The nuclear receptor superfamily is responsible for regulating the expression of genes involved in development, reproduction and metabolism. These transcription factors control the expression of their target genes through the binding of small molecule regulators to their ligand binding domains. Classical nuclear receptors include the steroid receptors, which bind endocrine hormones and have been important targets of pharmaceutical intervention. However, approximately one half of the human nuclear receptors remain orphans and are without known cognate ligands. Focusing on the Drosophila orthologues of these orphan receptors, this project used mass spectrometry to identify the chemical diversity associated with the receptors following expression in recombinant systems. In a genome-wide screen of Drosophila nuclear receptors, this approach identified co-purifying molecules with a number of receptors. The physiological relevance of these putative ligand/receptor pairs was determined through biochemical analysis, in vivo characterization and structure determination. Ligand(s) or the ligand state was identified for the Drosophila receptors: DHR3, DHR96, E75, Ftz-f1 and USP. Of these, three were validated through the efforts of this project, and independent groups confirmed the remaining two. The most significant findings were the discoveries that the fly nuclear receptor E75 is regulated by heme, gas and redox, and that there is a similar regulatory scheme in the human orthologues, Reverbα and β. Furthermore, crystallization of the heme-bound Rev-erbβ ligand binding domain was also achieved, and this provided key insights into the mechanism of ligand regulation for the Rev-erbs. This project highlighted the role of nuclear receptors in metabolic surveillance. The ligands/signals identified in association with these receptors include: cholesterol, dehydrocholesterol, heme, NO, CO, redox and phospholipids. Unlike the classical steroid hormones, these are not dedicated signaling molecules, but instead are key substrates or products of metabolism. In the context of nuclear receptor signaling, I hypothesize that these metabolites serve as metabolic indicators/signals in the regulation of development and metabolism. Furthermore, four of these Drosophila receptors comprise the ecdysone-response pathway in the developing fly. Taken together, this suggests that both the metabolic state of the organism and steroid hormones drive nuclear receptor regulation of development.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2016-04-08T23:04:24Z No. of bitstreams: 1 PauloVitorSilva_TESE.pdf: 16564605 bytes, checksum: d1310555faa2a858e9c72cbd78a64731 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2016-04-11T22:58:46Z (GMT) No. of bitstreams: 1 PauloVitorSilva_TESE.pdf: 16564605 bytes, checksum: d1310555faa2a858e9c72cbd78a64731 (MD5) / Made available in DSpace on 2016-04-11T22:58:46Z (GMT). No. of bitstreams: 1 PauloVitorSilva_TESE.pdf: 16564605 bytes, checksum: d1310555faa2a858e9c72cbd78a64731 (MD5) Previous issue date: 2015-05-11 / A necessidade constante de novas fontes de energias renov?veis vem promovendo cada vez mais o aumento de investimentos nessa ?rea. Dentre essas fontes, a energia e?lica vem tomando grande destaque. Torna-se importante promover a busca pelo aprimoramento das tecnologias envolvidas nas topologias de aerogeradores, buscando alternativas que aumentem o rendimento obtido, apesar da irregularidade da velocidade do vento. Este trabalho apresenta um novo sistema para controle de velocidade, aqui aplicado em aerogeradores, o Regulador Eletromagn?tico de Frequ?ncia (REF). Um dos dispositivos mais utilizados em algumas topologias s?o as caixas de engrenagens mec?nicas que, al?m de uma vida ?til curta, representam frequentemente, fontes de ru?do e defeitos. O REF dispensa essas caixas de transmiss?o, representando um avan?o tecnol?gico, utilizando para isso uma m?quina de indu??o adaptada, na qual o estator passa a ser m?vel, solid?rio ao eixo da turbina. Na topologia utilizada neste trabalho, o REF tamb?m permite dispensar o uso de conversores eletr?nicos para estabelecer o acoplamento entre o gerador e a rede el?trica, raz?o pela qual tamb?m proporciona a possibilidade de obten??o de gera??o em corrente alternada, com tens?o e frequ?ncia constantes, onde n?o exista a rede el?trica. Respons?vel pelo controle da velocidade mec?nica do gerador, o REF pode ser ?til em outros sistemas de transmiss?o onde o controle de velocidade mec?nica de sa?da seja o objetivo. Al?m disso, por operar atrav?s da combina??o de duas entradas, uma mec?nica e outra el?trica, o REF multiplica as possibilidades de aplica??es por ser apto ao acoplamento sin?rgico entre energias de matrizes diferentes, e, por tais motivos, possibilita que as diversas fontes de energia envolvidas sejam desacopladas da rede, sendo o gerador s?ncrono o respons?vel pela conex?o do sistema com a rede el?trica, simplificando as estrat?gias de controle quanto ? pot?ncia injetada na mesma. Resultados de simula??o e experimentais s?o apresentados no decorrer do trabalho, voltados a um aerogerador, validando a proposta em rela??o a efici?ncia no controle de velocidade do sistema para diferentes condi??es de vento. / The constant necessity for new sources of renewable energy is increasingly promoting the increase of investments in this area. Among other sources, the wind power has been becoming prominent. It is important to promote the search for the improvement of the technologies involved in the topologies of the wind turbines, seeking for alternatives which enhance the gotten performance, despite the irregularity of the wind speed. This study presents a new system for speed control, in this case applied to the wind turbines - the Electromagnetic Frequency Regulator (EFR). One of the most used devices in some topologies is the mechanical gearboxes which, along with a short service life, often represent sources of noise and defects. The EFR does not need these transmission boxes, representing a technological advancement, using for that an adapted induction machine, in which the stator becomes mobile, supportive to the axis of the turbine. In the topology used in this study, the EFR also allows us to leave out the usage of the eletronic converters to establish the coupling between the generator and the electrical grid. It also the reason why it provides the possibility of obtaining the generation in alternating current, with constant voltage and frequency, where there is no electrical grid. Responsable for the mechanical speed control of the generator, the EFR can be useful in other transmission systems in which the mechanical speed control output is the objective. In addition, the EFR operates through the combination of two inputs, a mechanical and other electrical. It multiplies the possibilities of application because it is able to synergistic coupling between different arrays of energy, and, for such reasons, it enables the various sources of energy involved to be uncoupled from the network, being the synchronous generator responsible for the system connection with the electrical grid, simplifying the control strategies on the power injected in it. Experimental and simulation results are presented through this study, about a wind turbine, validating the proposal related to the efficience in the speed control of the system for different wind conditions.

Diploma thesis called "Financial analysis of the industry of musical instruments" deals with analysis of financial health of producers of musical instruments during the years 2008-2013. There were chosen three Czech and three European companies. It was also made their comparision with the European industry of musical instruments. The thesis is divided into three main parts. In the first part there is described selected methodology which includes horizontal and vertikal analysis, ratio analysis, bankruptcy and credibility models and correlation analysis. In the next part there are presented the specifics of musical instruments industry. After that individual companies are introduced and followed by application of the methodology defined in the first part. The main outcome of analysis is that companies and industry are very sensitive to global economic situation. The impacts of financial crisis in 2008 really damaged this companies and industry of musical instruments.

<div> <p>E-commerce organizations, these days, need to keep striving for constant innovation. Customers have a massive impact on the performance of an organization, so industries need to have solid customer retention strategies. Various big data analytics methodologies are being used by organizations to improve overall online customer experience. While there are multiple techniques available, this research study utilized and tested a framework proposed by Laux et al. (2017), which combines Big Data and Six Sigma methodologies, to the e-commerce domain for identification of issues faced by the customer; this was done by analyzing online product reviews and ratings of customers to provide improvement strategies for enhancing customer experience. </p> <p>Analysis performed on the data showed that approximately 90% of the customer reviews had positive polarity. Among the factors which were identified to have affected the opinions of the customers, the Rating field had the most impact on the sentiments of the users and it was found to be statistically significant. Upon further analysis of reviews with lower rating, the results attained showed that the major issues faced by customers were related to the product itself; most issues were more specifically about the size/fit of the product, followed by the product quality, material used, how the product looked on the online portal versus how it looked in reality, and its price concerning the quality.</p> </div> <br>

Au cours des dernières décennies, le développement de nouveaux matériaux a progressé pour les applications liées à la mécanique. De nouvelles générations de composites ont été développées, qui peut offrir des avantages par rapport aux tapis unidirectionnels renforcés de fibres couramment utilisés les matériaux prennent alors le nom de woven fabrics. Le comportement de ce matériau est fortement influencé par la micro-structure du matériau. Dans la thèse, les modèles mécaniques et les schémas numériques capables de modéliser les comportement des tissus et des matériaux de réseau généraux ont été développés. Le modèle prend en compte la micro-structure au moyen d'une technique d'homogénéisation. Les fibres dans le réseau ont été traités comme des micro-poutres, ayant une rigidité à la fois en extension et en flexion, avec différents types de connexions. La procédure développée a été appliquée pour obtenir les modèles mécaniques homogénéisés pour certains types de réseaux de fibres biaxiaux et quadriaxiaux, simulant soit des réseaux de fibres (en ce cas a été supposé parmi les fibres) ou des tissus avec une interaction négligeable entre les faisceaux de fibres et en empêchant tout glissement relatif (dans ce cas, les connexions ont été simulés au moyen de pivots). Différentes géométries ont été analysées, y compris la cas dans lesquels les fibres ne sont pas orthogonales. On obtient généralement un premier milieu à gradient mais, dans certains cas, la procédure d'homogénéisation lui-même indique qu'un continuum d'ordre supérieur est mieux adapté pour représenter la déformation de la micro-structure. Des résultats spéciaux ont été obtenus dans le cas de fibres reliées par pivots. Dans ce cas, un matériau orthotrope à module de cisaillement nul a été obtenu. Un tel matériau a un tenseur constitutif elliptique, il peut donc conduire à des concentrations de contrainte. Cependant, il a été montré que certaines considérations sur le comportement physique de tels réseaux indiqué que les termes d'ordre supérieur inclus dans l'expansion des forces internes et des déformations, de sorte qu'un matériau de gradient de déformation a été obtenu. Les résultats obtenus peuvent être utilisés pour la conception de matériaux spécifiques nécessitant des propriétés. Bien que le modèle de référence soit un matériau de réseau, les résultats obtenus peuvent être appliqué à d'autres types similaires de microstructures, comme des matériaux pantographiques, des micro-dispositifs composé de micro-poutres, etc. Ils étaient limités à la gamme d'élasticité linéaire, qui est petite déformation et comportement élastique linéaire. Ensuite, les simulations numériques ont été axées sur les tests d'extension et les tests de biais. Le obtenu configurations déformées sont conformes aux tests expérimentaux de la littérature, tant pour tissus équilibrés et non équilibrés. De plus, une comparaison entre les premier et deuxième gradients des prédictions numériques ont été effectuées. Il a été observé que les prédictions de deuxième gradient mieux simuler les preuves expérimentales. / In the past decades there has been an impressive progress in the development of new materials for mechanical related applications. New generations of composites have been developed, that can offer advantages over the unidirectional fibre-reinforced mats commonly used then materials take the name of woven fabrics. The behaviour of this material is strongly influenced by the micro-structure of the material. In the thesis mechanical models and a numerical scheme able to model the mechanical behaviour of woven fabrics and general network materials have been developed. The model takes in to account the micro-structure by means of a homogenization technique. The fibres in the network have been treated like microbeams, having both extensional and bending stiffness, with different types of connection, according to the pattern and detail of the network. The developed procedure was applied for obtaining the homogenized mechanical models for some types of biaxial and quadriaxial networks of fibres, simulating either fibre nets (in this case rigid connection were assumed among the fibres) or tissues with negligible interaction between the fibre bundles, and with relative sliding prevented (in this case the connections were simulated by means of pivots). Different geometries were analysed, including the cases in which the fibres are not orthogonal. A first gradient medium is usually obtained but, in some cases, the homogenization procedure itself indicates that a higher order continuum is better fit to represent the deformation of the micro-structure. Special results were obtained for the case of fibres connected by pivots. In this cases an orthotropic material with zero shear modulus was obtained. Such a material has a not elliptic constitutive tensor, thus it can lead to strain concentrations. However, it was shown that some considerations about the physical behaviour of such networks indicated that higher order terms had to be included in the expansion of the internal forces and deformations, so that a strain gradient material was obtained. The results obtained can be used for the design of specific materials requiring ad-hoc properties. Although the reference model is a network material, the results obtained can be applied to other similar kinds of microstructures, like pantographic materials, micro devices composed by microbeams etc. They have been limited at the range of linear elasticity, that is small deformation and linear elastic behaviour. Then, numerical simulations were focused on extension tests and bias tests. The obtained deformed configurations are consistent with the literature experimental tests, both for balanced and unbalanced tissues. Moreover, a comparison between first and second gradient numerical predictions was performed. It was observed that second gradient predictions better simulate the experimental evidences.

Plusieurs pays tels que la France, la Belgique et la Suisse prévoient de confiner leurs déchets radioactifs de moyenne et haute activités à vie longue dans des installations souterraines sises au sein de formations argileuses profondes. Ces formations constituent en effet de très bonnes barrières ultimes contre la dispersion des radionucléides, de par leur grande capacité de rétention et leur très faible perméabilité. Néanmoins, la dégradation de certains colis de déchets devrait libérer d’importantes quantités de sels nitratés et sulfatés solubles. Ainsi, ces panaches salins en déséquilibre chimique avec l’encaissant devraient conduire à des phénomènes de dissolution/colmatage, faisant évoluer localement la structure porale de la roche argileuse. Aussi, pour estimer la performance de telles installations souterraines, l’évolution des propriétés de confinement de ces roches en réponse à ces processus physicochimiques se doit d’être étudiée, et ce, sur des échelles de temps et d’espace représentatives du stockage. Cela est réalisé à l’aide de codes couplés chimie-transport basés sur une approche continue, avec la définition de volumes élémentaires représentatifs (VER). Cependant, ces codes s’appuient pour leurs simulations sur des relations empiriques, telles la relation d’Archie, utilisée pour décrire l’effet de rétroaction de la chimie sur les propriétés de transport diffusif. De ce fait, il est primordial, avant les simulations long-termes de tester la robustesse de ces relations. Dans ce cadre, le présent travail de thèse s’est intéressé au développement d’expériences de diffusion réactives pour estimer (i) l’impact de la précipitation de minéraux sur les propriétés de confinement de matériaux poreux “modèles” et (ii) la capacité des codes de chimie-transport à reproduire ce jeu de données expérimentales.La mise au point de ces expériences simplifiées a nécessité de se focaliser sur trois matériaux poreux « modèles », de la craie, de la kaolinite et de l’illite, choisis pour décrire une propriété spécifique des roches argileuses (charges de surface des argiles ou structure du réseau poreux). Par ailleurs, deux minéraux sulfatés, gypse et barytine, ont été sélectionnés comme minéraux susceptibles de précipiter car ils représentent deux extrêmes vis-à-vis de leur cinétique de précipitation et de leur solubilité. Dans un premier temps, les propriétés initiales de chaque matériau « modèle » ont été déterminées (distribution de taille de pores, coefficient de diffusion effectif (De) des traceurs de l’eau (HTO ou HDO) ou d’un traceur des anions (36Cl-)). La précipitation de la barytine a été étudiée sur les trois matériaux « modèles », tandis que celle du gypse uniquement au travers des échantillons de craie. Durant ces expériences de diffusion réactives, l’évolution des concentrations des réactifs dans les deux réservoirs enserrant l’échantillon poreux a été suivie, et, après un temps t, le 36Cl- et/ou les traceurs de l’eau ont été injectés dans le réservoir amont pour diffuser au travers des échantillons déjà impactés par la précipitation. En complément des essais de diffusion, des caractérisations des échantillons par micro-tomographie X (µCT) et par observation au Microscope électronique à Balayage (MEB) ont permis de préciser le rôle joué par la structure porale initiale du matériau « modèle » et celui des propriétés intrinsèques du minéral précipitant. Enfin, l’estimation de la robustesse des codes de chimie transport a été réalisée à l’aide de deux codes, HYTEC et CrunchTope, à l’aide de simulations 1D et 2D. / Several countries such as France, Belgium and Switzerland have proposed to host a deep geological facility to confine high and mid-level long lived radioactive waste into argillaceous formations. Such formations are considered as a potential host-rock, because of their very high containment properties, i.e. high retention capacity and very low permeability. However, decay of some radioactive waste are expected to release large amount of soluble salts of nitrate and/or sulfate nature. These saline plumes should generate physicochemical imbalance and, by enhancing mineral dissolution/clogging, could make evolve the local rock porous network. Thus, for safety assessment of such facility the evolution of rock containment properties in response to these physicochemical phenomena over large time and space scale needs to be investigated. This can be done by using diffusion-reaction numerical simulators based on equivalent (macroscopic) continuum approach considering representative elementary volume (REV). But, these codes rely on empirical relationships, such as the Archie’s law used to describe the feedback of chemistry on the diffusive transport properties. Thus, prior to long-term prediction, it is essential to create a data to test and improve the description of the feedback of chemistry on transport. In this view, this thesis work dealt with developing such reactive diffusion experiments to estimate mineral precipitation impacts on containment properties of porous materials under diffusive transport regime and the capability of REV chemistry transport codes to reproduce such an experimental dataset.In order to design these simplified experiments, three proxy porous materials (micritic chalk, compacted kaolinite and compacted illite) were chosen to address specific property describing claystones (clay surface charge, pore size distribution). Two sulfate-alkali minerals, namely barite and gypsum were selected as precipitating minerals, since they present two extremities in reference to their kinetic rate of precipitation and solubility. In a first step, intact properties of each proxy material were determined (pore size distribution, effective diffusion coefficient (De) of water tracers (HTO & HDO) and anionic tracer, 36Cl-). Barite precipitation was studied in all the proxy materials and gypsum precipitation was studied in chalk only. During these through diffusion experiments, we monitored the reactant concentration evolution in the reservoirs at both ends of the sample and, after a known experimental time, 36Cl- and/or water tracers were allowed to diffuse through the porous samples impacted by precipitation. In addition to diffusive testing, the combined impact of pore structure and intrinsic property of mineral (solubility and kinetic rate of precipitation) on final evolution of mineral in each proxy material was also quantified using X-ray tomography (µCT) and Scanning Electron Microscopy (SEM). Finally, to test the robustness of chemistry transport codes, the results from the reactive diffusion experiments where barite or gypsum precipitated in chalk were numerically described in 1D and 2D using two codes namely HYTEC and CrunchTope.

La première partie de mon travail de thèse a été de définir le rôle joué par le récepteur nucléaire Rev-erb alpha dans les mécanismes de synchronisation par la nourriture d'une horloge circadienne putative, non encore localisée, appelée " horloge alimentaire ". La seconde partie de mon travail a consisté à étudier la participation de Rev-erb alpha dans les régulations des métabolismes glucidique et lipidique. L'ensemble de nos données indique que le répresseur transcriptionnel Rev-erb alpha joue un rôle charnière dans les fonctions circadiennes ainsi que dans le métabolisme. En effet, d'un point de vue circadien, l'absence de Rev-erb alpha altère la synchronisation à l'heure des repas - démontré par une réduction des sorties comportementales et physiologiques de l'horloge alimentaire, ainsi que par l'absence d'ajustement du rythme de la protéine d'horloge PER2 dans l'oscillateur cérébelleux. Sur le plan métabolique, la délétion de ce gène modifie notamment le métabolisme des lipides - démontré par une accumulation excessive de tissu adipeux, une utilisation préférentielle des acides gras, ainsi qu'une perte de contrôle de l'expression de la Lipoprotéine lipase.

HIV-1 has evolved with a great deal of functional complexity contained within a very small genome by encoding small, but critical viral proteins within larger viral genes and dividing the replication cycle into early and late phases to differentially produce all proteins leading to efficient replication and virion release. Early replication is restricted by the host spliceosome that processes HIV-1 vRNA transcripts so only the small intragenomic proteins are produced, one of which is Rev (Regulator of Virion Expression). Rev in turn governs the transition from early to late replication by interacting with a highly structured region of vRNA termed the Rev Response Element (RRE). The binding of Rev to the RRE is believed to cause a change in the vRNA tertiary structure and inhibition of splicing of the vRNA. Once, a Rev:RRE complex is formed, a nuclear export signal within Rev facilitates the export of partially spliced and unspliced vRNA to the cytoplasm. During late replication the partially spliced and unspliced vRNA is translated to polyproteins and is packaged into a budding virion where the viral aspartyl protease (HIV-1 PR) autocatalytically excises itself from the larger polyprotein and processes the remaining polyproteins to release all viral structural and functional proteins to form a mature and infectious virion. Since the vRNA salvaged by Rev is translated to the polyproteins containing HIV-1 PR, the inhibition of Rev function will reduce the amount of HIV-1 PR available and thereby reduce the amount of HIV-1 PR therapeutics required to elicit a clinical effect. Therfore a combination approach to HIV-1 treatment using suitably developed therapeutics that inhibit Rev and HIV-1 PR function represents an attractive synergistic approach to treating HIV-1 infection in vivo. The work of this thesis was divided into two parts, the first part was concerned with HIV-1 PR structural biology and addressing problems encountered with inhibitor design. A bicyclic peptide (based on inhibitors of analogous structure) was co-crystallised with active HIV-1 PR to develop an enzyme-product (E-P) complex and with a catalytically inactive mutant HIV-1 PR to provide an analogy to the enzyme-substrate (E-S) complex. Both structures of the E-P and E-S complexes were solved to 1.6Å resolution and were compared to a hydroxyethylamine isostere enzyme-inhibitor complex (E-I), highlighting the similarity of binding mode for all ligands. The inhibitor in the E-I complex was translated towards the S1 - S3 pockets of the substrate binding cleft relative to the substrate in the E-S complex due to the increased length of the hydroxylethylamine isostere compared to the peptide backbone, although the inhibitor "puckered" the isostere linkage and maintains a binding mode similar to the substrate with very little overall differences in the position of the ligands and surrounding protein. The similarity of the E-S, E-I and E-P complexes was attributed to the macrocyclic ligands ordering the surrounding protein environment, especially the protein -strand "flap" structures that form a roof over the ligands in the active site but were not found to close more tightly in any of the trapped catalytic states. The new structures allowed refinement of details of the mechanism of peptide hydrolysis. The mechanism relies on the optimal nucleophilic attack of a water molecule on the scissile amide bond with concerted acid-base catalysis of the active site aspartyl residues intitiated by D125. The alignment and intrinsic position of the N-terminus of the bicyclic substrate was interpreted as being critical to facilitate efficient electron transfer with the bicyclic substrate. An N-terminal cyclic inhibitor, similar to the N-terminal portion of the bicyclic substrate, was used to address a major problem in HIV-1 PR drug design termed "cooperativity," where the sequential optimisation of an inhibitor (or substrate) to individual pockets of the substrate binding cleft, can negatively impact on adjacent and downfield subsites and thereby alter the binding mode of the "optimised" inhibitor. The technique referred to here as "templating" uses the N-terminal cycle to lock the binding mode into a known conformation, probing the S1' and S2' pockets. The structure activity relationship suggested that by viewing the S1' - S3' pockets as a single trough, bulky aromatic groups attached to an N-alkyl sulfonamide could be directed along the line of the trough without adverse interactions with the tops of the S1' and S3' pockets, providing very potent inhibitors. It was also found that specificity and potency of an inhibitor can be maintained with smaller functionalities that carry their bulk low and close to the inhibitor backbone in the S2' pocket, making the P2 functionalities more substrate-like. The second part of the thesis was concerned with establishing suitable surface plasmon resonance assays for testing potential inhibitors of Rev function. Recombinant Rev and its minimal RNA aptamer target (stem loop II of the RRE termed RBE3), were expressed, purified, and used to develop BIAcore-based assays and test potential inhibitors of their interaction. The system was applied to screening of aminoglycoside antibiotics and other small molecules in a competitive assay, and also to quantitative assay of Neomycin and moderate sized analytes: Rev and three peptidic analogues of the high-affinity binding site of Rev - the native peptide, succinylated form of the peptide and a form incorporating a novel helix-inducing cap. The peptide and protein assay was undertaken to test the proposition that helix induction of the high-affinity binding site of Rev can increase affinity for the biologically important RNA target and thereby form the basis of a new class of inhibitors. The screen of small molecule antagonists found that Neomycin was the best inhibitor of the Rev:RBE3 interaction and that efficacy of other aminoglycosides was due to the neamine-base structure presenting charge to bind to the RNA and blocking interaction with Rev. The quantitative assay was optimised to reduce non-specific interactions of Rev protein to allow reliable studies of the analytes with RBE3 by the sytematic testing of buffers and modifiers. It was found that mutliple analytes bound to the RBE3 aptamer and a comparison of the KD values found that the native and capped peptides had similar affinity for RBE3 RNA (native slightly greater at 21 ± 7nM cf capped 41 ± 10nM) that was greater than the Rev protein (101 ± 19nM), however the succinylated peptide exhibited stronger binding with a KD ≤8nM and Neomycin had the lowest affinity (KD 13 ± 3M). The similarity of the native and capped peptides may be due to the high concentration of salt in the assay buffers and was necessary for the stability of the Rev protein, but is sufficient to influence secondary structure of the peptides. Therefore, it could not be stated that the helix-inducing cap increased the affinity of the native peptide for the biologically important therapeutic target. The work conducted in this thesis firmly establishes foundations for the continued development of inhibitors against both Rev and HIV-1 PR that play key roles in the HIV-1 replication strategy. It is envisaged this work could lead to a novel synergistic therapeutic approach to treating HIV-1 infection.

För att infrastrukturen, gällande trafiken, ska fungera krävs det att vägnätet uppfyller sin funktion. Även ansvarsfördelningen mellan de olika parterna, Trafikverket, kommunerna och enskilda fastighetsägare, är viktig och det är lätt att det uppstår konflikter eller meningsskiljaktigheter. Vilka det är som ska sköta drift och underhåll till vilken väg samt vad lagarna säger om respektive väg är ibland otydligt. Trafikverket, kommuner och enskilda fastighetsägare är de tre olika parter som kan vara ansvariga vad gäller drift och underhåll, även kallat väghållare, av vägar. Om vägen är belägen inom eller utom planlagt område har stor betydelse vad gäller vilken lagstiftning som ska tillämpas. Plan- och bygglagen (PBL) och Väglagen (VägL) är de lagar som används inom planlagt område, men också Anläggningslagen (AL) vid bildande av gemensamhetsanläggningar (GA). Det är främst PBL som tillämpas eftersom den reglerar planläggningen, vägen kan då benämnas gata enligt PBL, men kommunerna kan också välja att planlägga vägen enligt VägL. I de fall VägL tillämpas är processen mer omfattande och för att denna lag ska gälla måste det vara en allmän väg. Utanför planlagt område finns ingen lag som styr på liknande sätt som PBL, här tillämpas AL för de enskilda vägar och då främst gällande bildandet av en GA. Det är vid upprättandet av denna som stadgarna sätts, anläggningsbeslutet reglerar driften och underhållet av vägen som berör de enskilda fastighetsägarna. Studien omfattas av en enkätundersökning som skickades ut till 49 kommuner i Västra Götalands län. Fyra av dessa kommuner valdes sedan ut för vidare fördjupade intervjuer. Därtill intervjuades Trafikverket och Riksförbundet enskilda vägar (REV) intervjuades, detta för att få en övergripande syn och inte endast information från olika kommuner. Resultatet baseras främst på intervjuerna men även svar från enkäterna. Informationen som framkommit under intervjuerna med kommunerna skiljer sig från de svar som erhållits från enkäten vad gäller frågan om kommunerna upplever problem med förvaltning av enskilda vägar. Kommunerna menar istället att det generellt är mer problem inom planlagt område än utom. Resultatet tyder även på en variation mellan kommunerna medan Trafikverket har tydliga ramar för kriterier vad gäller val av väghållare. REV tar upp att det förekommer problematik kring svårigheten att få personer att ordna styrelsearbetet rörande enskild väg. Definitionen av allmän och enskild väg skiljer sig mellan de olika intervjuade parterna. I studien reflekteras det kring tydligare riktlinjer för när en väg ska vara allmän respektive enskild för att tydliggöra gränsen för när ett väghållaransvar tar vid och ett annat tar slut. / In terms of traffic, in order for the infrastructure to work it requires that the road network fulfills its function. Communication between the different parties, the Swedish Transport Administration (Trafikverket), municipalities and individual property owners is important, and situations with conflicts and disagreement are easy to evolve. Which part should manage the operation and maintenance to a certain road and what the laws say about each road are sometimes unclear. The Swedish Transport Administration, municipalities and individual property owners are the three different parties that can be responsible for operation and maintenance, also known as road holders, by roads. If the road is located within or outside of the planned area depends on which legislation is applicable. Planning and building act (PBL) and road legislation (VägL) are the laws used inside the planned area, but so is the plant legislation (AL) in the formation of community facilities. It is mainly the planning and building act that is applied because it regulates the planning, the road is then named street according to PBL, but in some cases the municipalities may choose to plan the road according to the road legislation. In cases where the road legislation is applied the process is more extensive and for this law to apply, the roads must be public. Outside planned area there is no law that regulates in the same way as the planning and building act, instead the plant legislation is applied for the individual roads mainly to establish a community facility. It is at the establishment of the community facilities the statutes are set, these regulate the operation and maintenance of the road which concerns the individual property owners. The study includes a survey conducted to 49 municipalities in the county Västra Götaland. Four of these municipalities were then selected for a further interview. The Swedish Transport Administration and National federation individual roads (Riksförbundet enskilda vägar) were also interviewed, this in order to get an overall view and not only information from different municipalities.The result is based primarily on the interviews but also the answers from the survey. The information that emerged from the interviews with the municipalities differs from the answers received from the survey whether the municipalities experience problems with managing individual roads. Instead, the municipalities mean that there are generally more problems in the planned area than outside. The result also indicates a variation between the municipalities, while the Swedish Transport Administration has clear limits on the selection of road holders. REV addresses the problem of the difficulty of getting people to arrange  ii Allmänna och enskilda vägar – Väghållaransvar utanför detaljplanelagt område  the board work on an individual road. The definition of public and individual road differs between the different interviewed parties. The study reflects on distinct guidelines for when a road should be public respectively individual in order to clarify the boundary of when road maintenance takes over and another ends.

Dans la cellule, les ARN pré messagers contenant des introns sont normalement retenus au noyau par leur interaction avec des facteurs d’épissage. Cependant, les ARN partiellement et non épissés des rétrovirus doivent entrer dans le cytoplasme pour servir de matrice pour la synthèse de certaines protéines telles que Env, Gag et Gag-Pol ainsi que d’ARN génomique qui sera empaqueté dans les nouveaux virions. Un mécanisme post-transcriptionnel utilisé par les lentivirus pour éviter la séquestration nucléaire de ces ARNm dépend d’une protéine virale appelée Rev. Pour assurer sa fonction d’exportation, Rev doit transiter entre le noyau et le cytoplasme et doit aussi pouvoir former des multimères. Par conséquent, Rev est dotée de domaines fonctionnels lui procurant ces habiletés. On retrouve le domaine riche en arginines qui contient le domaine de liaison à l’ARN et le signal de localisation nucléaire (NLS), un second domaine, riche en leucines, porte le signal d’exportation nucléaire (NES) et finalement le domaine de multimérisation. Bien que les protéines Rev du virus de l’immunodéficience humaine de type 1 (VIH-1) et bovine (VIB) aient été caractérisées, aucune étude n’a été réalisée pour la protéine Rev du virus de la maladie de Jembrana (JDV) et très peu sur le virus de l’immunodéficience féline (VIF). Comme les domaines fonctionnels et la voie d’importation des protéines Rev déjà caractérisées sont différents, nous supposons que chaque protéine Rev possède une organisation qui lui est propre et que les mécanismes de transport nucléo-cytoplasmique diffèrent entre les virus. Ce projet a pour objectif de caractériser ces domaines pour la protéine Rev du JDV et ceux du VIF ainsi que les mécanismes permettant leur transport nucléaire. L’utilisation de mutants de la protéine Rev de ces virus couplés à la protéine de fluorescente verte (EGFP) exprimés dans des cellules appropriées et observés par microscopie a permis d’identifier des séquences NLS et NES différentes de celles déjà caractérisées. Le NLS de la protéine Rev du JDV a été identifié et est composé des résidus arginines de la séquence 76-RRPARRPPIRR-87 avec un NoLS composé des mêmes résidus en plus des arginines R74, R103 et R104. Son NES est composé des résidus hydrophobes de la séquence 116-MAELEERFEDLAL-128 et est du type de l’inhibiteur de la protéine kinase (PKI pour « protéine kinase inhibitor »). Pour la protéine Rev du VIF, son NLS est composé des résidus basiques de la séquence 84-KKKRQRRRRKKKAFKK-99. Le NoLS est composé des mêmes acides aminés en plus du résidu K82. De plus, les essais d’importation nucléaires et d’interaction semblent indiquer que les voies d’importation utilisées diffèrent entre les virus et que plusieurs voies peuvent être utilisées. Ces travaux pourront éventuellement servir de base pour identifier de nouvelles cibles thérapeutiques contre les lentivirus. / In the cell, pre-messenger RNAs containing introns are normally retained in the nucleus by their interaction with splicing factors. However, the partially and unspliced ​​RNAs of retroviruses must enter the cytoplasm to serve as a template for the synthesis of certain proteins such as Env, Gag and Gag-Pol as well as genomic RNA to be packaged in the new virions. A post-transcriptional mechanism used by lentiviruses to prevent nuclear sequestration of these mRNAs depends on a trans-activator, the viral protein Rev. To ensure its export function, Rev must be able to shuttle between the nucleus and the cytoplasm and to form multimers. As a result, Rev has functional domains that provide these abilities: the arginine-rich domain, which contains the RNA binding domain and the nuclear localization signal (NLS), a second domain, rich in leucine, corresponding to the nuclear export signal (NES) and finally the multimerization domain. Although the Rev proteins of the human and bovine immunodeficiency virus (HIV-1 and BIV respectively) have been characterized, no studies have been performed for the Jembrana disease virus (JDV) Rev protein and very little on the feline immunodeficiency virus (FIV). Since the functional domains and import pathway of the already characterized Rev proteins are different, we assume that each Rev protein has its own organization and that the nucleo-cytoplasmic transport mechanisms differ between viruses. The goal of this project is to characterize these domains for the JDV and FIV Rev proteins as well as to elucidate mechanisms for their nuclear transport. The use of Rev mutants fused to the EGFP expressed in appropriate cells and observed by microscopy has identified NLS and NES sequences that differ from those already characterized. JDV Rev NLS is composed of arginine residues in the 76-RRPARRPPIRR-87 sequence with a NoLS composed of the same residues with the addition of arginine R74, R103 and R104. JDV Rev NES is composed of hydrophobic residues in the 116-MAELEERFEDLAL-128 sequence and is of the protein kinase inhibitor type (PKI). For the FIV Rev protein, its NLS is composed of basic residues in the 84-KKKRQRRRRKKKAFKK-99 sequence. FIV Rev NoLS is composed of the same residues with the addition of the lysine at position 82. In addition, the nuclear import and interaction tests suggest that the import routes used by Rev differ between the different viruses studied and that more than one import pathway may be used. This work could serve as a basis for identifying new therapeutic targets against lentiviruses.