京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21736号 / 工博第4553号 / 新制||工||1710(附属図書館) / 京都大学大学院工学研究科社会基盤工学専攻 / (主査)教授 木村 亮, 教授 岸田 潔, 准教授 木元 小百合 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

[pt] O campo de estudo com estruturas origami tem crescido nos últimos anos como soluções inovadoras para problemas em ciência e engenharia. As primeiras aplicações aproveitaram a ideia de que um sistema estrutural pode ser dobrado de forma compacta e subsequentemente estendido, ou que a automontagem pode ser usada para construir uma estrutura tridimensional inspirada em uma folha fina. O presente trabalho apresenta comparações entre o modelo de barra e dobradiça com os modelos mais simples possíveis de elementos finitos híbridos de placa e casca para a representação de painéis de estruturas origami. O modelo de barra e dobradiça traz uma abordagem baseada na modelagem do estado parcialmente dobrado de painéis com uma estrutura de treliça articulada, onde as dobras são as barras, e os vértices as articulações. Os modelos de casca e placa utilizam a formulação híbrida dos elementos finitos, que tem como base o potencial de Hellinger-Reissner, que permite a aproximação dos campos de tensões satisfazendo a equação de equilíbrio do problema de elasticidade, e campos de deslocamento que atendem a compatibilidade no contorno. Exemplos numéricos mostram o comportamento mecânico dessas estruturas e a energia para realização das dobras através de autovalores dos respectivos automodos. É feita também uma avaliação dinâmica dos modelos para montagens estruturais com uma, duas e quatro células. / [en] Research works involving structural origami have grown in recent years, especially applied to science and engineering problems. Early applications took advantage of the idea that a system can be folded compactly and subsequently deployed, and that self-assembly can be used to construct a three dimensional structure by starting from a thin sheet. The present work compares bar and hinge models with the simplest hybrid finite element models for plate and shell in order to represent origami structure panels. The hybrid finite element formulation is based on the Hellinger-Reissner potential for an approximation of the stress field, thus satisfying the equilibrium equation of the elasticity problem in the domain. The bar and hinge model approach, as given in the literature, is based on folded patterns as pin-jointed truss frameworks. Each vertex in the folded sheet is represented by a pin-joint, and every fold line by a bar element. Numerical examples show the mechanical behavior of these structures and the folding energy using eigenvalues of the respective eigenmodes. Dynamic analysis of the models is also carried out for structure assemblages with one, two and four cells.

ABSTRACT Fabrication and Characterization of Torsional Micro-Hinge Structures Mike Marrujo There are many electronic devices that operate on the micrometer-scale such as Digital Micro-Mirror Devices (DMD). Micro actuators are a common type of DMD that employ a diaphragm supported by torsional hinges, which deform during actuation and are critical for the devices to have high stability and reliability. The stress developed within the hinge during actuation controls how the actuator will respond to the actuating force. Electrostatically driven micro actuators observe to have a fully recoverable non-linear viscoelastic response. The device consists of a micro-hinge which is suspended by two hinges that sits inside a micro machined well. To achieve a specific angle of rotation when actuated, the mechanical forces need to be characterized with a range of different forces applied to the edge of the micro-hinge. This research investigates the mechanical properties and the amount of force needed to rotate to specific angles by comparing theoretical performance to experimentally measured values. Characterizing the mechanical forces on the micro-hinge will further the understanding of how the device operates under a specific applied force. The material response to the amount of stress within the hinges will control the amount of actuation that is achieved by that force. The test devices were fabricated using common semiconductor fabrication techniques. The micro-hinge device was created on a 500µm, double-sided polished, single crystal (100) silicon wafer. In order to create this device, both wet etching and dry etching techniques were employed to produce an 8µm thick plate structure. The bulk etching of 480µm was achieved by wet etching down into the silicon (Si) to create the wells. Dry etching was used for its high precision to release the micro-hinge structure. Once fabricated, the micro-hinge actuators were tested using a Technics turntable arm with a built in micrometer that applied a constant force while measuring the displacement of the actuator. The rotation of the hinge was measured by reflecting a Helium-Neon (HeNe) laser beam off a mirror, which is attached to the pivot of the arm that’s applying the force, and any type of displacement was recorded with a Photo Sensitive Device (PSD). The test stand applied a small force which replicated the amount of electrostatic forces needed to achieve a specific degree of rotation. Results indicate that the micro-hinge achieved a repeatable amount of rotation when forces were applied to it. The micro-hinge would endure deformation when too much force would be applied and yield a maximum amount of force allowed.

No description available.

O objetivo deste estudo foi comparar o eixo de rotação verdadeiro com o anatômico em ambiente virtual 3D, e seus efeitos sobre dois pontos anatômicos mandibulares. O eixo verdadeiro foi determinado em 14 indivíduos por meio de axiografia, e transferido para o ambiente virtual por TCFC, e posteriormente determinado anatomicamente, onde foram medidas as distâncias entre ambos. Foram simuladas rotações de 2º, 5º e 8º da mandíbula nos dois eixos, tanto para abertura como fechamento, e quantificadas as diferenças nos pontos da linha média inferior (LMI) e pogônio (Pg). O teste t pareado foi utilizado para examinar as diferenças entre as médias nas posições desses pontos (p<0,05). Os eixos verdadeiros localizaram-se dentro de um raio de 5 mm do anatômico em 67,86% da amostra. A distância absoluta média entre os eixos foi de 4,79 mm, enquanto que a vetorial foi de 2,33 no plano horizontal e 3,03 mm no vertical, resultando na direção anteroinferior em 71,43% dos eixos verdadeiros. Houve diferença estatisticamente significante na posição dos pontos LMI e Pg para todas as magnitudes e direções, entre os eixos. O eixo verdadeiro está localizado na direção anteroinferior em relação ao anatômico. Os efeitos na mandíbula são significantes e diferentes em todas as amplitudes, tanto para abertura como fechamento, porém com possível pequena relevância clínica. / The aim of this study was to compare the true hinge axis to the anatomic one in a virtual 3D environment, and also their respective effects on two mandibular anatomic points. The true axis has been determined in 14 individuals by means of axiography, and later transferred to a virtual environment by CBTC, where the anatomical axis was determined, and measured the distances between them. Mandibular rotation of 2º, 5º and 8º in both axes were performed, both for opening and closing, as well as the quantification of the difference found in the points of the lower midline (LM) and pogonion (Pg). Paired t-test was used to examine differences between the average values in the position of those points (p<0,05). The true axis was located within a 5mm-radius of the anatomic axis throughout 67.86% of the sample. The average absolute distance between the axes was 4.79 mm, while the vector distance was 2.33 mm in the horizontal plane e 3.03mm in the vertical plane, amounting to an anteriorinferior direction of 71.43% of the true axis. There was significant difference in the position of points LM and Pg to all magnitudes and directions within the axes. The true hinge axis is located in the anterior-inferior direction in relation to the anatomic axis. The effects observed onto the mandible are significant and different in all amplitudes, both for opening and closing positions, however they present small clinical relevance.

The analytical tools used in displacement based design and assessment procedures require accurate strain limits to define the performance levels. Additionally, recently proposed changes to modeling and acceptance criteria in seismic regulations for both flexure and shear dominated reinforced concrete structural walls proves that a comprehensive study is required for improved limit state definitions and their corresponding values. This is due to limitations in the experimental setups, such that most previous tests used a single actuator at the top of the wall, which does not reflect the actual loading condition, and infeasibility of performing tests of walls of actual size in actual structural configuration. This study utilizes a well calibrated finite element modeling tool to investigate the relationship between the global drift, section rotation and curvature, and local concrete and steel strains at the extreme fiber of rectangular structural walls. Functions defining more exact limits of modeling parameters and acceptance criteria for rectangular reinforced concrete walls were developed. This way a strict evaluation of the requirements embedded in the Turkish Seismic Code and other design guidelines has become possible. Several other aspects of performance evaluation of structural walls were studied also. Accurate finite element modeling strategies and analytical models of wall and frame-wall systems were developed for seismic response calculations. The models are able to calculate both the static and dynamic characteristics of wall type buildings efficiently. Seismic responses of wall type buildings characterized with increasing wall area in the plan were analyzed under design spectrum compatible normal ground motions.

Βασικός σκοπός της παρούσης διατριβής είναι η ανάπτυξη μιας διαδικασίας προσδιορισμού της βέλτιστης στρατηγικής ενίσχυσης ενός υφιστάμενου ανεπαρκούς κτιρίου, αξιοποιώντας τα δεδομένα που προκύπτουν από την αποτίμησή του με χρήση της ανελαστικής στατικής ανάλυσης και συνεκτιμώντας τις καμπύλες που αντιστοιχούν σε εναλλακτικές λύσεις επέμβασης και προσδιορίζονται προσεγγιστικά. Επειδή η καμπύλη αντίστασης του αρχικού φορέα αποτελεί τη σημαντικότερη πληροφορία που αξιολογείται στα πλαίσια της διαδικασίας που αναπτύχθηκε, ένας έμμεσος στόχος της παρούσης διατριβής είναι η αξιολόγηση των προσομοιωμάτων συμπεριφοράς στοιχείων οπλισμένου σκυροδέματος που συμπεριλαμβάνονται στο σχέδιο του Ελληνικού Κανονισμού Επεμβάσεων (ΚΑΝ.ΕΠΕ.), τα οποία χρησιμοποιήθηκαν στα πλαίσια των αναλύσεων που πραγματοποιήθηκαν. Για τις ανάγκες της διερεύνησης των προσομοιώματων του ΚΑΝ.ΕΠΕ. και την ανάπτυξη της μεθόδου επιλογής στρατηγικής επέμβασης διενεργήθηκαν ανελαστικές αναλύσεις σε κτίρια που τα οποία είχαν μορφωθεί και διαστασιολογηθεί με βάση της επικρατούσες πριν το 1985 αντιλήψεις. Στο πρώτο κεφάλαιο της εργασίας γίνεται μια εισαγωγή στο θέμα αποτίμησης και ενίσχυσης υφιστάμενων κατασκευών. Εντοπίζονται οι δυσκολίες και οι απαιτήσεις του προβλήματος της μελέτης υφιστάμενων κτιρίων και σχολιάζονται σύντομα τα υπάρχοντα κανονιστικά σχέδια για την αποτίμηση υφιστάμενων κατασκευών. Στο δεύτερο κεφάλαιο παρουσιάζονται οι μέθοδοι που χρησιμοποιούνται για την αποτίμηση υφιστάμενων κτιρίων. Οι μέθοδοι διακρίνονται σε ελαστικές και ανελαστικές. Η στατική ανελαστική ανάλυση παρουσιάζεται εκτενέστερα, καθώς χρησιμοποιείται για τις αναλύσεις που πραγματοποιούνται. Συγκεκριμένα αναφέρονται οι παραδοχές στις οποίες βασίζεται και παρουσιάζονται τρεις διαφορετικές διαδικασίες για τον προσδιορισμό της στοχευόμενης μετατόπισης. Ιδιαίτερη αναφορά γίνεται στη μέθοδο των ανελαστικών φασμάτων απαίτησης, στην οποία βασίζεται η διαδικασία επιλογής στρατηγικής επέμβασης που αναπτύχθηκε. Το τρίτο κεφάλαιο αφορά τα προσομοιώματα συμπεριφοράς στοιχείων Ο/Σ που χρησιμοποιούνται σε ανελαστικές αναλύσεις. Συγκεκριμένα παρουσιάζεται αναλυτικά το προσομοίωμα του ΚΑΝ.ΕΠΕ. που υιοθετείται στη συνέχεια για τις ανάγκες της προσομοίωσης των κτιρίων που αναλύονται. Σύντομη αναφορά γίνεται και σε άλλα προσομοιώματα, τα οποία προτείνονται σε σχέδια κανονισμών ή ερευνητικές εργασίες. Στο τέταρτο κεφάλαιο εξετάζονται αναλυτικά τα προτεινόμενα από τον ΚΑΝ.ΕΠΕ. προσομοιώματα συμπεριφοράς των δομικών στοιχείων και η χρήση τους για την σεισμική αποτίμηση με χρήση της μη-γραμμικής στατικής ανάλυσης. Οι προτεινόμενες σχέσεις χρησιμοποιούνται για την προσομοίωση των μελών δύο ιδεατών κτιρίων και ενός πραγματικού. Εξετάζονται πιθανές αποκλίσεις μεταξύ των διατιθέμενων σχέσεων, καθώς και η επιρροή διαφορετικών παραδοχών για τις τιμές του μήκους διάτμησης και του ανηγμένου αξονικού φορτίου στα προσδιοριζόμενα μεγέθη. Τέλος ελέγχεται η επίδραση των ίδιων παραμέτρων στην τελική μορφή της καμπύλης τέμνουσας βάσης – μετατόπισης και στα συμπεράσματα της διαδικασίας αποτίμησης. Το πέμπτο κεφάλαιο αναφέρεται στις στρατηγικές ενίσχυσης υφιστάμενων κτιρίων. Συγκεκριμένα αρχικά γίνεται η διάκριση μεταξύ στρατηγικής και τεχνικής επέμβασης. Στη συνέχεια αναφέρονται και σχολιάζονται διαδικασίες για την επιλογή της κατάλληλης στρατηγικής επέμβασης που αναπτύχθηκαν παλαιότερα. Ακολούθως παρουσιάζεται η προτεινόμενη διαδικασία για την επιλογή της κατάλληλης στρατηγικής επέμβασης. Αναφέρονται οι βασικές παραδοχές που λαμβάνονται και τα βήματα υπολογισμών που πραγματοποιούνται. Τέλος η προτεινόμενη διαδικασία εφαρμόζεται σε δύο ιδεατά κτίρια και παρουσιάζονται αναλυτικά τα αποτελέσματα και τα συμπεράσματα που αφορούν τις απαιτούμενες ενισχύσεις στα κτίρια που αναλύονται. Στο έκτο κεφάλαιο επιχειρείται η σύνδεση των εκτιμώμενων καμπυλών συμπεριφοράς για τα ενισχυμένα κτίρια, οι οποίες προκύπτουν από τη διαδικασία επιλογής στρατηγικής επέμβασης που αναπτύχθηκε, με τις απαιτούμενες επεμβάσεις στα μέλη. Σκοπός είναι να προκύψει μια διαδικασία προδιαστασιολόγησης των ενισχύσεων. Ορίζονται αδιάστατες παράμετροι που συσχετίζουν τα χαρακτηριστικά του φορέα με αυτά των μελών. Αναλύονται φορείς που προκύπτουν από υλοποίηση εναλλακτικών ενισχύσεων στα κτίρια στα οποία εφαρμόστηκε η προτεινόμενη διαδικασία και εξετάζεται πως μεταβάλλεται η τιμή των παραμέτρων που ορίστηκαν. Με βάση τα αποτελέσματα των ανελαστικών αναλύσεων και των υπολογισμών που παρουσιάζονται, διατυπώνονται απλοί κανόνες για την αρχική διαστασιολόγηση των επεμβάσεων στα μέλη. Στο τελευταίο κεφάλαιο γίνεται μια προσπάθεια ερμηνείας των αποτελεσμάτων των διερευνήσεων που παρουσιάσθηκαν ώστε να προκύψουν γενικότερα συμπεράσματα για τη διαδικασία επιλογής στρατηγικής επέμβασης που αναπτύχθηκε. Από τη διερεύνηση που πραγματοποιήθηκε προκύπτει πως με χρήση της προτεινόμενης μεθόδου εκτιμώνται με ικανοποιητική ακρίβεια οι καμπύλες συμπεριφοράς των ενισχυμένων κατασκευών για δύο ακραίες περιπτώσεις επέμβασης (αύξηση αντοχής - δυσκαμψίας και αύξηση πλαστιμότητας), η αξιολόγηση των οποίων μπορεί να οδηγήσει σε μια αξιόπιστη επιλογή της βέλτιστης στρατηγικής ενίσχυσης, χωρίς να απαιτούνται εμπειρικού χαρακτήρα εκτιμήσεις. Τέλος είναι δυνατόν να γίνει μια συντηρητική εκτίμηση των απαιτούμενων επεμβάσεων στα μέλη, η οποία κατευθύνει σε μεγάλο βαθμό την τελική επιλογή λύσης. / The main aim of the present thesis is the development of a procedure to determine the optimum retrofit strategy for an existing building, using the results obtained from the assessment of the building via non-linear static analysis and evaluating the capacity curves that correspond to the application of different strengthening solutions for the building under consideration. The latter curves are approximated without further analysis. Because of the significance of the capacity curve of the original building which is taken into consideration in the proposed strategy selection procedure, a second aim of this thesis is the evaluation of the analytical models for the behavior of Reinforced Concrete (R/C) members which are included in the first and second draft versions of the Greek Retrofitting Code (GRECO). These were used to create the numerical models of the buildings that have been analyzed. In order to develop the proposed procedure for the selection of the optimum retrofit strategy, as well as to evaluate the proposed models that are referred above, three buildings have been analyzed via non-linear static analysis (pushover analysis). The dimensioning of members of these buildings complies with the regulatory demands of the prior to 1985 Greek building Codes. In the first chapter of the present thesis, a brief introduction to the topic of assessment and strengthening of existing buildings is conducted. The basic difficulties of the problem of analyzing existing structures are pointed. Finally, draft codes that have been developed for the assessment and rehabilitation of existing buildings are reviewed briefly. In the second chapter, the basic analysis procedures that are used for the assessment of existing buildings are presented. The available procedures are separated into linear and non-linear. Non-linear static (pushover) analysis is presented thoroughly since it is used for the analyses of the buildings referred above. The basic assumptions of this analysis procedure are described as well as three different methods to determine the target displacement (or performance point). The capacity spectrum method is presented in detail, since it is the basis for the development of the proposed strategy selection procedure. The third chapter refers to the analytical models for the behavior of R/C members that are used in non-linear analyses. The models proposed in GRECO, which have been used in terms of the analyses of the buildings that were examined in this thesis, are presented thoroughly. Other models included in draft codes (such as FEMA 356) or proposed by researchers are briefly reviewed. In the fourth chapter, the analytical models for the behavior of R/C members that are included in GRECO are presented in detail. Moreover, the application of the above models in the assessment of existing buildings using pushover analysis is examined. The proposed equations are applied to model the behavior of the members of three buildings, in order to examine whether the use of different equations leads to significantly different results for the inelastic deformation capacities of the members. Furthermore, the impact of different assumptions for parameters, such as the non-constant axial load, to the results of the above equations is discussed. Finally, the effect of the above parameters in the capacity curve of the building, which is being analyzed, is examined. In the fifth chapter, the strategies for the retrofit of existing buildings are discussed. The difference between retrofit techniques and retrofit strategies is stated. Available procedures for the selection of the optimum retrofit strategy are reviewed and commented. Furthermore, the procedure proposed in this thesis is presented. The basic assumptions and the required calculations are stated. Finally, the procedure is applied for the selection of the optimum retrofit strategy of two of the buildings analyzed earlier in the present thesis. The results and main conclusions are referred briefly. In the sixth chapter, the estimated capacity curves of the strengthened buildings, which arise from the strategy selection procedure that has been developed, are correlated with the required rehabilitation measures for the members. The buildings under consideration are analyzed taking into account the application of different rehabilitation scenarios and several parameters such as the strength or ductility of the retrofitted members in regard with that of the entire building are evaluated. Finally a simplified procedure for the estimation of the needed rehabilitation measures for the members in order to achieve the targeted capacity curve for the structure is proposed. In the final chapter, the results concerning the proposed procedure for the estimation of the optimum retrofit strategy for an existing building are reviewed and the main conclusions are presented. The use of the proposed procedure results in the estimation of the capacity curve of the rehabilitated building with acceptable accuracy, considering two “extreme” retrofit scenarios (system strengthening and stiffening or increasing the ductility of the building). The evaluation of these two curves leads to the selection of the optimum retrofit strategy for a building, which usually combines the effects of the above scenarios. Finally, it is possible to estimate the required retrofit measures for the members of the structure under consideration, although the results are conservative and can be used only for the needs of the initial evaluation discussed in this thesis.

O objetivo deste estudo foi comparar o eixo de rotação verdadeiro com o anatômico em ambiente virtual 3D, e seus efeitos sobre dois pontos anatômicos mandibulares. O eixo verdadeiro foi determinado em 14 indivíduos por meio de axiografia, e transferido para o ambiente virtual por TCFC, e posteriormente determinado anatomicamente, onde foram medidas as distâncias entre ambos. Foram simuladas rotações de 2º, 5º e 8º da mandíbula nos dois eixos, tanto para abertura como fechamento, e quantificadas as diferenças nos pontos da linha média inferior (LMI) e pogônio (Pg). O teste t pareado foi utilizado para examinar as diferenças entre as médias nas posições desses pontos (p<0,05). Os eixos verdadeiros localizaram-se dentro de um raio de 5 mm do anatômico em 67,86% da amostra. A distância absoluta média entre os eixos foi de 4,79 mm, enquanto que a vetorial foi de 2,33 no plano horizontal e 3,03 mm no vertical, resultando na direção anteroinferior em 71,43% dos eixos verdadeiros. Houve diferença estatisticamente significante na posição dos pontos LMI e Pg para todas as magnitudes e direções, entre os eixos. O eixo verdadeiro está localizado na direção anteroinferior em relação ao anatômico. Os efeitos na mandíbula são significantes e diferentes em todas as amplitudes, tanto para abertura como fechamento, porém com possível pequena relevância clínica. / The aim of this study was to compare the true hinge axis to the anatomic one in a virtual 3D environment, and also their respective effects on two mandibular anatomic points. The true axis has been determined in 14 individuals by means of axiography, and later transferred to a virtual environment by CBTC, where the anatomical axis was determined, and measured the distances between them. Mandibular rotation of 2º, 5º and 8º in both axes were performed, both for opening and closing, as well as the quantification of the difference found in the points of the lower midline (LM) and pogonion (Pg). Paired t-test was used to examine differences between the average values in the position of those points (p<0,05). The true axis was located within a 5mm-radius of the anatomic axis throughout 67.86% of the sample. The average absolute distance between the axes was 4.79 mm, while the vector distance was 2.33 mm in the horizontal plane e 3.03mm in the vertical plane, amounting to an anteriorinferior direction of 71.43% of the true axis. There was significant difference in the position of points LM and Pg to all magnitudes and directions within the axes. The true hinge axis is located in the anterior-inferior direction in relation to the anatomic axis. The effects observed onto the mandible are significant and different in all amplitudes, both for opening and closing positions, however they present small clinical relevance.

The aim of the thesis is to create an analytical 2D model of a multi-storey building and its consequent loading until the point of collapse which occures due to formation of plastic hinges. The first part is going to present a linear analysis of the problem; it focuses on location and level of load when the plastic hinges are formed. Then a nonlinaer analysis is going to be performed by RFEM programme using postcritical analysis and dynamic relaxation. Differences between the results of mentioned types and methods of analysis are going to be compared and an impact of variables is going to be evaluated .

Abstract The aim of this PhD-study was to better understand the structure-function relationship of triosephosphate isomerase (TIM) and to use this expertise to change its substrate specificity. TIM is an important enzyme of the glycolytic pathway which catalyzes the interconversion of D-glyceraldehyde phosphate (D-GAP) and dihydroxyacetone phosphate (DHAP). Two main subjects are discussed: the engineering of monomeric TIM to create new substrate specificity and the structure-function relationship studies of the catalytically important mobile loop6. The starting point for the protein engineering project was the monomeric ml8bTIM, with an extended binding pocket between loop7 and loop8. Rational protein engineering efforts have resulted in a new variant called A-TIM that can competently bind wild type transition state analogues. A-TIM was also able to bind citrate, a compound that the wild type TIM does not bind. This A-TIM citrate complex structure is a good starting point for future protein engineering efforts. Based on the assumption that it would be beneficial for the monomeric forms of TIM to have loop6 closed permanently to increase the population of competent active sites, two point mutation variants, A178L and P168A were generated and characterized. The A178L-mutation was made to favor the closed conformation of loop6 through steric clashes in the open conformation. The P168A variant was made to stabilize the closed conformation of loop6 by removing strain. The A178L mutation induced some features of the closed conformation, but did not result in a closed conformation in the absence of ligands. Our structural studies also show that the P168A mutation does not favor the closed conformation either. However, the structures of the unliganded and liganded P168A variant, together with other known TIM structures show that the substrate binding first induces closure of loop7. This conformational switch subsequently forces loop6 to adopt its closed conformation. The protein engineering project was successful, but the efforts to find variants with a permanently closed loop6 did not fully succeed. In the context of this thesis a monomeric variant of TIM, with new binding properties, was created. Nevertheless, A-TIM still competently binds the inhibitors and transition state analogues of wild type TIM. Also, when combined, results discussed in the context of this thesis indicate that in wild type TIM the closure of loop7 after ligand binding is the initial step in the series of conformational changes that lead to the formation of the competent active site. / Tiivistelmä Tämän väitöskirjatyön tarkoituksena oli oppia paremmin ymmärtämään trioosifosfaatti-isomeraasin (TIM) toimintamekanismeja sen rakenteen perusteella ja käyttää tätä tietämystä samaisen proteiinin muokkaamiseen uusiin tarkoituksiin. TIM on keskeinen entsyymi solun energian tuotannossa ja sen toiminta on välttämätöntä kaikille eliöille. Tämän vuoksi on tärkeää oppia ymmärtämään miten se saavuttaa tehokkaan reaktionopeutensa ja miksi se katalysoi vain D-glyseraldehydi-3-fosfaattia (D-GAP) ja dihydroksiasetonifosfaattia (DHAP). TIM:n toiminta mekanismien ymmärtämiseksi sen aminohapposekvenssiä muokattiin kahdesta kohtaa (P168A ja A178L) ja seuraukset todettiin mittaamalla tuotettujen proteiinien stabiilisuutta optisesti eri lämpötiloissa ja selvittämällä niiden kolmiulotteinen rakenne käyttäen röntgensädekristallografiaa. Mutaatioita tehtiin dimeeriseen villityypin TIM:in (wtTIM) ja jo aikaisemmin muokattuun monomeeriseen TIM:in (ml1TIM). Näiden mutaatioiden tarkoituksena oli suosia entsyymin aktiivista konformaatiota, jossa reaktion kannalta välttämätön vapaasti liikkuva peptidisilmukka numero 6 on suljetussa konformaatiossa. Monomeerisissä TIM:ssa peptidisilmukka numero 6:n ei ole välttämätöntä aueta. Tulokset mutaatiokokeista olivat osittain lupaavia. P168A-mutaatio lisäsi D-GAP:in sitoutumista, mutta rikkoi tärkeän mekanismin suljetussa, ligandia sitovassa, konformaatiossa. A178L-mutaatio aiheutti muutoksia avoimeen konformaatioon ja teki siitä suljettua konformaatiota muistuttavan jopa ilman ligandia, mutta samalla koko proteiini muuttui epävakaammaksi. Näistä kahdesta mutaatiosta A178L voisi olla hyödyllinen muokattujen TIM-versioiden ominaisuuksien parantamiseksi. Lisäksi yhdessä jo aikaisemmin julkaistujen yksityiskohtien kanssa nämä tulokset tekevät mahdolliseksi esittää tarkennusta siihen miten TIM toimii kun ligandi saapuu sen lähettyville. Tämän väitöskirjatyön yksi tavoite oli myös muokata edelleen monomeeristä TIM versiota (ml8bTIM), joka on suunniteltu siten, että se voi mahdollisesti sitoa uudenlaisia ligandeja. Tämä projekti vaati onnistuakseen 20 eri versiota ml8bTIM:n sekvenssistä ja noin 30 rakennetta. Uusia ligandeja sitova muoto (A-TIM) sitoi onnistuneesti sitraattia ja villityypin TIM:n inhibiittoreita. Erityisen lupaavaa oli, että A-TIM sitoi myös bromohydroksiasetonifosfaattia (BHAP), joka sitoutuu ainoastaan toimivaan aktiiviseen kohtaan. Nämä tulokset osoittavat, että A-TIM kykenee tarvittaessa katalysoimaan isomerisaatio reaktion uudenlaisille molekyyleille. Esimerkiksi katalysoimaan isomerisointireaktiota sokerianalogien tuotannossa.