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Deploying Monitoring Trails for Fault Localization in All-optical Networks and Radio-over-Fiber Passive Optical NetworksMaamoun, Khaled M. 24 August 2012 (has links)
Fault localization is the process of realizing the true source of a failure from a set of collected failure notifications. Isolating failure recovery within the network optical domain is necessary to resolve alarm storm problems. The introduction of the monitoring trail (m-trail) has been proven to deliver better performance by employing monitoring resources in a form of optical trails - a monitoring framework that generalizes all the previously reported counterparts. In this dissertation, the m-trail design is explored and a focus is given to the analysis on using m-trails with established lightpaths to achieve fault localization. This process saves network resources by reducing the number of the m-trails required for fault localization and therefore the number of wavelengths used in the network. A novel approach based on Geographic Midpoint Technique, an adapted version of the Chinese Postman’s Problem (CPP) solution and an adapted version of the Traveling Salesman’s Problem (TSP) solution algorithms is introduced. The desirable features of network architectures and the enabling of innovative technologies for delivering future millimeter-waveband (mm-WB) Radio-over-Fiber (RoF) systems for wireless services integrated in a Dense Wavelength Division Multiplexing (DWDM) is proposed in this dissertation. For the conceptual illustration, a DWDM RoF system with channel spacing of 12.5 GHz is considered. The mm-WB Radio Frequency (RF) signal is obtained at each Optical Network Unit (ONU) by simultaneously using optical heterodyning photo detection between two optical carriers. The generated RF modulated signal has a frequency of 12.5 GHz. This RoF system is easy, cost-effective, resistant to laser phase noise and also reduces maintenance needs, in principle. A revision of related RoF network proposals and experiments is also included. A number of models for Passive Optical Networks (PON)/ RoF-PON that combine both innovative and existing ideas along with a number of solutions for m-trail design problem of these models are proposed. The comparison between these models uses the expected survivability function which proved that these models are liable to be implemented in the new and existing PON/ RoF-PON systems. This dissertation is followed by recommendation of possible directions for future research in this area.
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Cysteine residues of the mammalian GET receptor: Essential for tail-anchored protein insertion?Schaefer, Moritz 30 May 2017 (has links)
No description available.
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Modelování závislosti mezi hydrologickými a meteorologickými veličinami měřenými v několika stanicích / Modelling dependence between hydrological and meteorological variables measured on several stationsTurčičová, Marie January 2012 (has links)
Title: Modelling dependence between hydrological and meteorological variables measured on several stations Author: Bc. Marie Turčičová Department: Department of Probability and Mathematical Statistics Supervisor: Prof. RNDr. Daniela Jarušková CSc., Czech Technical University in Prague, Faculty of Civil Engineering, Department of Mathematics Abstract: The aim of the thesis is to explore the dependence of daily discharge averages of the Opava river on high daily precipitation values in its basin. Three methods are presented that can be used for analyzing the dependence between high values of random variables. Their application on the studied data is also given. First it is the tail-dependence coefficient that measures the dependence between high values of two continuous random variables. The model for the high quantiles of the discharge at a given precipitation value was first determined non-parametrically by quantile regression and then parametrically through the peaks-over-threshold (POT) method. Keywords: extremal dependence, tail-dependence coefficient, quantile regression, peaks over threshold method
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Měření systémového rizika v časově-frekvenční doméně / Measuring systemic risk in time-frequency domainMuzikářová, Ivana January 2015 (has links)
This thesis provides an analysis of systemic risk in the US banking sector. We use conditional value at risk (∆CoVaR), marginal expected shortfall (MES) and cross-quantilogram (CQ) to statistically measure tail-dependence in return series of individual institutions and the system as a whole. Wavelet multireso- lution analysis is used to study systemic risk in the time-frequency domain. De- composition of returns on different scales allows us to isolate cycles of 2-8 days, 8-32 days and 32-64 days and analyze co-movement patterns which would oth- erwise stay hidden. Empirical results demonstrate that filtering out short-term noise from the return series improves the forecast power of ∆CoVaR. Eventu- ally, we investigate the connection between statistical measures of systemic risk and fundamental characteristics of institutions (size, leverage, market to book ratio) and conclude that size is the most robust determinant of systemic risk.
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Évaluation du lien entre la caudophagie et le microbiote intestinal chez le porcRabhi, Nassima 07 1900 (has links)
No description available.
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Desenvolvimento de um sistema modificado de suspensão do rato pela cauda, como modelo de osteopenia / Development of a modified system of tail suspension of the rat as a model of osteopenia.Falcai, Mauricio José 19 August 2011 (has links)
Introdução: A suspensão do rato pela cauda é método usado para simular os efeitos da microgravidade e hipoatividade física sobre o sistema musculoesquelético e outros sistemas. O método convencional usa a tração cutânea para a fixação da cauda do animal ao sistema de suspensão, sendo idealmente aplicado durante até três semanas. Depois desse período surgem lesões cutâneas, situações estressantes e soltura dos animais. Estes fatos limitam observações por períodos mais longos. O objetivo deste trabalho foi propor e avaliar um sistema de suspensão do rato pela cauda que utiliza tração esquelética com fio de Kirschner atravessado na vértebra caudal, comparando sua eficiência como modelo de osteopenia com a tração cutânea convencional, durante três e seis semanas. Metodologia: 60 ratas foram distribuídas em seis grupos (n=10): GI - três semanas-suspensão pela cauda em tração esquelética; GII - três semanas-suspensão em tração cutânea; GIII - três semanas sem suspensão; GIV - seis semanas-suspensão em tração esquelética; GV - seis semanas-suspensão em tração cutânea; GVI - seis semanas sem suspensão. Avaliação foi clínica com preenchimento de lista diária de achados de estresse e exame post-mortem com determinação dos níveis de corticosterona plasmática e estado da mucosa gastroesofágica. Avaliação dos efeitos da suspensão sobre osso ocorreu por meio da determinação da densidade mineral óssea, ensaio mecânico e histomorfometria, realizados tanto no fêmur, quanto no úmero. Resultados: não houve diferença estatisticamente significante entre os grupos suspensos observados durante três semanas, para quaisquer dos parâmetros investigados. Entretanto, em seis semanas, sete animais (70%) em tração cutânea foram perdidos por lesões de pele e, na tração esquelética, apenas um (10%). Quanto ao ganho de peso corporal e os outros parâmetros clínicos não houve diferenças entre os grupos suspensos por seis semanas. A densidade mineral óssea, força máxima, rigidez e parâmetros histomorfométricos dos fêmures diminuíram até três semanas quando os animais suspensos foram comparados com os controles. Entretanto, depois estabilizaram, tanto para os animais suspensos pela tração cutânea, quanto esquelética, sem diferenças entre eles. No úmero não houve diferenças importantes entre os animais suspensos e os controles. Conclusão: O sistema de tração esquelética foi mais eficiente para manter os animais suspensos até seis semanas, quando o número de complicações foi menor que na tração cutânea. A eficiência de ambos os métodos de suspensão em termos de enfraquecimento ósseo foi semelhante em ambos grupo. / Background: Suspension of the rat by the tail is a method that is used to simulate the effects of microgravity and physical hypoactivity on the musculoskeletal system and also on other systems. The conventional suspension method uses the skin traction for fixing the animal to the suspension system and it is ideally applied for three weeks. After this period of time, skin lesions, stressful conditions and animal loosening may occur. These facts limit observations for longer periods of time. The aim of the present study was to propose and evaluate a rat tail suspension system using skeletal traction with a crossing Kirschner wire in the tail vertebra and to compare it with the conventional skin traction method, during three and six weeks. Methods: 60 rats allocated in six groups (n = 10): GI - three-week tail suspension in skeletal traction; GII - three week skin traction-suspension; GIII - three weeks without suspension; GIV - six-week suspension skeletal traction; GV - six weeks in cutaneous traction, GVI - six weeks without any suspension. Clinical evaluation was made filling up a daily list of findings of stress indicators and, at the end of the experimental period, by post-mortem examination, with determination of plasma corticosterone levels and status of the gastroesophageal mucosa. Evaluation of the effects of suspension on bone was carried out by the determination of bone mineral density, histomorphometry and mechanical tests that were conducted both in femurs and humerus. Results: no statistically significant difference was observed between groups for three weeks for the suspended animals, for any of the parameters investigated. In six weeks, seven suspended animals in skin traction were lost by skin lesions (70%) and, in skeletal traction, one (10%). As for the weight gain and other clinical parameters no differences was observed between the suspended groups. Comparison between suspended and control animals showed that bone mineral density, maximum strength, stiffness and histomorphometric parameters of the femur of suspended decreased in three weeks and then stabilized for both groups suspended by the skin traction and skeleton, with no differences between them. Humerus presented no significant differences between suspended animals and controls. Conclusion: The system of skeletal traction was more efficient to keep the animal suspended for six weeks, when the number of complications was lower than in the skin traction group. Both suspension methods had the same efficiency to weaken the bone.
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Avaliação da qualidade óssea da tíbia de ratas submetidas à hipoatividade através da suspensão pela cauda e tratadas com exercícios de natação / Assessment of the tibia bone quality in rats subjected to hypoactivity by tail suspension and treated with swimming exercisesSilva, Adriana Valadares da 21 February 2014 (has links)
O osso requer que alguns de seus elementos detectem estímulos mecânicos e os transformem em sinais celulares adequados para que o tecido ósseo possa manter a eutrofia e adaptar-se às solicitações mecânicas a que é submetido. Assim, a estrutura óssea pode torna-se mais resistente quando a demanda mecânica aumenta e, da mesma forma, menos resistente em condições de hipoatividade como imobilizações, sequelas motoras, repouso prolongado ou em ambientes de microgravidade como as experimentadas por astronautas. Dessa interação podem resultar ossos com massa diminuída, o que caracteriza situações de osteopenia ou osteoporose. Uma das indicações do exercício de natação é estimular e manter o trofismo do tecido ósseo. No entanto, a relação entre natação e qualidade óssea em pessoas com osteopenia, não foi suficientemente estudada de forma a embasar recomendações clínicas. Assim, o objetivo desta pesquisa foi investigar a eficácia da natação na recuperação da qualidade óssea avaliada em tíbias de ratas com osteopenia induzida por suspensão dos membros posteriores. Foram utilizadas 50 ratas da raça Wistar divididas em cinco grupos experimentais (n=10/grupo); dois controles e três experimentais. Houve um grupo suspenso pela cauda durante 21 dias consecutivos (S) e seu controle que permaneceu em gaiolas comuns pelo mesmo período (CI). No grupo suspenso e treinado os animais permaneceram em suspensão durante 21 dias, e depois foram submetidos a 20 sessões de natação (ST). No grupo suspenso não treinado os animais foram suspensos por 21 dias e soltos em gaiolas comuns por 30 dias (SNT). No grupo controle II as ratas foram observadas em gaiolas comuns por 51 dias, sem intervenção (CII). A avaliação da qualidade óssea foi feita pela densitometria (DXA) e ensaio mecânico realizados na tíbia direita e histomorfometria da região metafisária proximal da tíbia esquerda. Para análise estatística todos os valores foram considerados diferentes significativamente em p<0,05. Os ensaios mecânicos mostraram que, para o grupo S os valores de força máxima (-14,03%, p=0,0003), rigidez (-21,68%, p=0,0055), DMO (-17,62%, p=0,019) e percentual de osso trabecular (-57,2%, p=0,0001), diminuíram significativamente em comparação com o grupo CI. A comparação das propriedades mecânicas simultâneas entre os grupos avaliados após 51 dias (STxCIIxSNT) mostrou diferença estatística significante entre os grupos para força máxima de (p=0,0014), rigidez (p=0,0010), DMO (p= 0,0095) e percentual de osso trabecular (p<0,0001). O grupo ST apresentou aumento significativo na força máxima (+10,23%, p<0,05), rigidez (+21,91%, p<0,001), DMO (+9,46%, p<0,05) e percentual de osso trabecular (+48,82%, p<0,001) em comparação com o grupo SNT. Na comparação dos grupos SNT e CII também houve diminuição significativa (-14,4%, p<0,05) para força máxima, rigidez (-25,21%, p<0,005), DMO (-13,34%, p<0,05) e percentual de osso trabecular (-52,06%, p<0,001). Finalmente, a comparação entre os grupos ST e CII não apresentou diferença estatística significante (p>0,05) para os valores de força máxima, rigidez, DMO e percentual de osso trabecular. Em relação à análise histológica qualitativa o grupo S apresentou ossos com características histológicas condizentes com osteopenia, com trabéculas ósseas bem mais delgadas em quantidade muito menor tanto na epífise quanto na metáfise, quando comparado ao grupo CI. O grupo suspenso apresentou ainda, diminuição na espessura do tecido ósseo compacto e redução significativa na quantidade de tecido esponjoso em sua porção mais interna. A espessura do tecido ósseo compacto se mostrou similar nos diferentes grupos (ST, SNT e CII). Todavia, a espessura e a quantidade de trabéculas do osso esponjoso diferiram expressivamente nos diferentes grupos. O grupo ST apresentou uma aparente maior quantidade de trabéculas ósseas, bastante delgadas, ao passo que o grupo CII apresentou, comparativamente, uma menor quantidade de trabéculas bem mais espessas. Comparado a estes últimos, o grupo SNT apresentou uma menor quantidade de trabéculas com delgada espessura. Com a suspensão por três semanas os parâmetros mostraram que houve deterioração da qualidade óssea expressa pela diminuição da densidade mineral óssea, enfraquecimento mecânico do osso e perda de massa óssea trabecular. A quantidade de osso trabecular na região metafisária foi o parâmetro mais afetado pela hipoatividade, mas também o elemento que apresentou a resposta mais rápida para a atividade motora. A conclusão foi que a natação reverteu mais rapidamente a deterioração osteopênica causada pela hipoatividade, com recuperação completa da qualidade óssea. / The bone is able to detect mechanical stimuli and to transform them into cell response so that the bone tissue can maintain its health and to adapt to different mechanical demands (mechanotransduction). Thus, the bone structure become more resistant when the mechanical solicitations are increased and, conversely, less resistant when in hypoactivity conditions as occurring during immobilizations, in locomotor sequela, prolonged bed rest or in a microgravity environment as experienced by astronauts. In general, hypoactivity leads the bone to develop a decreased and deteriorated mass, thus characterizing conditions of osteopenia or osteoporosis. Thus, physical activity such as swimming is indicated as one of the factors that contribute to the bone health. However, the relation between swimming and bone quality in individuals with osteopenia has not been sufficiently studied to fully support clinical recommendations. So, the aim of this study was to investigate the effectiveness of swimming in recovering bone quality evaluated in osteopenic tibias of rats that spent three weeks in hindlimb suspension. Fifty Wistar rats were used and divided into five groups (n = 10/group); two control and three experimental groups. There was a group suspended by the tail for 21 consecutive days (S) and its control kept in regular cages for the same period (CI). In the suspended and trained group, animals were suspended for 21 consecutive days and then underwent a 20 swimming sessions (ST). In the suspended, not trained group, animals were suspended for 21 days and released in regular cages for 30 days (SNT). In the control group II rats were kept in regular cages for 51 days without intervention (CII). The bone quality was assessed by densitometry (BMD), bending mechanical testing and histomorphometry. Statistical significance was set at p<0.05. Mechanical tests showed that for the suspended group there was a decrease of the maximum force (-14.03%, p = 0.0003), stiffness (-21.68%, p = 0.0055), BMD (-17.62 %, p = 0.019) and the percentage of trabecular bone (-57.2%, p = 0.0001) in comparison with the control group. Comparison among the groups after 51 days (STxCIIxSNT) showed statistically significant differences between groups for maximum force (p = 0.0014), stiffness (p = 0.0010), BMD (p = 0. 0095) and the percentage of trabecular bone (p<0.0001). The suspended and trained group showed a significant increase in maximum force (+10.23%, p <0.05), stiffness (+21.91%, p<0.001), BMD (+9.46%, p<0.05) and the percentage of trabecular bone (+48.82%, p <0.001) compared to the suspended and not trained group. When the suspended but not trained group was compared to control CII, there was also a significant decrease (- 14.4%, p<0.05) for maximum force, stiffness (-25.21%, p<0.005), BMD (-13.34%, p<0.05) and the percentage of trabecular bone (-52.06%, p<0.001). Finally, the comparison between group suspended and trained ad its control and CII showed no statistically significant difference (p>0.05) for all the parameters. Considering the qualitative histological analysis, suspended group presented bones with histological characteristics consistent with osteopenia, with thinner trabecular bone and in much less quantity both in the epiphysis and in the metaphysis. The suspended and trained group had a higher quantity of trabecular bone, with thinner trabeculae, while the CII group showed a comparatively smaller amount of much thicker trabeculae. Compared to the latter, the suspended and not trained group had an apparent smaller amount of trabeculae with a thinner thickness. In conclusion, the three week suspension caused a marked deterioration of the tibia bone tissue that was completely reverted by swimming and partially reverted with spontaneous cage activities taking in consideration all the parameters analyzed.
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Évaluation d'un substitut osseux résorbable porteur de cellules souches : approche cellulaire pour la régénération osseuse in vivo / Evaluation of a Resorbable Bone Substitute carrying Stem Cells : Cell-Based Approach for Bone RegenerationRenaud, Matthieu 22 November 2018 (has links)
Malgré le développement de biomatériaux de plus en plus nombreux dans le domaine des greffes osseuses et de la préservation alvéolaire, les résultats sont toujours insuffisants pour assurer des reconstructions ad integrum du tissu osseux. Les techniques d’ingénieries osseuses semblent être la piste à privilégier pour améliorer nos techniques chirurgicales. Le silicium poreux est un matériau prometteur pour l’ingénierie tissulaire et notamment pour la régénération osseuse. En effet, son état de surface permet une adhésion cellulaire importante et ses propriétés non toxique et résorbable en fond un matériau porteur de cellules souches intéressant. Les cellules souches de la pulpe dentaire (DPSC) sont des cellules facilement accessibles dans la cavité buccale. Leurs capacités de prolifération et de différenciation associées au silicium poreux semblent être un atout pour les applications thérapeutique pour la régénération osseuse. Des résultats d’études ultérieures in vitro ont montré leur fort intérêt à une application in vivo. Dans ce travail thèse, nous avons tester l’association silicium poreux et cellules souches de la pulpe dentaire, aux même caractéristiques énoncées dans l’étude de référence in vitro, chez l’animal. Pour cela, le matériau a été produit sous forme de particules de manière a être utilisé comme moyen de comblement osseux, associé ou non à des DPSC. Le modèle de queue de rat a été développé et tester pour diminuer le nombre d’animaux nécessaire à l’étude tout en conservant la puissance statistique des résultats. Les études ont montré la possibilité d’utiliser ce modèle pour la régénération de défauts osseux crées chirurgicalement. De plus, il semblerait que ce modèle puisse également être utile pour les études sur l’ostéointégration de système implantables et sur la régénération osseuse autour de ces implants. Le silicium poreux a ensuite été testé dans ces conditions, associé ou non aux DPSC, en comparaison avec un témoin positif et un témoin négatif. Cette association est apparue comme une piste prometteuse pour la régénération osseuse in vivo. / Despite the development of biomaterials in the field of bone grafts and alveolar preservation, the results are no sufficient to made reconstructions ad integrum of bone tissue. Bone engineering techniques seem to be the preferred way to improve our surgical techniques. Porous silicon is a promising material for tissue engineering and especially for bone regeneration. Indeed, its surface allows cell adhesion. And then, it’s a non-toxic and bioresorbable interesting material properties carrying stem cells. Dental pulp stem cells (DPSC) are easily accessible cells in the oral cavity. Their proliferation and differentiation capacities associated with porous silicon appear to be attractive for therapeutic applications in bone regeneration. The results of the in vitro studies have shown the interest for in vivo application. In this thesis, we have tested the combination of porous silicon and dental pulp stem cells in vivo experimentation, using the same characteristics of the in vitro reference study. For this, the material was produced in particle form to be used as bone filling material, associated or not with DPSC. The rat-tail model was developed and tested to reduce the number of animals needed for the study while maintaining the statistical power of the results. Studies have shown the possibility of using this model for bone regeneration defects surgically created. In addition, it seems that this model can also be useful for studies on osseointegration of implantable systems and bone regeneration around these implants. Then, the porous silicon was tested under these conditions, with or without DPSC, in comparison with a positive control and a negative control. This association has emerged as a promising approach for bone regeneration in vivo.
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Podcasting jako fenomén nových médií a jeho současný stav v České republice / Podcasting as a New Media Phenomenon and its Current State in the Czech RepublicSvobodová, Markéta January 2019 (has links)
This MA thesis is devoted to podcasting, one of the phenomena of new media. It describes its origins and development; in greater detail it deals with the present state and characteristic features of podcasts from the viewpoint of their contents, forms, user practice, ways of financing, and also technological perspectives. Attention is focused on elements which interconnect podcasting with radio and radio programmes and also on their differences. The phenomenon is presented in the context of chosen concepts related to web 2.0. Several chapters focussed generally on the development and present state of podcasting are devoted to the situation in the Czech Republic. The research part then provides insight into the present situation from the point of view of independent podcast creators, with whom half-structured interviews were carried out. On the basis of a qualitative analysis of these interviews, several types of attitudes to podcasting and a number of factors related to production of podcasts were identified - primarily motivations to create them as well as some discouraging aspects. Further results of the analysis concern motivation of the authors to listen to podcasts of other creators, their view of the present state of affairs in the sphere of podcasting in the Czech Republic, and their...
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Estudos termodinâmicos e estruturais da interação cabeça-cauda da , alpha-tropomiosina muscular / Thermodynamic and structural studies of the head-to-tail complex of the muscular alpha-TropomyosinFernando Corrêa 20 June 2008 (has links)
Tropomiosina (Tm) é uma das proteínas que compõe o filamento fino (actina, Tm, Troponina) do sistema muscular esquelético e desempenha um importante papel na regulação da contração muscular. Tm é um coiled-coil de 284 resíduos que forma longos homopolímeros lineares através da sobreposição de onze resíduos entre os terminais de Tms adjacentes (Interação cabeça-cauda) em condições de baixa força iônica. A presença de vários resíduos carregados (D2, K5, K6, K7, D275, H276 e D280) nas extremidades da Tm sugere que contatos intermoleculares eletrostáticos entre estes aminoácidos podem ter um importante papel na estabilidade dos polímeros. Entretanto, a estrutura do complexo cabeça-cauda demonstra que a maioria dos contatos intermoleculares na interface é de natureza hidrofóbica. A fim de analisarmos a contribuição dos grupos carregados para a estabilidade do complexo cabeça-cauda, construímos fragmentos recombinantes correspondentes à metade amino (ASTm1-142 ) e carboxi (Tm143-284(5OHW269)) terminais da proteína contendo mutações pontuais daqueles resíduos para alanina, e adicionalmente H276 para Glu. Medimos a afinidade entre todas as possíveis combinações destes fragmentos na ausência e presença de íons Mg2+, visto que este cátion está sempre presente em condições fisiológicas e é importante para estabilizar a interação entre Tm e actina. Os efeitos das mutações foram analisados por simulações de docking, desnaturações térmicas e ciclos de duplos mutantes. Os resultados demonstram que os aminoácidos K5, K7 e D280 presentes na interface formam contatos intermoleculares essenciais para a estabilidade do complexo. Enquanto, D2, K6, D275 e H276 não participam na formação de contatos intermoleculares, no entanto, contribuem para a estabilidade da interação cabeça- cauda através de suas interações intramoleculares que atuam na estabilidade das hélices individuais. Os aumentos na estabilidade da metade C-terminal da Tm (Tm143-284(5OHW)) induzidos por Mg2+ foram dependentes das mutações neste trecho da proteína sugerindo a presença de um sítio de ligação para este íon na extremidade carboxi terminal da molécula no trecho que forma a interação cabeça- cauda. Construímos um fragmento menor do C-terminal (Tm259-284(W269)) para acompanharmos mudanças no deslocamento químico induzidas pela ligação do íon usando ressonância magnética nuclear. Os resultados obtidos comprovaram nossa hipótese e nos permitiram definir pela primeira vez que a estrutura da Tm tem um ou mais sítios de ligação Mg2+ em uma região próxima ao resíduo H276 que está localizado entre vários resíduos carregados negativamente que participam da interação cabeça-cauda. Por último, estudamos os efeitos de solventes cosmótropicos (TFE e glicerol) nas estabilidades dos fragmentos da Tm, uma vez que a instabilidade (flexibilidade) da extremidade C-terminal é importante para a formação do complexo cabeça-cauda. Observamos que TFE, porém não glicerol, reduziu a afinidade entre os terminais. Ambos os co-solventes induziram aumentos na estabilidade dos fragmentos, no entanto, apenas TFE induziu um aumento no conteúdo de α-hélice e causou uma redução significativa na cooperatividade de desenovelamento das proteínas. Estes resultados indicam que estes compostos orgânicos estabilizam as estruturas dos fragmentos individuais da Tm de maneiras diferentes e que estas diferenças podem estar relacionadas aos diferentes efeitos observados na formação da interação cabeça-cauda. / Tropomyosin (Tm) is a protein component of the skeletal muscle thin filament (actin, Tm, Troponin) which has an important role in the regulation of muscle contraction. Tm is a dimeric coiled-coil (284 aminoacids) which forms long linear homopolymers through the overlap of eleven residues of adjacent Tm termini (Head- to-tail interaction) in low ionic strength conditions. The presence of several charged amino acids (D2, K5, K6, K7, D275, H276 e D280) in Tm extremities suggests that electrostatic contacts among those residues may have an important role in the stability of the polymers. Nevertheless, the solution structure of the head-to-tail complex demonstrated that most of the contacts in the interface are hydrophobic. In order to study the contribution of these charged residues to the stability of the head- to-tail complex, we built recombinant fragments corresponding to the amino (ASTm1-142) and carboxy (Tm143-284(5OHW269)) termini containing single mutations of those amino acids to alanine, and additionally a substitution of H276 for Glu. We measured the binding affinities among all possible combinations of wild-type and mutant fragments in the absence or presence of Mg2+ ions. This cation is always physiologically present in the muscle and it is known to strengthen the binding of Tm to actin. The effects of the mutations were analyzed by protein-protein docking, thermodynamic cycles and thermal denaturations. The results show that residues K5, K7 and D280 are essential to the stability of the complex. Though D2, K6, D275 and H276 are exposed to the solvent and do not participate in intermolecular contacts in the NMR structure, they may contribute to the complex stability by modulating the stability of the helices at the Tm termini. Mg2+-induced increases in stability of the C- terminal were sensitive to mutations in residues located in the head-to-tail overlap region, suggesting that Mg2+ ions may bind specifically to the carboxy extremity of the protein. We produced a small peptide (Tm259-284(W269)) to follow amide chemical shift perturbations upon Mg2+ binding by nuclear magnetic resonance measurements. The results obtained with this peptide allowed us to define for the first time that the Tm structure has one or more Mg2+ binding sites in a region centered in the vicinity of H276 in which are located several negatively charged residues that participate in the head-to-tail interaction. We also studied the effects of kosmotropic co-solvents (TFE and glycerol) in the stability of Tm fragments, as the instability (flexibility) of the C- terminal region has been pointed as important for the formation of the head-to-tail complex. We observed that TFE, but not glycerol, reduces the affinity between the termini. Both TFE and glycerol increased the stability of the isolated N- and C- terminal fragments; however, only TFE caused an increase in the helical content and a significant reduction in the cooperativity of unfolding of the proteins. Our results show that these two co-solvents stabilize the structures of individual Tm fragments in different manners and that these differences may be related to their different effects on head-to-tail complex formation.
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