This paper summarizes and extends the modeling state of the art of magnetostrictive energy harvesters with a focus on the pick-up coil design. The harvester is a one-sided clamped galfenol unimorph loaded with two brass pieces each containing a permanent magnet to create a biased magnetic field. Measurements on different pick-up coils were conducted and compared with results from an analytic model. Resistance, mass and inductance were formulated and proved by measurements. Both the length for a constant number of turns and the number of turns for a constant coil length were also modeled and varied. The results confirm that the output voltage depends on the coil length for a constant number of turns and is higher for smaller coils. In contrast to a uniform magnetic field, the maximal output voltage is gained if the coil is placed not directly at but near the fixation. Two effects explain this behavior: Due to the permanent magnet next to the fixation, the magnetic force is higher and orientates the magnetic domains stronger. The clamping locally increases the stress and forces the magnetic domains to orientate, too. For that reason the material is stiffer and therefore the strain smaller. The tradeoff between a higher induced voltage in the coil and an increasing inductance and resistance for every additional turn are presented together with an experimental validation of the models. Based on the results guidelines are given to design an optimal coil which maximizes the output power for a given unimorph.

Among global steel markets in recent years, China steel industry expands the fastest with the largest capacity and most fiercest competitions, also bringing the greatest impacts to global and Taiwan steel markets. A few aggressive Taiwan mills have set up their production facilities in China and commissioned. This study conducts in-depth analysis on the leading Taiwan middlestream galvanizers & coil coaters (the products are hot-dip galvanized & pre-painted steel sheets, hereinafter called ¡§galvanized & pre-painted steel¡¨). Besides, the steel works are categorized into Taiwan works, China works-Taiwan capital, China works-foreign capital, and China local works based on different capital sources. Analysis and evaluation are done in light of every activity in value chain. Six types of galvanizers & coil coaters are categorized based on different facilities and processes. Type I: Integrated galvanizers & coil coaters Type II: CSP galvanizers & coil coaters Type III: Independent galvanizers Type IV: Independent coil coaters Type V: Independent galvanizers & coil coaters Type VI: Independent full-process galvanizers & coil coaters Owing to their different market environments, development backgrounds, technologies, human resources and management, this study will analyze their strengths, strategies adopted, possible future development trends and potential challenges. This study especially focuses on one issue: in the atmosphere of competition and cooperation between steel mills in Taiwan and China, how China independent galvanizers & coil coaters-Taiwan capital and Taiwan parent company upgrade their competitiveness is crucial. Synthesizing related literature & theories, market & mill information and interviews, verifying by comparison, four conclusions are obtained as follows. 1. The more value chain activities a mill has, the more profits and stronger competitiveness it acquires. 2. Taiwan markets of galvanizing & pre-painted steel are already saturated. The competitiveness & development strategy a globalization enterprise should adopt is to increase its export percentages and establish offshore production bases. 3. For those mills setting up independent galvanizers & coil coaters in both China and Taiwan, the marketing strategies they should employ are described as follows. 3.1 Both China and Taiwan works receive orders and manufacture for their domestic markets. In terms of exports, only Taiwan parent company receives orders for both. But they export through Taiwan¡¦s existing channels globally. 3.2 To ally with different downstream industries and benefit each other. 3.3 China products are exported to ASEAN 10 nations, or 10+3, or 10+4, or any country that hasn¡¦t accuse China mills of dumping, whereas Taiwan products are sold to China, or any country that hasn¡¦t accuse Taiwan mills of dumping. 3.4 Products should be differentiated. Order receiving, production planning, lead-time and customer service should be flexible and elastic. 3.5 The chosen customers and suppliers must be big and strong in their region. Thus, the overall enterprise competitiveness is excellent. 4. In the past, steel companies self-expand to grow whereas today they grow via merger and acquisition. Any enterprise must have its value-creation strategy, i.e. it must have a growth strategy to react to the fierce market competitions. This study provides six recommendations for the competitiveness & growth strategies which could be adopted by domestic Taiwan independent galvanizers & coil coaters. 1. For the investors who invest in independent galvanizers & coil coaters for the first time, the first choice should be Type VI: independent full-process galvanizers & coil coaters. The second choice should be Type V: Independent galvanizers & coil coaters. Next, to set up an independent galvanizer is superior to an independent coil coater. 2. Independent Galvanizers & Coil Coaters should adopt the growth strategy that horizontal developing to a certain scale at the outset is the priority. Next, they should develop toward upstream instead of downstream. In order to eliminate capacity, they should ally with different downstream industries. 3. In the market of demand exceeding supply, mills usually self-expand to achieve larger scale. Nevertheless, when the market is oversupplied, it is recommended to adopt acquisitions. 4. Independent galvanizers & coil coaters choose the most beneficial investment items & scale and start their oversea development based on their growth strategies. 5. In the initial phase of plant construction, independent galvanizers & coil coaters should negotiate with upstream raw material suppliers regarding raw material supply matters. A good relationship should be built in order to secure stability of raw material sources. 6. To manage China mills-Taiwan capital and compete with other mills, the following measures are recommended. 6.1 To take advantage of the enterprise strength, develop specialized products and promote marketing features. 6.2 To promptly establish market reaction mechanism via the internationalization strategy of ¡§Integration-Responsiveness¡¨. 6.3 To employ human resources management to aid internationalization strategy. 6.4 Integration model created by overseas plant construction or acquisition can be adopted to integrate an enterprise¡¦s organization, culture and resources via dynamics management system.

L’objectif de la présente thèse est d’évaluer les performances des micro-antennes, en cas d’emploi en spectroscopie in vivo et de prévoir les éventuelles améliorations, tout en respectant les contraintes en termes d’encombrement et de biocompatibilité imposées par des applications cérébrales sur modèle animal. Par conséquent, dans ce travail, il fallait également évaluer la sensibilité tout en comprenant le fonctionnement particulier de ces micro-capteurs, points qui n’avaient pas encore été explorés. Ce document est organisé en cinq chapitres, le Premier est un aperçu sur la situation du sujet par rapport à l’existant et un rappel technique sur les travaux déjà effectués en matière de réalisation et en rapport avec les différents métabolites «cibles». Le Chapitre II présente les développements instrumentaux entrepris en vue de l’amélioration des conditions de détection: optimisation de l’adaptation en puissance et de l’adaptation en bruit à l’entrée du spectromètre prise aussi en considération. La difficulté du maintien du modèle dans un espace restreint est solutionnée en proposant différents montages de réglages déportés. L’introduction d’un préamplificateur à faible bruit est également étudiée et réalisée sur la micro-antenne et une antenne de surface fait maison. La question importante de la limite de détection est abordée sous un angle théorique et expérimental dans le Chapitre III. L’intérêt de cette analyse est l’évaluation des performances de la micro-antenne, on verra que cette nouvelle notion permet également de qualifier une installation de spectroscopie (capteur associé à un spectromètre pour une expérience donnée). Une étude d’amélioration apportée sur la limite de détection (LOD) par la technique de pondération sera traitée et validée sur des acquisitions spectroscopiques. Le Chapitre IV est destinée dans un première partie, à travers les modélisations, à discuter les variations de la limite de détection pour certains changements structurels (la dimension et le positionnement de la micro-bobine). Dans la deuxième partie, une mise en application de la limite de détection, à travers les expériences spectroscopiques sera présentée dans le but de comparer les performances de capteurs existants: principalement une antenne commerciale, la micro-antenne et une antenne fait maison. La question de la biocompatibilité de ces microantennes implantables est traitée dans le Chapitre V. La réalisation de cette étude a été effectuée en amont des travaux théoriques et instrumentaux et a permis leurs développements avec une démarche plus pragmatique / The objective of this thesis is to evaluate the micro-coil performance, in spectroscopy in vivo and provide any improvements, while respecting the constraints in terms of obstruction and biocompatibility imposed by applications on brain of animal models. Therefore, this work had also to assess the sensitivity understanding of these particular micro-sensors, a notion that has not yet been explored. This document is organized into five chapters: the first one is an "overview" of state of art techniques and a reminder of the work already done in terms of production and in relation to different metabolite "targets". Chapter II presents the instrumental developments undertaken to improve the detection: optimization of adaptation in power and noise matching at the spectrometer input, were also taken into account. The difficulty of maintaining the animal model in a reduced space is solved by providing different configurations of remote settings. The introduction of a low-noise preamplifier is also studied and implemented. The important question of the limit of detection is approached from a theoretical point of view in Chapter III. The interest of this analysis is to assess the performance of micro-coil, we will point out that this new concept enables to describe a spectroscopy facility (sensor associated with a spectrometer for a given experiment). A study of enhancement of the limit of detection by the apodization technique will be addressed and validated on spectroscopy acquisitions. Chapter IV is an implementation in the first part, through modelling, to discuss changes in the limit of detection for some structural changes (size and positioning of the micro-coil). In the second part, an implementation of the limit of detection through spectroscopic experiments will be presented in order to compare the performance of existing coils: a commercial coil and a micro-coil. The biocompatibility of implantable micro-coil treated in Chapter V. The completion of this study was carried out upstream of the theoretical and instrumental aspects in chapter III and IV and that helped their development with a more pragmatic approach

Diploma thesis deals with geothermal foundations. The aim of the thesis is to find optimal way of pipe winding for heat-carrying liquid in foundation piles. The problem is solved by CFD simulations in ANSYS Fluent. Determinative parameters like the pressure loss, heat fluxes and U-value in one pile circuit with two ways of pipe installations – Single coil, where reverse pipe is led in the middle of pile and Duplex Coil with reverse pipe in spiral shape are observed.

Nukleinsäuren und Peptide erlauben es, Kontrolle über molekulare Prozesse auszuüben. In dieser Arbeit werden strukturgebende Elemente wie Coiled-Coil-Peptide oder PNA∙DNA-Strukturen genutzt, um Rezeptoren auf lebenden Zellen zu markieren und in ihrem Verhalten zu modulieren, oder cytosolische Proteine in ihrem Bindungsverhalten zu steuern. Im ersten Konzept wird die Interaktion des Coiled-Coil-Paars K3/E3 genutzt, um eine Transferreaktion, in welcher eine PNA-Sequenz vom K3-Donor auf den E3-Akzeptor übertragen wird, zu induzieren. Durch die Fusion des Akzeptorpeptids mit einem Rezeptor werden kovalente PNA-Rezeptorkonjugate auf der Oberfläche lebender Zellen geschaffen. Die Reaktion zwischen Thiol und Thioester erlaubt dabei einen schnellen Transfer. So wurden Rezeptoren aus der Familie der GPCR sowie der EGFR mit einem PNA-Strang versehen und durch fluoreszente PNA oder DNA selektiv markiert. Zusätzlich wurden verzweigte DNA-Architekturen mit mehreren Fluorophoren genutzt, um die Helligkeit der Markierung quantitativ zu erhöhen. Die PNA-EGFR-Konjugate wurden durch eine zwei Rezeptoren verbrückende Cy3-DNA adressiert und so zeitgleich markiert und dimerisiert. Dadurch wurde die Rezeptoraktivität gesteigert, was über Western Blot-, Immunofluoreszenz- und Fluoreszenzmikroskopieanalyse belegt wurde. In weiteren Ansätzen wurden Coiled-Coil-Systeme genutzt, um i) parallel zwei verschiedene Akzeptorpeptide mit verschiedenen Fluorophoren zu markieren und ii) Coiled-Coil-Peptide schaltbar zu machen. Durch die asymmetrische Verlängerung von K3/E3-Paaren mit Coiled-Coil-Sequenzen kann die Interaktion der Peptide an und aus geschaltet werden. Dies wurde sowohl in einem Fluoreszenzassay als auch in einer direkten Anwendung an der Syk-Kinase demonstriert. Die Liganden der Kinase wurden an den schaltbaren Peptiden angebracht und so die Affinität zur Syk-Kinase kontrolliert. / Nucleic acids and peptides can be used to obtain control over molecular processes within living cells. In this work, structural elements as coiled-coil peptides or PNA∙DNA-structures were used to label and modulate receptor behavior on living cells and to control ligand binding of cytosolic proteins. For the first concept the K3/E3-coiled-coil peptide pair was used to establish a proximity-guided, covalent transfer of a PNA strand from a K3-donor peptide onto the complementary E3-acceptor peptide. By fusion of the acceptor peptide to a receptor, PNA-receptor-conjugates were generated selectively on living cells. The native chemical ligation type of reaction allowed a fast PNA-transfer within minutes. Receptors from the family of GPCRs and the EGFR were tagged with a PNA-sequence and subsequently labeled by the addition of a fluorescent DNA or PNA. By recruiting branched DNA architectures which were decorated with several fluorophores, the total brightness of the labeling was increased quantitatively. A twice complementary Cy3-DNA was used to simultaneously label and dimerize the EGFR. Thereby, an artificially induced increase in receptor activity could be achieved, which was shown in Western Blot and immunofluorescence analysis as well as in fluorescence microscopy. In two other approaches coiled-coil peptides were used to i) label two different acceptor peptides simultaneously with two different dyes and ii) introduce coiled-coil peptides as part of a dynamic switchable system. Using an asymmetric coiled-coil elongation on the K3/E3 pair the interaction of both can be turned on and off. This was demonstrated in a fluorescence assay and applied to the Syk kinase, were Syk ligands were attached to the switchable peptides. Those ligands were changed from a bi- to a monovalent presentation status and thus the affinity of the Syk kinase towards its ligands can be controlled.

Nesse trabalho, são investigadas as propriedades dinâmicas de modelos da mecânica estatística na criticalidade. Inicialmente, trabalhando com modelos de spin e utilizando os conceitos de persistência global e de dimensão anômala da magnetização inicial, mostramos que o modelo de Baxter-Wu não está na mesma classe de universalidade dos modelos de Potts com quatro estados e de Ising com interação de três spins em uma direção, todos bidimensionais. Na segunda parte da tese, estudamos o fenômeno de crescimento da superfície gerada pela deposição segundo as regras que definem os autômatos celulares probabilísticos propostos por Grassberger (modelos A e B). Esses dois autômatos não pertencem à classe de universalidade de Domany-Kinzel e apresentam novos expoentes críticos, cuja origem se deve à conservação de paridade. Determinamos o expoente de crescimento beta w, válido em tempos curtos, assim como os outros expoentes críticos associados ao crescimento de superfície (alfa e z). Nossas estimativas se comparam bem com os resultados obtidos a partir de razões de inteiros propostas por Jensen para os expoentes beta, ni paralelo e ni perpendicular. Finalmente, investigamos a transição de fase entre o estado helicoidal e o estado desordenado (random coil) da polialanina e do fragmento peptídico PTH(1-34), que corresponde aos resíduos 1 a 34 da região aminoterminal do hormônio das paratireóides. Nosso cálculo, que leva em conta as interações entre todos os átomos da molécula, está baseado em uma abordagem de tempos curtos. Os resultados dessa análise indicam que a transição helix-coil das polialaninas e do PTH(1-34) é de segunda ordem e apontam para uma classe de universalidade para a transição helix-coil em homopolímeros e proteínas (partindo de um estado helicoidal). / In this work we investigated dynamic properties of statistical mechanical models at criticality. At first, using the concepts of global persistence and anomalous dimension of initial magnetization, we showed that the Baxter-Wu model does not belong to the same universality class as 4-state Potts model and Ising with multispin interaction in one direction. In the sequence, we studied the roughening behavior generated by deposition governed by rules defined by probabilistic cellular automata proposed by Grassberger (A and B models). Those models are known do not belong to the Domany-Kinzel universality class. They are characterized by different exponents which are related to the parity conserving (PC). We estimated the growth exponent beta w, in short-time regimen, such as, other critical exponents associated to the surface growth (alpha and z). Our results are in good agreement with those expected for parity conserving universality class. At last we studied the phase transition between the completely helical state and the random coil of the polyalanine, such as, for the 34-residue human parathyroid fragment PTH(1-34). Our short-time simulations of the helix-coil transition are based on a detailed all-atom representation of proteins. The results indicate that helix-coil transition in polyalanine and PTH(1-34) is a second-order phase transition and suggest a universality class to the helix-coil transition in homopolymer and (helical) proteins.

Um die Funktion von Proteinen in ihrer natürlichen Umgebung zu verstehen, ist es unerlässlich ihre Lokalisation und Bewegung im lebenden System durch z.B Fluoreszenz-markierung sichtbar zu machen. Eine ideale Markierungsmethode zeichnet sich dadurch aus, dass sie das Zielprotein (protein of interest, POI) selektiv und in kurzer Zeit mit einer maßgeschneiderten Reportergruppe ausstattet, ohne die Proteinfunktion und -lokalisation zu beeinflussen. Dabei ist die Größe der Erkennungssequenz von großer Bedeutung. In dieser Arbeit wird die Entwicklung einer Markierungsstrategie beschrieben, bei der die Ausbildung eines parallelen Coiled-Coil-Motivs den Transfer einer Reportergruppe auslöst. Untersucht wurde dabei die Übertragung eines Sulfonat-gebundenen Fluorophors auf ein Cystein in der Erkennungssequenz durch nukleophile Substitution. Ebenfalls untersucht wurde der Transfer verschiedener Thioester-verknüpfter Reporter auf ein N-terminales Cystein der Erkennungssequenz durch eine Acyltransferreaktion. Beide Strategien zeichnen sich durch eine hohe Selektivität und einen geringen Massenzuwachs am Zielprotein aus. Der Acyltransfer mit Arylthioestern zeigte zudem eine bemerkenswerte Reaktivität und erlaubte eine Markierung innerhalb weniger Minuten Reaktionszeit. Die Vielfältigkeit dieser Methode wurde anhand der Fluoreszenzmarkierung von sieben verschiedenen G-Protein gekoppelten Membranrezeptoren auf der Oberfläche lebender Zellen demonstriert. Die markierten Rezeptoren blieben dabei funktional und konnten ihren entsprechenden Liganden mit hoher Affinität binden. / In order to understand the function of proteins in their native environment, it is crucial to visualize their localization and trafficking in living cells by means of e.g. fluorescence labeling. An ideal labeling method adds a custom reporter group to the protein of interest (POI) in a selective and fast manner without disturbing the POIs function and localization. Hence the size of the recognition sequence is of major concern. This work describes the development of a labeling strategy in which the formation of a parallel coiled coil motif triggers the transfer of a reporter group. The transfer of a sulfonate-linked fluorescence dye onto the cysteine-modified recognition sequence via a nucleophilic substitution reaction was tested. Also the transfer of various thioester-linked reporters onto the N-terminal cysteine of the recognition sequence via an acyl transfer reaction was investigated. Both strategies are characterized by a high selectivity and low mass increase at the target protein. The acyl transfer with aryl thioesters also showed a remarkable reactivity and allowed labeling reactions to proceed within minutes. The versatility of this method was demonstrated by applying it to the labeling of seven different G-protein coupled membrane receptors on the surface of living cells. The labeled receptors remained functional and were able to bind their respective ligand with high affinity.

The main histological feature of Alzheimer\'s disease is the presence of amyloid plaques in the patient\'s brain. The most abundant element of these plaques is the β-amyloid peptide (Aβ). Initially soluble, the peptide exhibits in solution an intricate equilibrium among monomeric, oligomeric (some of which are regarded as the toxic species) and fibrillar states, which prevents its crystallization and subsequent structural determination by X-ray diffraction. High-pressure nuclear magnetic resonance (NMR) spectroscopy has been used by our group to detect rare, high-energy monomeric Aβ (1-40) states, coexisting in equilibrium with oligomers and fibrils. This work aims to characterize the thermodynamics and the structure of the rare excited states of the Aβ peptide through the use of high pressure NMR. A large collection of NMR spectra of the Aβ (1-40) peptide as a function of pressure was recorded and analyzed. Secondary structure predictions revealed that the Aβ peptide adopts extended β-strand-like structures, similar to those found in amyloid-fibril structures. From the pressure curves of chemical shifts and cross-peak volumes, at least three monomeric states could be detected, which were thermodynamically characterized by the calculation of the variation of their Gibbs free energy (ΔGij) and molar partial volumes (ΔVij). The study of nuclear Overhauser effects (NOEs) and 3JHα-HN NMR couplings reinforces the existence of extended structures with β-strand propensity, both at ambient (0.1 MPa) and high (275 MPa) pressures. The interaction between the Aβ peptide and the D-peptides RD2 and RD2D3, D-enantiomeric fibril inhibitors, was also characterized. Our results indicate that the D-peptides recognize and bind to a more compact conformation of Aβ. The formation of the Aβ-D-peptide heterodimers ultimately prevents the formation of toxic oligomers, therefore representing a potential therapy against Alzheimer´s disease. Additionally, in the second chapter, we present results on the coiled-coils (CC) from group-III human septins (SEPT1, SEPT2, SEPT4 and SEPT5) also studied by NMR spectroscopy. Septins are GTP-binding proteins present in most eukaryotic organisms and capable of forming filaments, which are essential in cell division. In this study, we used 1H-1H-NOESY spectra to detect the orientation and helix pairings adopted by the C-terminal coiled-coils in solution. The NOE analysis, aided by back-calculated spectra, showed that the only sequence to show an antiparallel structure was SEPT2CC; all the others are parallel. However, the disappearance of specific peaks in the NMR spectrum of SEPT5CC caused by the attachment of a paramagnetic spin label indicates an antiparallel orientation, contrary to our other NMR result. A simple evaluation of the coiled-coil heptameric positions, based on the occurrence of each amino acid residue occupying each position, revealed that both orientations are equally stable. Despite being far less stable compared to other coiled-coils, both could exist physiologically. Other results from the group also suggest that these peptides could have the ability to form both parallel and antiparallel coiled-coils. We speculate that the antiparallel conformation might be related to cross-linking between filaments. / A principal característica histológica da doença de Alzheimer é a presença de placas amiloides no cérebro de pacientes. O constituinte mais abundante dessas placas é o peptídeo β-amiloide (Aβ). Inicialmente solúvel, o peptídeo exibe em solução um intrincado equilíbrio entre estados monoméricos, oligoméricos (alguns deles tidos como as espécies tóxicas) e fibrilares, o que impossibilita sua cristalização e posterior determinação estrutural por difração de raios-X. A espectroscopia de ressonância magnética nuclear (RMN) de alta pressão foi utilizada por nosso grupo para detectar estados monoméricos raros e de alta energia do Aβ(1-40), coexistindo em equilíbrio com oligômeros e fibras. Esse trabalho visa caracterizar a termodinâmica e a estrutura dos estados excitados raros do peptídeo Aβ através do uso da RMN de alta pressão. Uma grande coleção de espectros de RMN do peptídeo Aβ(1-40) em função da pressão foi coletada e analisada. Predições de estrutura secundária revelaram que o peptídeo Aβ adota estruturas estendidas do tipo fitas-β, similares àquelas encontradas em estruturas de fibras amiloides. A partir das curvas de deslocamento químico e volume de pico pela pressão, ao menos três estados monoméricos puderam ser detectados, os quais foram termodinamicamente caracterizados através do cálculo da variação das suas energias livres de Gibbs (ΔGij) e volumes parciais molares (ΔVij). O estudo de efeitos Overhauser nucleares (NOEs) e de acoplamentos 3JHα-HN de RMN reforçam a existência de estruturas estendidas com propensão a fitas-β, tanto a pressão ambiente (0,1 MPa) quanto em alta pressão (275 MPa). A interação entre o peptídeo Aβ e os D-peptídeos RD2 e RD2D3, inibidores D-enatioméricos de fibras, também foi caracterizada. Nossos resultados indicam que os D-peptídeos reconhecem e se ligam a uma conformação mais compacta de Aβ. A formação dos heterodímeros Aβ-D-peptídeo previne, por fim, a formação dos oligômeros tóxicos, representando uma potencial terapia contra a doença de Alzheimer. Adicionalmente, no segundo capítulo, apresentamos resultados sobre os coiled-coils (CC) das septinas humanas do grupo III (SEPT1, SEPT2, SEPT4 e SEPT5) também estudados por espectroscopia de RMN. Septinas são proteínas ligantes de GTP presentes na maioria dos organismos eucarióticos e capazes de formar filamentos, os quais são essenciais à divisão celular. Nesse estudo, utilizamos espectros 1H-1H-NOESY a fim de detectar a orientação e o pareamento de hélices adotados pelos coiled-coils em solução. A análise dos NOEs, auxiliada por espectros retrocalculados, mostrou que a única sequência a mostrar uma estrutura antiparalela foi SEPT2CC; todas as outras são paralelas. Entretanto, o desaparecimento de picos específicos no espectro de RMN de SEPT5CC causado pela presença de um marcador paramagnético de spin indica uma orientação antiparalela, contrário ao nosso outro resultado de RMN. Uma avaliação simples das posições heptaméricas dos coiled-coils, baseada na ocorrência de cada resíduo de aminoácido em ocupar cada posição, revelou que ambas as orientações são igualmente estáveis. Apesar de serem bem menos estáveis comparadas a outros coiled-coils, ambas poderiam existir fisiologicamente. Outros resultados do grupo também sugerem que esses peptídeos poderiam formar tanto coiled-coils paralelos quanto antiparalelos. Nós especulamos que a conformação antiparalela pode estar relacionada a ligações cruzadas entre filamentos.

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.

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.