatography techniques have enabled the successfully generation of a cell-free bacterial translation system composed of highly pure and active components. This system provided an ideal platform for better elucidating the mechanism of each individual step of the prokaryotic protein biosynthesis and the function of the translation factors involved in the process. In doing so, we have discovered that the N-terminal domain or complete deletions of the ribosomal protein L11 reduced the termination efficiency of RF1 on cognate stop codons by four to six folds. The L11 deletions also conferred a two folds decrease in the missense error suggesting the increased nonsense termination accuracy of RF2 by two folds, which would clarified previous in vivo observations. The versatility of the cell-free system has provided the additional possibility to study the effects of class II release factor RF3 mutations in mediating fast dissociation of class I release factors RF1 and RF2 from the post-termination ribosome complexes. The results show a series of mutations within RF3 conferring considerable reduction of the class I release factors recycling rate. These observations together with sequence alignment studies suggest the possible location on RF3 of the class I release factors interaction site. In addition, the utilization of the cell-free system has made it possible to develop a new biotechnological approach for continuous production of polypeptides, based on gel filtration chromatography. The pilot trials have so far resulted in a six fold production increase of the MFTI test peptide compared to the conventional batch method.

This composite dissertation explores mediation in internal armed conflicts from a bargaining perspective. Four separate essays investigate why mediation occurs, why it is successful, and why peace guarantors’ commitments are credible. Essay 1 examines the conditions under which mediation takes place. The study argues that whereas it is costly for governments to accept international mediation, it is a less costly intervention tool for potential third parties. This argument implies that mediation will be more likely when and where negotiated settlements are least likely to be reached, a contention that is supported by empirical tests. Essay 2 reviews the contemporary debate on what types of mediators that can disseminate information in a credible manner, and formulates a set of testable hypotheses on mediation partiality. The analysis shows that negotiated settlements are more likely if biased or interested mediators intervene, while neutral mediators are not associated with mediation success. Essay 3 elaborates on the role of biased mediators. It proposes that rebels face a commitment problem when negotiated settlements are to be reached, which government-biased mediators can mitigate. The study finds that such types of mediators outperform rebel-biased mediators in terms of helping combatants to settle the armed conflict. Essay 4 deals with the commitment problem that comes to pass between, on the one hand the primary parties, and on the other, the potential peace guarantors. The study probes the requests and promises for third-party security guarantees and suggests that the reputation of the United Nations (UN) enhances its credibility as peace guarantor compared to non-UN actors. It finds that although the UN is more restrictive with its promises, it is more likely that peacekeeping forces will be provided if the UN is one of the guarantors. In sum, utilizing unique data from two time-periods (post World War II and post Cold War), this dissertation arrives at new insights on the role of mediators in bringing about negotiated settlements of internal armed conflicts.

The chimpanzee is humankind’s closest living relative and the two species diverged ~6 million years ago. Comparative studies of the human and chimpanzee genomes and transcriptomes are of great interest to understand the molecular mechanisms of speciation and the development of species-specific traits. The aim of this thesis is to characterize differences between the two species with regard to their genome sequences and the resulting transcript profiles. The first two papers focus on indel divergence and in particular, indels causing premature termination codons (PTCs) in 8% of the chimpanzee genes. The density of PTC genes is correlated with both the distance to the telomere and the indel divergence. Many PTC genes have several associated transcripts and since not all are affected by the PTC we propose that PTCs may affect the pattern of expressed isoforms. In the third paper, we investigate the transcriptome divergence in cerebellum, heart and liver, using high-density exon arrays. The results show that gene expression differs more between tissues than between species. Approximately 15% of the genes are differentially expressed between species, and half of the genes show different splicing patterns. We identify 28 cassette exons which are only included in one of the species, often in a tissue-specific manner. In the fourth paper, we use massive parallel sequencing to study the chimpanzee transcriptome in frontal cortex and liver. We estimate gene expression and search for novel transcribed regions (TRs). The majority of TRs are located close to genes and possibly extend the annotations. A subset of TRs are not found in the human genome. The brain transcriptome differs substantially from that of the liver and we identify a subset of genes enriched with TRs in frontal cortex. In conclusion, this thesis provides evidence of extensive genomic and transcriptomic variability between human and chimpanzee. The findings provide a basis for further studies of the underlying differences affecting phenotypic divergence between human and chimpanzee.

Until the developments in computer technology, linear test administrations within classical test theory framework is mostly used in testing practices. These tests contain a set of predefined items in a large range of difficulty values for collecting information from students at various ability levels. However, placing very easy and very difficult items in the same test not only cause wasting time and effort but also introduces possible extraneous variables into the measurement process such as possibility of guessing, chance of careless errors induced by boredom or frustration. Instead of administering a linear test there is another option that adapts the difficulty of test according to the ability level of examinees which is named as computerized adaptive test. Computerized adaptive tests use item response theory as a measurement framework and have algorithms responsible for item selection, ability estimation, starting rule and test termination. The present study aims to determine the applicability of computerized adaptive testing (CAT) to Turkish Pupil Monitoring System&rsquo / s (PMS) mathematics assessments. Therefore, live CAT study using only multiple choice items is designed to investigate whether to obtain comparable ability estimations. Afterwards, a Monte Carlo simulation study and a Post-hoc simulation study are designed to determine the optimum CAT algorithm for Turkish PMS mathematics assessments. In the simulation studies, both multiple-choice and open-ended items are used and different scenarios are tested regarding various starting rules, termination criterion, ability estimation methods and existence of exposure/content controls. The results of the study indicate that using Weighted Maximum Likelihood (WML) ability estimation method, easy initial item difficulty as starting rule and a fixed test reliability termination criterion (0.30 standard error as termination rule) gives the optimum CAT algorithm for Turkish PMS mathematics assessment. Additionally, item exposure and content control strategies have a positive impact on providing comparable ability estimations.

Darbe nagrinėjami apsaugos nuo nepagrįsto atleidimo iš darbo klausimai. Tema nėra nauja, tačiau nepakankamai išsamiai nagrinėta. Apsaugos nuo nepagrįsto atleidimo iš darbo analizei teisinėje literatūroje skiriamas nepakankamas dėmesys. Siekiant įrodyti iškeltą hipotezę, kad Lietuvos teisės aktai, reglamentuojantys apsaugą nuo nepagrįsto atleidimo iš darbo, atitinka tarptautinius standartus, analizuojamos tarptautinės ir nacionalinės apsaugos nuo nepagrįsto atleidimo iš darbo teisės normos. / There are researched questions of protection from unjustified dismissal in this work. This theme is not new, but it is not properly studied. There is given too little attention for analysis of protection from unjustified dismissal in law literature. In pursuance of hypothesis, that Lithuanian law, which regulate protection from unjustified dismissal, are parallel to international standards; there are analyzed international and national law standards for protection from unjustified dismissal. In the first part of work there is analyzed conception of unjustified dismissal, which is consolidated in International Law Organization, Europe Union as international regional organization and national law acts. After accomplishing of law analysis of standard acts it is concluded, that there is not given definition of unjustified dismissal in the researched international and national law acts. Legitimacy of dismissal is related with criteria of important reasons. In the second part of work there is discussed and by comparative aspect analyzed measures of protection from unjustified dismissal, which are consolidated in international and Europe Union member states national law acts. In the third part generalised the practice of Lithuanian Highest Court in cases of unjustified dismissal. There is given the interpretation of Labour Code of Lithuanian law standards by Lithuanian Highest Court, because judiciary practice is promoting application of fair and equal law standards. Also reviewing... [to full text]