The primary objective of this study is to evaluate the operational effects of U-turn movement at signalized intersections. More specifically, the research objectives include the following parts: To identify the factors affecting the operational performance of U-turning vehicles. In this case, we are particularly interested in the U-turn speeds of U-turning vehicles. To evaluate the impacts of U-turns on capacity of signalized intersections, and To simulate U-turn movement at signalized intersections using Synchro and validate the simulation results. To achieve the research objectives, extensive field data collection work was conducted at sixteen selected sites at Tampa Bay area of Florida. The data collected in the field include: U-turning speed Left turning speed Turning radius Queue discharge time Control delay Hourly traffic volume, and Percentage of U- turning vehicles in left turn lane. Based on the collected field data, a linear regression model was developed to identify the factors affecting the turning speeds of U-turning vehicles at signalized intersections. The model shows the turning speed is significantly impacted by the turning radius and the speed of U-turning vehicles increases with the increase of turning radius. On the basis of field data field data collection, a regression model was developed to estimate the relationship between the average queue discharge time for each turning vehicle and the various percentages of U-turning vehicles in the left turn traffic stream. Adjustment factors for various percentages of U-turning vehicles were also developed by using the regression model. The adjustment factors developed in this study can be directly used to estimate the capacity reduction due to the presence of various percentages of U-turning vehicles at a signalized intersection. The developed adjustment factors were used to improve the simulation of U-turn movement at signalized intersection by using Synchro. The simulation model was calibrated and validated by field data. It was found that using the developed adjustment factors will greatly improve the accuracy of the simulation results for U-turn movement.

The No-U-Turn Sampler (NUTS) is a relatively new Markov chain Monte Carlo (MCMC) algorithm that avoids the random walk behavior that common MCMC algorithms such as Gibbs sampling or Metropolis Hastings usually exhibit. Given the fact that NUTS can efficiently explore the entire space of the target distribution, the sampler converges to high-dimensional target distributions more quickly than other MCMC algorithms and is hence less computational expensive. The focus of this study is on applying NUTS to one of the complex IRT models, specifically the two-parameter mixture IRT (Mix2PL) model, and further to examine its performance in estimating model parameters when sample size, test length, and number of latent classes are manipulated. The results indicate that overall, NUTS performs well in recovering model parameters. However, the recovery of the class membership of individual persons is not satisfactory for the three-class conditions. Also, the results indicate that WAIC performs better than LOO in recovering the number of latent classes, in terms of the proportion of the time the correct model was selected as the best fitting model. However, when the effective number of parameters was also considered in selecting the best fitting model, both fully Bayesian fit indices perform equally well. In addition, the results suggest that when multiple latent classes exist, using either fully Bayesian fit indices (WAIC or LOO) would not select the conventional IRT model. On the other hand, when all examinees came from a single unified population, fitting MixIRT models using NUTS causes problems in convergence.

This thesis investigates the influence of attitude on the life of an individual and the role that it can play inside and outside today’s organizations. The purpose of the thesis is to gain insight on the subject of attitude and its significance both in the life of today’s employee and on the management and leadership of today’s organizations. I will endeavor to investigate the importance of cultivating a positive attitude and the consequences of yielding to a negative attitude. I will also discuss why attitude not only can influence the firm’s progress but also the external image and advancement of the organization as well.With the abduction and narrative approaches, I will endeavor to explain the importance of maintaining a positive attitude and ways of understanding individuals’ attitudes and their influence on their overall performance. I will also attempt to address how employees in a given organization can deal with a customer who has a sour attitude. Moreover, I’ll address some of the possible causes of negative attitudes. The theoretical research will help expound on the subject matter. Through empirical research, I’ll attempt to address the fact that despite an employee’s skills and competence, positive attitude can be an added advantage.With an understanding based of my personal life experience, I’ll explain how I struggled with negative attitudes especially during my early years in Sweden and how I eventually made an attitude adjustment. My personal experience at the Sheraton will be used as a case study on the subject of attitude. Furthermore, I’ll examine some other life experiences such as the lessons I learned from my father on the subject. Besides, I’ll discuss the challenge of attitude on leadership as well. Finally, I will conclude with a discussion on the fact that attitude needs to be addressed in a good-enough approach thereby circumventing the trap of making attitude appear to be the principle aspect as this may lead to absolutism. / Uppsatsnivå: D

<p>Antisense RNAs regulate gene expression in many bacterial systems. The best characterized examples are from prokaryotic accessory elements such as phages, plasmids and transposons. Many of these antisense RNAs have been identified as plasmid copy number regulators where they regulate the replication frequency of the plasmid by negative feedback. Instability and fast binding kinetics is crucial for the regulatory efficiency of these antisense RNAs. </p><p>In this thesis, the interaction of the cis-encoded antisense RNA CopA with its target CopT was studied in detail using <i>in vivo</i> reporter gene fusion expression and different <i>in vitro </i>methods, such as surface plasmon resonance, fluorescence resonance energy transfer, and gel-shift assays.</p><p>Formation of inhibitory complexes differs from simple hybridization reactions between complementary strands. E.g., the binding pathway of CopA and CopT proceeds through a hierarchical order of steps. It initiates by reversible loop-loop contacts, resulting in a helix nucleus of two or three base pairs. This is followed by rapid unidirectional helix progression into the upper stems, resulting in a four-way helical junction structure. It had been suggested that the loop of CopT carries a putative U-turn, a structure first found in tRNA anticodon loops. We showed that this putative U-turn is one of the structural elements of CopA/CopT required to achieve fast binding kinetics. Furthermore, the hypothetical U-turn structure determines the direction of helix progression when the kissing complex progresses to a four-way helical junction structure. Another structural element in CopT is the helical stem adjacent to the recognition loop. This stem is important to present the recognition loop appropriately to provide a scaffold for the U-turn.</p><p>Furthermore, the role of protein Hfq in the interaction of antisense/target RNA was investigated, since several trans-encoded antisense RNAs had been shown to need this protein to exert their function. In contrast, studies of two cis-encoded antisense RNA systems showed that these antisense RNAs do not rely on Hfq for activity. In this study it was also shown that MicF, a trans-encoded antisense RNA which is dependent on Hfq, is greatly stabilized by this protein.</p>

Antisense RNAs regulate gene expression in many bacterial systems. The best characterized examples are from prokaryotic accessory elements such as phages, plasmids and transposons. Many of these antisense RNAs have been identified as plasmid copy number regulators where they regulate the replication frequency of the plasmid by negative feedback. Instability and fast binding kinetics is crucial for the regulatory efficiency of these antisense RNAs. In this thesis, the interaction of the cis-encoded antisense RNA CopA with its target CopT was studied in detail using in vivo reporter gene fusion expression and different in vitro methods, such as surface plasmon resonance, fluorescence resonance energy transfer, and gel-shift assays. Formation of inhibitory complexes differs from simple hybridization reactions between complementary strands. E.g., the binding pathway of CopA and CopT proceeds through a hierarchical order of steps. It initiates by reversible loop-loop contacts, resulting in a helix nucleus of two or three base pairs. This is followed by rapid unidirectional helix progression into the upper stems, resulting in a four-way helical junction structure. It had been suggested that the loop of CopT carries a putative U-turn, a structure first found in tRNA anticodon loops. We showed that this putative U-turn is one of the structural elements of CopA/CopT required to achieve fast binding kinetics. Furthermore, the hypothetical U-turn structure determines the direction of helix progression when the kissing complex progresses to a four-way helical junction structure. Another structural element in CopT is the helical stem adjacent to the recognition loop. This stem is important to present the recognition loop appropriately to provide a scaffold for the U-turn. Furthermore, the role of protein Hfq in the interaction of antisense/target RNA was investigated, since several trans-encoded antisense RNAs had been shown to need this protein to exert their function. In contrast, studies of two cis-encoded antisense RNA systems showed that these antisense RNAs do not rely on Hfq for activity. In this study it was also shown that MicF, a trans-encoded antisense RNA which is dependent on Hfq, is greatly stabilized by this protein.

This study employs Bayesian Hidden Markov Models as method to explore vehicle traffic flow conditions in an urban area in Stockholm, based on sensor data from separate road positions. Inter-arrival times are used as the observed sequences. These sequences of inter-arrival times are assumed to be generated from the distributions of four different (and hidden) traffic flow states; nightly free flow, free flow, mixture and congestion. The filtered and smoothed probability distributions of the hidden states and the most probable state sequences are obtained by using the forward, forward-backward and Viterbi algorithms. The No-U-Turn sampler is used to sample from the posterior distributions of all unknown parameters. The obtained results show in a satisfactory way that the Hidden Markov Models can detect different traffic flow conditions. Some of the models have problems with divergence, but the obtained results from those models still show satisfactory results. In fact, two of the models that converged seemed to overestimate the presence of congested traffic and all the models that not converged seem to do adequate estimations of the probability of being in a congested state. Since the interest of this study lies in estimating the current traffic flow condition, and not in doing parameter inference, the model choice of Bayesian Hidden Markov Models is satisfactory. Due to the unsupervised nature of the problematization of this study, it is difficult to evaluate the accuracy of the results. However, a model with simulated data and known states was also implemented, which resulted in a high classification accuracy. This indicates that the choice of Hidden Markov Models is a good model choice for estimating traffic flow conditions.

Les interactions ARN/ARN de type kissing-loop sont des éléments de structure tertiaire qui jouent souvent des rôles clés chez les ARN, tant au niveau fonctionnel que structural. En effet, ce type d’interaction est crucial pour plusieurs processus dépendant des ARN, notamment pour l’initiation de la traduction, la reconnaissance des ARN antisens et la dimérisation de génome rétroviral. Les interactions kissing-loop sont également importantes pour le repliement des ARN, puisqu’elles permettent d’établir des contacts à longue distance entre différents ARN ou encore entre les domaines éloignés d’un même ARN. Ce type d’interaction stabilise aussi les structures complexes des ARN fonctionnels tels que les ARNt, les riborégulateurs et les ribozymes. Comme d’autres ARN fonctionnels, le ribozyme VS de Neurospora contient une interaction kissing-loop importante. Celle-ci est impliquée dans la reconnaissance du substrat et se forme entre la tige-boucle I (stem-loop I, SLI) du substrat et la tige-boucle V (stem-loop V, SLV) du domaine catalytique. Des études biochimiques ont démontré que l’interaction kissing-loop I/V, dépendante du magnésium, implique trois paires de bases Watson-Crick (W-C). De plus, cette interaction est associée à un réarrangement de la structure du substrat, le faisant passer d’une conformation inactive dite unshifted à une conformation active dite shifted. Les travaux présentés dans cette thèse consistent en une caractérisation structurale et thermodynamique de l’interaction kissing-loop I/V du ribozyme VS, laquelle est formée de fragments d’ARN représentant les tige-boucles I et V dérivées du ribozyme VS (SLI et SLV). Cette caractérisation a été réalisée principalement par spectroscopie de résonance magnétique nucléaire (RMN) et par titrage calorimétrique isotherme (isothermal titration calorimetry, ITC) en utilisant différents complexes SLI/SLV dans lesquels l’ARN SLV est commun à tous les complexes, alors que différentes variations de l’ARN SLI ont été utilisées, soit en conformation shiftable ou preshifted. Les données d’ITC ont permis de démontrer qu’en présence d’une concentration saturante de magnésium, l’affinité d’un substrat SLI preshifted pour SLV est extrêmement élevée, rendant cette interaction plus stable que ce qui est prédit pour un duplexe d’ARN équivalent. De plus, l’étude effectuée par ITC montre que des ARN SLI preshifted présentent une meilleure affinité pour SLV que des ARN SLI shiftable, ce qui a permis de calculer le coût énergétique associé au réarrangement de structure du substrat. En plus de confirmer la formation des trois paires de bases W-C prédites à la jonction I/V, les études de RMN ont permis d’obtenir une preuve structurale directe du réarrangement structural des substrats SLI shiftable en présence de magnésium et de l’ARN SLV. La structure RMN d’un complexe SLI/SLV de grande affinité démontre que les boucles terminales de SLI et SLV forment chacune un motif U-turn, ce qui facilite l’appariement W-C intermoléculaire. Plusieurs autres interactions ont été définies à l’interface I/V, notamment des triplets de bases, ainsi que des empilements de bases. Ces interactions contribuent d’ailleurs à la création d’une structure présentant un empilement continu, c’est-à-dire qui se propage du centre de l’interaction jusqu’aux bouts des tiges de SLI et SLV. Ces études de RMN permettent donc de mieux comprendre la stabilité exceptionnelle de l’interaction kissing-loop I/V au niveau structural et mènent à l’élaboration d’un modèle cinétique de l’activation du substrat par le ribozyme VS. En considérant l’ensemble des données d’ITC et de RMN, l’étonnante stabilité de l’interaction I/V s’explique probablement par une combinaison de facteurs, dont les motifs U-turn, la présence d’un nucléotide exclu de la boucle de SLV (U700), la liaison de cations magnésium et l’empilement de bases continu à la jonction I/V. / Kissing loops are tertiary structure elements that often play key roles in functional RNAs. Their formation is central to many RNA-mediated processes, such as translation initiation, antisense recognition and retroviral dimerization. Kissing loops are also involved in RNA folding as they form long-range interactions between different RNAs or remote domains within the same RNA and stabilize the complex architecture of functional RNA, such as tRNA, riboswitch aptamers and ribozymes. Like several other functional RNAs, the Neurospora VS ribozyme contains an important kissing-loop interaction. The substrate recognition by the VS ribozyme depends largely on the formation of a magnesium-dependent kissing-loop interaction between stem-loop V (SLV) of the catalytic domain and stem-loop I (SLI) that defines the substrate domain. It has been shown from biochemical studies that the I/V kissing-loop interaction involves three Watson-Crick base pairs and is associated with a structural rearrangement of the SLI substrate from an unshifted and inactive to a shifted and active conformation. Here, we present a thermodynamic and structural characterization of the VS ribozyme I/V kissing-loop interaction using isolated stem-loop fragments (SLI and SLV). Both isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy studies were conducted with several SLI/SLV complexes using a common SLV, but either shiftable or preshifted SLI variants. From the ITC studies, we show that, under saturating amount of magnesium ions, the affinity of the preshifted SLI variants for SLV is remarkably high, the interaction being more stable than predicted for a comparable duplex. In addition, these ITC studies demonstrate that preshifted SLI variants have higher affinity for SLV than shiftable SLI variants, and these results allow us to evaluate the energetic cost of the conformational shift in SLI. From the NMR studies, we confirm formation of three Watson-Crick base pairs at the kissing-loop junction and provide direct evidence on the structural rearrangement of shiftable SLI variants in the presence of magnesium and SLV. The NMR structure of a high-affinity SLI/SLV complex demonstrates that both the SLI and SLV loops adopt U-turn structures, which facilitate intermolecular Watson-Crick base pairing. Several other interactions at the I/V interface, including base triples and base stacking help create a continuously stacked structure. These NMR studies provide a structural basis for the high stability of the kissing-loop interaction and lead us to propose a kinetic model for substrate activation by the VS ribozyme. Taken together, our ITC and NMR data suggest that the remarkable stability of the I/V interaction is likely provided by a combination of several elements, especially the presence of the U-turn motif, the presence of an extruded nucleotide in SLV (U700), the binding of magnesium ions and the extensive base stacking interactions at the junction.

Le ribozyme VS de Neurospora catalyse des réactions de clivage et de ligation d’un lien phosphodiester spécifique essentielles à son cycle de réplication. Il est formé de six régions hélicales (I à VI), qui se divisent en deux domaines, soit le substrat (SLI) et le domaine catalytique (tiges II à VI). Ce dernier comprend deux jonctions à trois voies qui permettent de reconnaître le substrat en tige-boucle de façon spécifique. Ce mode de reconnaissance unique pourrait être exploité pour cibler des ARN repliés pour diverses applications. Bien que le ribozyme VS ait été caractérisé biochimiquement de façon exhaustive, aucune structure à haute résolution du ribozyme complet n’a encore été publiée, ce qui limite la compréhension des mécanismes inhérents à son fonctionnement. Précédemment, une approche de divide-and-conquer a été initiée afin d’étudier la structure des sous-domaines importants du ribozyme VS par spectroscopie de résonance magnétique nucléaire (RMN) mais doit être complétée. Dans le cadre de cette thèse, les structures de la boucle A730 et des jonctions III-IV-V et II-III-VI ont été déterminées par spectroscopie RMN hétéronucléaire. De plus, une approche de spectroscopie RMN a été développée pour la localisation des ions divalents, tandis que diverses approches de marquage isotopique ont été implémentées pour l’étude d’ARN de plus grandes tailles. Les structures RMN de la boucle A730 et des deux jonctions à trois voies révèlent que ces sous-domaines sont bien définis, qu’ils sont formés de plusieurs éléments structuraux récurrents (U-turn, S-turn, triplets de bases et empilement coaxial) et qu’ils contiennent plusieurs sites de liaison de métaux. En outre, un modèle du site actif du ribozyme VS a été construit sur la base des similarités identifiées entre les sites actifs des ribozymes VS et hairpin. Dans l’ensemble, ces études contribuent de façon significative à la compréhension de l’architecture globale du ribozyme VS. De plus, elles permettront de construire un modèle à haute résolution du ribozyme VS tout en favorisant de futures études d’ingénierie. / The Neurospora VS ribozyme catalyzes the cleavage and the ligation of a specific phosphodiester bond, which is essential for its replication cycle. It is formed of six helical regions (I to VI) that are divided in two domains: the substrate (SLI) and the catalytic domain (stems II-VI). The latter contains two three-way junctions that allow recognition of the stem-loop substrate in a specific manner. This unique mode of substrate recognition could be exploited to target folded RNAs for diverse applications. Even though the VS ribozyme has been extensively characterized biochemically, no high-resolution structure of the complete ribozyme has been published yet and this limits our mechanistic understanding. A divide-and-conquer approach was previously initiated to study the structure of the important subdomains of the VS ribozyme by nuclear magnetic resonance (NMR), but this approach needs to be completed. In this thesis, the structures of the A730 loop, the III-IV-V junction and the II-III-VI junction were determined by heteronuclear NMR spectroscopy. Moreover, a unique NMR approach was developed for localizing divalent metal ions, whereas several isotope-labeling strategies were implemented to facilitate the study or large RNA molecules. The NMR structures of the A730 loop and the two three-way junctions reveal that these subdomains are well defined, that they are formed by several recurrent structural elements (U-turn and S-turn motifs, base triples and coaxial stacking) and that they contain several metal-binding sites. Interestingly, structural similarities were identified between the VS and hairpin ribozymes, which allowed the modeling of the VS ribozyme active site. In summary, these studies significantly contribute to a better understanding of the global architecture of the VS ribozyme. In addition, they will allow the construction of a high-resolution model of the complete VS ribozyme and facilitate future engineering studies.

This thesis attempts to evaluate the Markov Chain Monte Carlo (MCMC) methods Metropolis-Hastings (MH) and No-U-Turn Sampler (NUTS) to estimate systemic risk measures. The subject of analysis is an equity portfolio provided by a Nordic asset management firm, which is modelled using a vine copula. The evaluation considers three different crisis outcomes on a portfolio level, and the results are compared with a Monte Carlo (MC) benchmark. The MCMC samplers attempt to increase sampling efficiency by sampling from these crisis events directly, which is impossible for an MC sampler. The resulting systemic risk measures are evaluated both on the portfolio level as well as marginal level.  The results are divided. In part, the MCMC samplers proved to be efficient in terms of accepted samples, where NUTS outperformed MH. However, due to the practical implementation of the MCMC samplers and the vine copula model, the computational time required outweighed the gains in sampler efficiency - causing the MC sampler to outperform both MCMC samplers in certain settings. For NUTS, there seems to be great potential in the context of estimating systemic risk measures as it explores high-dimensional and multimodal joint distributions efficiently with low autocorrelation. It is concluded that asset management companies can benefit from both using vine copulas to model portfolio risk, as well as using MC or MCMC methods for evaluating systemic risk. However, for the MCMC samplers to be of practical relevance, it is recommended to further investigate efficient implementations of vine copulas in the context of MCMC sampling. / Detta examensarbete utvärderar Markov Chain Monte Carlo (MCMC)-metoderna No-U-Turn Sampler (NUTS) och Metropolis-Hastings (MH) vid uppskattning av systemiska riskmått. För att göra detta används en vine copula för att modellera en portfölj, baserad på empirisk data från ett nordiskt kapitalförvaltningsföretag. Metoderna utvärderas givet tre olika krishändelser och jämförs därefter med ett Monte Carlo (MC) benchmark. MCMC-metoderna försöker öka samplingseffektiviteten genom att simulera direkt från dessa krishändelser, vilket är omöjligt för en klassisk MC-metod. De resulterande systemiska riskmåtten utvärderas både på portföljnivå och på marginalnivå. Resultaten är delade. Dels visade sig MCMC-metoderna vara effektiva när det gäller accepterade samples där NUTS överträffade MH. Dock, med anledning av av den praktiska implementationen av MCMC-metoderna och vine copula modellen var beräkningstiden för hög trots effektiviteten hos metoden - vilket fick MC-metoden att överträffa de andra metoderna i givet dessa särskilda kontexter. När det kommer till att uppskatta systemiska riskmått finns det dock stor potential för NUTS eftersom metoden utforskar högdimensionella och multimodala sannolikhetsfördelningar effektivt med låg autokorrelation. Vi drar även slutsatsen att kapitalförvaltare kan dra nytta av att både använda riskmodeller baserade på vine copulas, samt använda MC- eller MCMC-metoder för att utvärdera systemisk risk. För att MCMC-metoderna ska vara av praktisk relevans rekommenderas det dock att framtida forskning görs där mer effektiva implementeringar av vine copula-baserade modeller görs i samband med MCMC-sampling.