Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting the upper and lower motor neurons causing muscle atrophy and paralysis followed by death. Aggregates containing superoxide dismutase (SOD1) are found as pathological hallmark in diseased ALS patients. Consequently ALS is regarded as a protein misfolding disorder like Alzheimer’s disease and Parkinson’s disease. So far, little is known about the cause and mechanism behind SOD1 aggregation but the inherent property of all polypeptide chains to form stable aggregated structures indicates that the protein misfolding diseases share a common mechanism. Our results show that SOD1 aggregation starts from the globally unfolded state, since fibrillation is fastest at full occupancy of denatured protein induced either by chemical denaturation or mutation. Even so, the fibrillation rate shows a surprisingly weak dependence on the concentration of globally unfolded SOD1 indicating fibril fragmentation as the dominant mechanism for aggregate formation. This is further supported by the observation that the SOD1 sample has to be mechanically agitated for fibrillation to occur.  Interestingly, we observe a similar SOD1 aggregation behaviour in vivo, where the survival times of ALS transgenic mice correlates with mutant stability, and aggregate growth depends weekly on the concentration of unfolded monomer. Additionally, in-cell NMR measurements reveal that in live cells the thermodynamic equilibrium is shifted towards the unfolded state of SOD1, which is also more fully extended than in vitro. This suggests that the globally unfolded aggregation competent protein is more abundant in the crowded environment in vivo than dilute in vitro conditions. Finally, antibody analysis of aggregates from ALS transgenic mice reveals the existence of aggregate strains involving different parts of the protein depending on mutation, which may offer an explanation for the various disease phenotypes observed in ALS. Altogether these findings provide important clues for understanding SOD1 aggregation with implications for ALS, as well as other protein misfolding diseases.

O setor de telefonia vem passando por transformações em seus canais de vendas nos últimos anos, o que tem afetado a forma como as competências organizacionais e funcionais são transmitidas para toda a rede de canais de distribuição das operadoras. A presente pesquisa visa a identificar e descrever quais são as principais lacunas existentes no desdobramento das competências organizacionais e funcionais de agentes autorizados que apresentam contrato de representantes comerciais junto a uma operadora de telefonia móvel do Estado do Rio Grande do Sul. Para tanto, a pesquisa envolveu a percepção tanto de proprietários quanto de funcionários da operadora, a fim de comparar tais visões e, assim, identificar pontos de convergência e divergência que pudessem se caracterizar em lacunas no desdobramento das competências. Foram entrevistadas 11 pessoas, sendo seis proprietários de agentes autorizados e cinco funcionários da operadora. A partir das entrevistas, foram identificadas 13 variáveis significativas quanto ao desdobramento das competências, que foram posteriormente reagrupadas em seis categorias de análise, realizando-se a identificação e descrição das percepções de cada um dos grupos de forma individualizada (em um primeiro momento), para posteriormente se proceder à comparação de tais visões. A partir desta comparação frente às seis categorias é que foi possível identificar as 22 lacunas que delas emergiram e que formam os principais fatores a serem desenvolvidos pela operadora para que haja o desdobramento das competências. / Over the last few years, the telephony industry has been experiencing transformations in its sales channels. Such adjustments have caused an impact on the way functional and organizational competences are transferred to the operator’s distribution channel networks. The present research intends to identify and delineate the main gaps that arise in the unfolding of functional and organizational competences with the authorized dealers who hold a business representative contract with a mobile telephony operator company in Rio Grande do Sul, Brazil. In order to identify the converging and diverging aspects that could constitute gaps in the unfolding of competences, this research confronted both the perceptions of the authorized dealer’s and the operator company employee’s. A total of 11 people were interviewed, 6 of whom were authorized dealers and 5 were employees of the operator company. From this interview’s, 13 important variables were initially identified and later regrouped into 6 categories for analysis. Individualized identification and description of the perceptions of each group was performed at first and comparison of the different points of view followed. From such comparison, it was possible to identify 22 points of divergence which comprise the main parameters to be improved by the operator so that the unfolding of competences can happen in a satisfactory manner.

An examination of the logical relationships between attitude statements suggests that attitudes can be ordered according to favourability, and can also stand in relationships of implication to one another. The traditional representation of attitudes, as points on a single dimension, is inadequate for representing both these relations but representing attitudes as intervals on a single dimension can incorporate both favourability and implication. An interval can be parameterised using its two endpoints or alternatively by its midpoint and latitude. Using this latter representation, the midpoint can be understood as the �favourability� of the attitude, while the latitude can be understood as its �generality�. It is argued that the generality of an attitude statement is akin to its latitude of acceptance, since a greater semantic range increases the likelihood of agreement. When Coombs� Theory of Unidimensional Unfolding is reformulated using the interval representation, the key question is how to measure the distance between two intervals on the dimension. There are innumerable ways to answer this question, but the present study restricts attention to eighteen possible �distance� measures. These measures are based on nine basic distances between intervals on a dimension, as well as two families of models, the Minkowski r-metric and the Generalised Hyperbolic Cosine Model (GHCM). Not all of these measures are distances in the strict sense as some of them fail to satisfy all the metric axioms. To distinguish between these eighteen �distance� measures two empirical tests, the triangle inequality test, and the aligned stimuli test, were developed and tested using two sets of attitude statements. The subject matter of the sets of statements differed but the underlying structure was the same. It is argued that this structure can be known a priori using the logical relationships between the statement�s predicates, and empirical tests confirm the underlying structure and the unidimensionality of the statements used in this study. Consequently, predictions of preference could be ascertained from each model and either confirmed or falsified by subjects� judgements. The results indicated that the triangle inequality failed in both stimulus sets. This suggests that the judgement space is not metric, contradicting a common assumption of attitude measurement. This result also falsified eleven of the eighteen �distance� measures because they predicted the satisfaction of the triangle inequality. The aligned stimuli test used stimuli that were aligned at the endpoint nearest to the ideal interval. The results indicated that subjects preferred the narrower of the two stimuli, contrary to the predictions of six of the measures. Since these six measures all passed the triangle inequality test, only one measure, the GHCM (item), satisfied both tests. However, the GHCM (item) only passes the aligned stimuli tests with additional constraints on its operational function. If it incorporates a strictly log-convex function, such as cosh, the GHCM (item) makes predictions that are satisfied in both tests. This is also evidence that the latitude of acceptance is an item rather than a subject or combined parameter.

Le Modèle Standard (MS) de la physique de particules avec les théories de jauge pour les interactions forte et électrofaible n'a pas encore été mis en défaut par les données expérimentales. Dans les recherches de nouvelle physique, au delà du MS, deux approches sont en général suivies : les recherches exploratoires à la frontière de haute énergie et des tests de précision à des énergies plus basses. Le but de cette thèse est dans le cadre de la deuxième approche, d'obtenir et utiliser des données précises des annihilations e+e− en hadrons, à des énergies de l'ordre 1 GeV. Ces données représentent un ingrédient important pour les tests du MS impliquant la polarisation du vide, comme par exemple la comparaison du moment magnétique du muon avec la théorie, ainsi que pour des tests de QCD et applications. Les différentes parties de cette thèse décrivent quatre aspects de mon travail dans ce contexte. (1) Les mesures de sections efficaces en fonction de l'énergie nécessitent la déconvolution des spectres de données des effets de détecteur. Je propose une nouvelle méthode itérative de déconvolution des données expérimentales, qui présente des améliorations par rapport aux outils existants. On peut déconvoluer, d'une manière dynamiquement stable, des spectres de données qui peuvent être fortement affectées par des fluctuations dans la soustraction du bruit de fond, et simultanément reconstruire des structures qui n'étaient pas initialement simulées. (2) Le coeur expérimental de cette thèse est constitué par l'étude du processus e+e− en K+K−, du seuil jusqu'à 5 GeV, utilisant la méthode de rayonnement dans l'état initial (ISR), par la mesure de e+e− en K+K− gamma avec le détecteur BABAR. Toutes les efficacités utiles sont mesurées utilisant les données expérimentales, et la normalisation absolue est fournie par la mesure simultanée du processus mu mu gamma. J'ai effectué l'analyse complète où une incertitude systématique de 0.7 % a été obtenue sur la résonance dominante, phi. Le facteur de forme du kaon chargé a été mesuré. Il présente une décroissance rapide au delà du phi et des structures distinctes dans la région 1.7-2.5 GeV, où on connaît l'existence de résonances de type vecteur. La dépendance en énergie, à haute masse, est comparée à la prédiction de QCD. On présente aussi les résultats du canal e+e− en pi+pi− , du seuil jusqu'à 3 GeV, pour lequel j'ai effectué la déconvolution des effets de détecteur et j'ai obtenu les résultats finals qui sont comparés avec les données existantes. (3) La prédiction pour le moment magnétique du muon (exprimée par l'intermédiaire de son 'anomalie', i.e. la déviation par rapport à la valeur de Dirac pour le rapport gyromagnétique égale à 2) est calculée dans le cadre su MS. Notre travail concerne seulement la contribution de polarisation hadronique du vide, obtenue à partir des données e+e− par une intégrale de dispersion. Comme la même information peut en principe être obtenue avec des données sur les désintégrations hadroniques du tau, on fait d'abord la mise à jour de la comparaison entre les utilisations des deux sources de données, et on trouve une différence réduite entre les évaluations correspondantes. Le nouveau résultat basé sur les données tau s'écarte de 1.9 déviations standard de la mesure directe. En suite, les nouvelles données précises de BABAR sont inclues dans une analyse combinée utilisant des outils (avec une procédure d'interpolation et moyennage de données améliorée, une propagation des incertitudes plus rigoureuse et une validation systématique) que j'ai développés. Avec les nouvelles données, le désaccord entre les résultats basés sur des données e+e− et tau pour le mode dominant 2 pi est réduit davantage, du 2.4 sigma précédent à 1.5 sigma, dans l'intégrale de dispersion, bien que des différences locales significatives entre les spectre persistent encore. Nous obtenons l'évaluation basée sur des données e+e− a_μ^had LO = (695.5 +- 4.1)*10^-10, où l'incertitude prend en compte toutes les sources. L'incertitude la plus grande pour la prédiction du MS est encore due à la polarisation hadronique du vide, mais elle est maintenant plus petite que l'erreur expérimentale. La comparaison actuelle entre la mesure directe et notre prédiction (basée sur les données e+e−) montre une indication intéressante de nouvelle physique (un effet à 3.2 sigma). (4) Les règles de somme de QCD sont des outils puissants pour obtenir des informations précises sur les paramètres de QCD, comme la constante de couplage fort α_S. Cette étude devrait faire usage de la mesure complète de e+e− en hadrons jusqu'à environ 2 GeV. Comme BABAR n'a pas encore mesuré complètement tous les processus hadroniques, j'ai travaillé sur une situation similaire utilisant les fonctions spectrales des désintégrations hadroniques du tau, mesurées par ALEPH. Je discute en détail la prédiction de QCD perturbatif, obtenue avec deux méthodes différentes : la théorie de perturbations à ordre fixe (FOPT) et la théorie de perturbations avec intégration de contour améliorée (CIPT). Les incertitudes théoriques correspondantes sont étudiées aux échelles de masse du tau et respectivement du Z. On trouve que la méthode CIPT est plus stable par rapport aux contributions manquantes d'ordre supérieur et par rapport à des changements de l'échelle de renormalisation. Il est également montré que FOPT souffre à cause de problèmes de convergence le long du contour d'intégration. La fiabilité d'une classe de modèles pour la fonction d'Adler, basée sur des renormalons, est étudiée dans le contexte de la comparaison entre CIPT et FOPT. On trouve que ces modèles ne sont pas suffisamment contraints pour qu'ils puissent identifier la meilleure méthode à utiliser pour la détermination de α_S(m_τ^2). La détermination de αS est mise à jour et une valeur très précise pour α_S(m_τ^2) est obtenue en utilisant CIPT (0.344 +- 0.005 (exp) +- 0.007 (th)). Une fois évoluée à la masse du Z, cette valeur est en accord avec α_S(m_Ζ^2) mesurée directement à partir de la largeur du Z. Ce résultat représente le test le plus précis du running de α_S en QCD.

<p>This thesis is based on studies, described in four papers, in which the folding energy landscape of MerP was investigated by various techniques. MerP is a water-soluble 72 amino acid protein with a secondary structure consisting of four anti-parallel β-strands and two α-helices on one side of the sheet in the order β1α1β2β3α2β4. </p><p>The first paper describes the use of CD and fluorescence analysis to examine the folding/unfolding process of MerP. From these experiments it was found that the protein folds according to a two-state model in which only the native and unfolded forms are populated without any visible intermediates. With a rate constant of 1.2 s^-1, the folding rate was found to be unusually slow for a protein of this size.</p><p>The studies presented in the second and third papers were based on measurements of native-state amide proton exchange at different temperatures (Paper II) and GuHCl concentrations (Paper III) in the pre-transitional region. In these studies partially unfolded forms were found for MerP which are essentially unrelated to each other. Thus, in the folding energy landscape of MerP, several intermediates seem to occur on different folding trajectories that are parallel to each other. The slow folding rate of MerP might be coupled to extensive visitation of these conformations. Hydrogen exchange in MerP did also reveal structure-dependent differences in compactness between the denatured states in GuHCl and H₂O.</p><p>In the last paper multivariate data analysis was applied to 2-dimensional NMR data to detect conformational changes in the structure of MerP induced by GuHCl. From this analysis it was suggested that regions involved in the most flexible part of the protein structure are disrupted at rather low denaturant concentrations (< 2.1 M GuHCl) while the native structures of the most stable parts are still not completely ruptured at 2.9 M GuHCl.</p><p>Finally, the stability, kinetics, contact order and folding nuclei of six proteins with similar topology (MerP, U1A, S6, ADA2h, AcP and HPr) were compared. In this analysis it was found that their folding properties are quite diverse, despite their topological similarities, and no general rules that have been formulated yet can adequately predict their folding behaviour.</p>

It is well known that folded proteins in water are destabilized by the addition of urea. When a protein loses its ability to perform its biological activity due to a change in its structure, it is said to denaturate. The mechanism by which urea denatures proteins has been thoroughly studied in the past but no proposed mechanism has yet been widely accepted. The topic of this thesis is the study of the mechanism of urea-induced protein denaturation, by means of Molecular Dynamics (MD) computer simulations and Nuclear Magnetic Resonance (NMR) spectroscopy. Paper I takes a thermodynamic approach to the analysis of protein – urea solution MD simulations. It is shown that the protein – solvent interaction energies decrease significantly upon the addition of urea. This is the result of a decrease in the Lennard-Jones energies, which is the MD simulation equivalent to van der Waals interactions. This effect will favor the unfolded protein state due to its higher number of protein - solvent contacts. In Paper II, we show that a combination of NMR spin relaxation experiments and MD simulations can successfully be used to study urea in the protein solvation shell. The urea molecule was found to be dynamic, which indicates that no specific binding sites exist. In contrast to the thermodynamic approach in Paper I, in Paper III we utilize MD simulations to analyze the affect of urea on the kinetics of local processes in proteins. Urea is found to passively unfold proteins by decreasing the refolding rate of local parts of the protein that have unfolded by thermal fluctuations. Based upon the results of Paper I – III and previous studies in the field, I propose a mechanism in which urea denatures proteins mainly by an enthalpic driving force due to attractive van der Waals interactions. Urea interacts favorably with all the different parts of the protein. The greater solvent accessibility of the unfolded protein is ultimately the factor that causes unfolded protein structures to be favored in concentrated urea solutions.

Anthraquinone (AQ) derivatives with relatively high reduction potentials have been synthesized to afford good candidates for electron transfer studies in DNA. Electron withdrawing groups on the anthraquinone ring gave derivatives with less negative reduction potentials. The anthraquinone imide (AQI) derivatives had reduction potentials less negative than AQ derivatives. The AQI ring system was subject to base-induced hydrolysis. Water-soluble sulfonated tetraarylporphyrins have been studied in a wide variety of contexts. Herein, we report the first synthesis of a pentasulfonated porphyrin bearing an internal cyclic sulfone ring. Treatment of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (TPPS4) with fuming H2SO4 gave a structure consistent with initial sulfonation followed by dehydration to give a sulfone bridge between an ortho-position of one of the phenyl groups and a β-pyrrole position on the porphine ring (TPPS4Sc). The structure was established by ESI-MS and 1HNMR. The Soret absorption is red shifted by about 32 nm compared to that of TPPS4. Streptococcus pyogenes obtains iron by taking up heme from the environment during infection. One of the heme uptake pathways is the Sia or Hts pathway. The initial protein in this pathway is Shr, which has two heme-binding NEAT domains, NEAT1 nearer the N-terminus, and NEAT2 nearer the C-terminus. We report biophysical characteristics of these two NEAT domains. To assess stability of this domain towards heme release, denaturation studies of the Fe(II) and Fe(III) forms were performed. For each domain, both the Fe(II) and the Fe(III) forms behave similarly in thermal denaturation and guanidinium denaturation. Overall, NEAT2 is more stable than NEAT1. Spectral signatures, sequence alignment and homology modeling for both domains suggest that one of the axial ligands is methionine. NEAT2 autoreduces as the pH increases and autooxidizes as the pH decreases. Heme uptake from the host environment is the only iron acquisition pathway in S. pyogenes; inhibition of this pathway might be an approach to infection control. Compounds that might inhibit the heme uptake pathway were selected via virtual screening.

Particle physics experiments with low event rates highly depend on background suppression methods. Neutron component of the ambient background radiation is especially problematic since neutrons are difficult to shield directly. TEXONO collaboration has employed a hybrid neutron detector composed of two different scintillating materials to measure the neutron background directly in the Kuo-Sheng Neutrino Laboratory. Detector is operated after calibration and optimization studies and from collected data, neutron flux is obtained using computational methods.

Atomic force microscopy (AFM) is a powerful technique that enables to study biological macromolecules and dynamic biological processes at different scales. It is an excellent tool for imaging of biological objects under various conditions at a nanometer resolution. Force mode of AFM, so called single molecule force spectroscopy (SMFS), allows for investigation of the strength of molecular interactions of different origins established between and within biological molecules. In the present work, SMFS was used to detect and locate structurally and functionally important interactions of sodium/glycine betaine transporter BetP of Corynebacterium glutamicum, which serves as a model system for this class of proteins. Mechanical pulling of BetP molecules embedded into the lipid membranes resulted in a step-wise unfolding of the protein and revealed insights into its structural stability. Effect of the lipid environment, N- and C-terminal extensions on inramolecular interactions of BetP as well as protein activation and ligand binding were investigated in great detail. In another part of this work, I demonstrate an application of the AFM based technique that can record unfolding of a protein under force-clamp conditions. This method directly measures the kinetics of the protein unfolding, allowing for the use of simple methods to analyze the data. For the first time the force-clamp technique was used to describe in detail unfolding kinetics of the membrane protein, i. e. Na+/H+-antiporter NhaA from Escherichia coli. Performed here experiments on NhaA in its functionally active and inactive states demonstrated the advantages of examining unfolding kinetics at the single-molecule level. It was possible to observe unfolding events for pH-activated conformation of NhaA that due to the low frequency of occurrence were not represented in the ensemble average of the single-molecule measurements. As mechanical unfolding, similarly to bond rupture, is a force-dependent process, force-clamp technique can allow for a more direct way of probing protein unfolding and is anticipated to be also useful to examine the folding/unfolding kinetics of other membrane proteins.

Assembly of the extracellular matrix (ECM) protein fibronectin (FN) is a mechanical process that involves cell binding to FN through cell surface integrin receptors and application of tensional forces generated in the cell's contractile actin cytoskeleton. Deformation-induced exposure of cryptic sites, defined as buried molecular recognition sites, in FN has been proposed as a mechanism by which cell tension drives FN fibrillogenesis. The primary integrin attachment site on FN is the RGD loop in the 10FNIII domain. In this thesis, I set out to define the molecular biophysical mechanism by which cell tension application at the RGD site promotes unfolding and thereby induces FN-FN self-assembly leading to matrix fibril formation. Chapter 1 of this dissertation provides an overview of the current knowledge behind the biophysical and molecular basis of FN assembly in the ECM and its key role in development and disease. In Chapter 2, steered molecular dynamic simulations show that the 10FNIII domain under force applied through its N-terminus and RGD loop (N-to-RGD) unfolds to a preferred kinetic intermediate with solvent-exposed N-terminal hydrophobic residues in a manner different from past analyses in the literature where force through the N- and C- termini leads to multiple unfolding pathways. Use of single-molecule atomic force spectroscopy in Chapter 3 experimentally reveals that a mechanically stable intermediate of 10FNIII exposed by N-to-RGD pulling shows a length extension that agrees with the predicted kinetic intermediate. Results of biochemical and cellular studies using synthetic peptides with sequences from the 10FNIII intermediate show in Chapter 4 that the twenty-three amino acid sequence that spans the unraveled N-terminus of the predicted intermediate mediates FN multimerization and contains a minimal seven amino acid sequence we call the multimerization motif that is sufficient to induce FN-FN multimer assembly. Finally, Chapter 5 summarizes the new insights supported by this work regarding the role that mechanical forces applied at the cell binding site in 10FNIII plays in the physiological unfolding of FN with respect to FN fibrillogenesis and ECM assembly.