Numerous troponin T (TnT) isoforms are generated by alternative RNA splicing primarily in its NH2-terminal hypervariable region, but the functions of these isoforms are not completely understood. In this dissertation work, calcium and terbium binding behavior of several forms of TnT were investigated by spectroscopic and radioactive techniques. Chicken breast muscle TnT binds calcium and terbium through its NH2-terminal Tx motif (HEEAH)n with high affinity (10-6 mM) and fast on-rate (106 - 107 M-1 s-1). Chicken leg muscle TnT and a human cardiac TnT NH2-terminal fragment, which both lack the Tx motif on their NH2-terminal regions, do not have affinities for calcium in the physiological range. Computational predictions on TnT N47 suggest that the TnT NH2-terminal region might fold into an elongated structure with at least one high affinity metal ion binding pocket comprised primarily of the Tx motif sequence and several lower affinity binding sites. In addition, calcium binding to TnT N47 might alter its conformation and flexibility. Luminescence resonance energy transfer measurements and other experimental observations are consistent with the computational predictions suggesting the computational simulated atomic model is reasonable. TnT mutations are responsible for 15% of familiar hypertrophic cardiomyopathy (FHC) cases with a phenotype of relatively mild hypertrophy, but a high incidence of sudden death. Detection of those genetic mutations would facilitate the clinical diagnosis and initiation of treatment at an early stage. This dissertation also investigated a novel hybridization proximity assay (HYPA) combining molecular beacon and luminescence resonance energy transfer (LRET) technologies. Experimental results suggest that a shared stem probe design produces a more consistent response upon hybridization, whereas the internally labeled probe was less consistent, but can yield the highest responses. Using the optimally designed molecular probes, the HYPA provides a detection of alterations in nucleic acid structure of as little as a single nucleotide. This novel HYPA is expected to expand its applications in the analysis and screening of genetic diseases.

Detection of single nucleotide substitutions (SNS) in DNA and RNA has a growing importance in biology and medicine. One traditional approach for recognition of SNS takes advantage of hybridization probes that bind target nucleic acids followed by measuring ?Tm, the difference in melting temperatures of matched and mismatched hybrids. The approach enables SNS differentiation at elevated temperatures (usually 40-65oC) often only in a narrow range of < 10oC and requires high-resolution melting devices. Here we demonstrate that a specially designed DNA probe (X sensor) can broaden ?Tm from ~10oC to ~16oC and distinguish SNS in the interval of ~5-40oC. Therefore, there is no need for heating or measuring Tm for accurate SNS differentiation. Our data indicate that this wide differentiation range is in part due to the non-equilibrium hybridization conditions. Further we explored the idea that it is possible to improve the performance of an X sensor operable in close to equilibrium conditions by shifting its operability to non-equilibrium conditions. One way to achieve this is to introduce as many as possible structured ligands in analyte's dissociated state. Here we show that by introducing the maximum possible conformational constraints in X probe it is possible to shift its operation to non-equilibrium conditions and to improve its selectivity at temperatures < 15oC. Thus, this work points towards a new strategy for the design of highly selective hybridization sensors which operate in non-equilibrium conditions at close to room temperature. The X sensors could be utilized in qPCR, microarrays, as well as RNA analysis in living cells and for ambient temperature point-of-care diagnostics. In the last part of this work, X sensors were used in real time detection of PCR products. The sensors were optimized to operate in PCR buffer with optimal Mg2+ concentration. They were able to detect the target amplicon together with nonspecific products. The results presented here suggest that X sensors might be adopted for real time PCR format.

Nukleinsäuretemplatkontrollierte Reaktionen, die häufig auf Ligations- oder Transferreaktionen basieren, unterliegen dem Effekt der Produktinhibierung. Dadurch können besonders niedrige Templatmengen nur schwer detektiert werden. Im Rahmen dieser Arbeit konnte erstmalig eine grundlegend neue Kategorie templatkontrollierter Reaktionen etabliert werden: templatkontrollierte Spaltungsreaktionen. Dazu wurde ein spaltbarer Linker auf N-Alkylpicoliniumbasis (NAP) entwickelt, der mit einfachen, orthogonalen Konjugationsmethoden (SPAAC, Maleimid-Thiol-Konjugation oder via Amidbinungsknüpfung) sowohl in PNA- als auch in DNA-Strukturen inkludiert werden kann. Die Templat-vermittelte Photoreduktion induziert eine C-O-Bindungsspaltung des Linkers. Daraus resultieren Produkte, die eine geringere Templataffinität besitzen als das Edukt, sodass die Reaktion keiner Produktinhibierung unterliegt. Dies konnte zum Beispiel mittels Triplex-bildender, spaltbarer PNA-Sonden realisiert werden, die eine rasche Spaltungsgeschwindigkeit aufweisen. Hierzu bindet die spaltbare PNA-Sonde auf dem Templat benachbart zu einer mit einem Ruthenium(II)-Komplex modifizierten Assistenzsonde, die die Photoreduktion lichtkontrolliert induzieren kann. In einem alternativen Ansatz wurde die Fluorophor-induzierte Photolyse von NAP-Derivaten näher untersucht und führte letztlich zur Entwicklung eines selbst-spaltenden Molecular Beacons (iMB). Dieser verhält sich wie ein konventioneller iMB, wodurch eine neue Klasse an Molecular Beacons vorgestellt werden konnte. Die templatkontrollierte Photolyse konnte nicht nur in wässrigem Milieu, sondern auch in komplexen Umgebungen wie Zellkulturmedium, Zelllysat und RNA-Extrakt durchgeführt werden. / Nucleic acid templated reactions, which are often based on ligation or transfer reactions, are limited by the phenomenon of product inhibition. As a result, the usage of catalytical amounts of target are up to date only applicable to a limited extend. In this work, a fundamentally new category of nucleic acid templated reactions could be established: nucleic acid templated cleavage reactions. For this purpose, a cleavable linker based on N-alkylpicolinium (NAP) was developed, which can be included in both PNA and DNA structures using simple, orthogonal conjugation methods (SPAAC, maleimide-thiol conjugation or via amide bond formation). A template-mediated photoreduction induces the C-O bond cleavage of the linker. The target affinity of the cleavage products is lower than the parental oligonucleotide prior to cleavage, hence providing a thermodynamical driving force for amplified nucleic acid detection. This could be realized, for example, using triplex-forming, cleavable PNA probes which have a fast cleavage rate. In a first approach various triplex-forming PNA probes were developed that would undergo a photo-reductive C-O-bond cleavage upon irradiation when placed on a template adjacent to an assistant probe equipped with a sensitizer (Ruthenium(II)-complex). In an alternative approach, the fluorophore-induced photolysis of NAP derivatives was investigated and ultimately led to the development of a self-immolative Molecular Beacon (iMB). The iMB behaves like a conventional MB, therefore a new class of Molecular Beacons was introduced. The template-controlled photolysis can be performed not only in aqueous environments, but also in various complex environments such as cell culture medium, cell lysate or RNA extract.

Submitted by FABERSON AUGUSTO FERRASI null (faberson.ferrasi@gmail.com) on 2017-05-28T17:26:24Z No. of bitstreams: 1 dissertassão_mestrado_Faberson Ferrasi_PPGMiT_2017.pdf: 1812047 bytes, checksum: 79b5df7e4bd32070c135e865cd18834d (MD5) / Rejected by Luiz Galeffi (luizgaleffi@gmail.com), reason: Solicitamos que realize uma nova submissão seguindo a orientação abaixo: O arquivo submetido não contém o certificado de aprovação com as assinaturas dos membros da banca (de acordo com as normas de sua unidade). Corrija esta informação e realize uma nova submissão com o arquivo correto. Agradecemos a compreensão. on 2017-05-31T16:24:51Z (GMT) / Submitted by FABERSON AUGUSTO FERRASI null (faberson.ferrasi@gmail.com) on 2017-05-31T17:36:12Z No. of bitstreams: 1 ferrasi_fa_me_bauru.pdf: 1814213 bytes, checksum: 548e7d51b663c5c3ae30e2fa3eafec17 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-05-31T17:43:58Z (GMT) No. of bitstreams: 1 ferrasi_fa_me_bauru.pdf: 1814213 bytes, checksum: 548e7d51b663c5c3ae30e2fa3eafec17 (MD5) / Made available in DSpace on 2017-05-31T17:43:58Z (GMT). No. of bitstreams: 1 ferrasi_fa_me_bauru.pdf: 1814213 bytes, checksum: 548e7d51b663c5c3ae30e2fa3eafec17 (MD5) Previous issue date: 2017-04-17 / Este trabalho tem por objetivo mostrar os resultados de um estudo exploratório sobre a evolução da Internet e conceituar sua fase atual denomina-se Internet das Coisas (Internet of Things - IoT). Tal conceito se apresenta como uma revolução tecnológica e oferece um grande potencial de inovação para diversas áreas. Nesta ocasião, são abordadas as possíveis inovações no uso dos dispositivos móveis alinhados ao seu potencial de comunicação com a Internet. Para isto, foi desenvolvido um experimento apoiado nas tecnologias, com o uso de dispositivos móveis, como smartphones e sensores de proximidade (beacons). Por meio do desenvolvimento de uma aplicação, são oferecidos recursos para captação autônoma de dados sobre a detecção da presença de pessoas em ambientes físicos. Através disso, oferecer um melhor gerenciamento dos dados obtidos e a possibilidade de outras formas de compartilhamento das informações geradas através da Internet, evidenciando, assim, o potencial de aplicação dos conceitos propostos na Internet das Coisas, foco central do projeto. / This work aims to show the results of an exploratory study on the evolution of the Internet and conceptualize its current phase is called Internet of Things (IoT). This concept presents itself as a technological revolution and offers great potential for innovation in several areas. This occasion, the possible innovations in the use of the mobile devices in line with their potential of communication with the Internet are approached. For this, an experiment was developed based on the technologies, with the use of mobile devices, such as smartphones and proximity sensors (beacons). Through the development of an application, resources are provided for autonomous capture of data on the detection of the presence of people in physical environments. Through this, offer a better management of the data obtained and the possibility of other forms of sharing of the information generated through the Internet, thus evidencing the potential of applying the concepts proposed in the Internet of Things, central focus of the project.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Applications that use sensor networks depends on knowing which position where each node is arranged in a particular geographic area. For this, there are algorithms that perform location calculations to estimate the exact position of each node. These algorithms use a different techniques and metrics to make it, for example: the measure of intensity of signal transmission between two nodes. In a sensor network with high density, it becomes infeasible to manually configuring each node positioning. To optimize this task, was developed different types of localization algorithms, with application in several areas, such as tracking, routing protocols, network density control, self organizing, monitoring remote areas, precision agriculture, among others. The basis for localization algorithms is to have a number of reference points, where from these, is calculated the position of each node. A network of sensors with a greater number of reference points, one can consider a network with higher accuracy for calculating the position of its nodes. This is due to being closer to the reference points. However, may result in a higher processing, thereby having a higher energy consumption. Developing a location algorithm with fewer reference points is not a trivial problem. This, because will increase the distance between the node and the reference point and may thereby inserting errors in the measurement of the criteria for the calculation of positioning. For calculation purposes, there are two types of algorithms, algorithms for centralized processing and distributed processing algorithms. Each with its advantages and disadvantages that will be discussed in this paper. The location algorithm is based on a calculation according to data coordinates in a coordinate system, which is raised according to the technique used to denote the distance between the sensor node and the reference points. The importance of knowing the location of each node, there is the need to know exactly where a particular event is occurring. For example, a forest that occupies a large geographic area is to principles of fire at different locations. / Aplicações que utilizam redes de sensores dependem de saber qual a posição onde cada nó encontra-se disposto em uma determinada área geográfica. Para isto, existem algoritmos de localização que efetuam cálculos a fim de estimar a posição exata de cada nó. Estes algoritmos utilizam-se de diferentes técnicas e medidas para realizar tal tarefa, como por exemplo: a medida de intensidade de sinal de transmissão entre dois nós. Em uma rede de sensores com alta densidade, torna-se inviável a configuração manual do posicionamento de cada nó. Para otimizar esta tarefa, foram desenvolvidos diferentes tipos de algoritmos de localização, com aplicação nas mais diversas áreas, como: rastreamento, protocolos de roteamento, controle de densidade da rede, auto organização, monitoramento de áreas remotas, agricultura de precisão, entre outros. A base para algoritmos de localização é possuir um número de pontos de referência, onde a partir destes, será calculado a posição de cada nó. Uma rede de sensores com maior número de pontos de referência, pode-se considerar uma rede com maior precisão para o cálculo de posição de seus nós. Isso, é devido aos nós estarem mais próximos aos pontos de referência. Porém, pode acarretar em um maior processamento, assim, tendo um maior consumo de energia. Projetar um algoritmo de localização com um menor número de pontos de referência não é um problema trivial. Isto porque irá aumentar a distância entre o nó e o ponto de referência, podendo desta forma, inserir erros na medição dos critérios para o cálculo de posicionamento. Para fins de cálculo, existem dois tipos de algoritmos, os algoritmos de processamento centralizado e os algoritmos de processamento distribuído. Cada um com suas vantagens e desvantagens que serão abordadas no decorrer deste trabalho. O algoritmo de localização baseia-se em um cálculo de acordo com as coordenadas em um dado sistema de coordenadas, que será levantada de acordo com a técnica utilizada para estipular a distância entre o nó sensor e os pontos de referência. A importância de conhecer a localização de cada nó, dá-se a necessidade de saber exatamente onde está ocorrendo determinado evento. Por exemplo, uma floresta que ocupa uma grande área geográfica está com princípios de incêndio em diferentes locais. É preciso avaliar quais são estes locais e qual significa um maior risco de destruição, para isto, é preciso saber a posição exata de cada local e assim tomar as devidas decisões de qual princípio deverá ser combatido primeiro. Neste caso, os nós sensores estariam estáticos, porém seria uma rede de sensores de alta densidade para cobrir uma área significativa de floresta. Pode-se também ter uma rede de sensores com nós móveis, onde então o objetivo é realizar o rastreamento da locomoção destes nós. A partir deste rastreamento, pode-se identificar o comportamento de um rebanho de animais por exemplo, verificando seu deslocamento, velocidade e direção. Com estes dados de comportamento, dependendo do animal pode-se deduzir se ele está doente, alimentando-se ou até mesmo morto se imóvel por um longo período de tempo.

Detection, imaging and quantification of gene expression in living cells can provide essential information on basic biological issues and disease processes. To establish this technology, we need to develop molecular probes and cellular delivery methods to detect specific RNAs in live cells with potential for in vivo applications. In this thesis work, the major focus is placed on the development of molecular beacons and biochemical approaches (peptides etc.) to deliver such probes to different cellular compartments. These approaches are then employed to study the expression and localization of mRNAs, co-localization of mRNAs with cytoplasmic organelles and cytoskeleton, and co-localization of RNA molecules in the nuclei of living cells. Further along this direction, we were interested in developing a better understanding of the functional states of mRNAs and the fluorescent signal observed in optical imaging experiments. To acheive this goal, we altered the translational process and studied its effect on the detection of mRNAs in living cells. The results of these studies indicate that the translational state of mRNAs favors the hybridization of molecular beacon with its target sequence. This study has also provided the evidence that molecular beacons are reversibly bound to target mRNAs and the repression of the translational process can prevent molecular beacon from binding to its target mRNA. Further, using these approaches in combination with FRAP based biophysical analysis, the dynamics of endogenous RNA in living cells are studied. These studies revealed the possible subcellular organization of RNA molecules and their dynamics in living cells. The results also demonstrated the role of cytoskeleton and ATP in the mobility of specific mRNAs in the cytoplasm. In addition to optical probes, studies have been carried out to develop an MRI contrast agent using iron-oxide nanoparticles for deep tissue molecular imaging. Specifically, we have functionalized magnetic nanoparticles that are water-soluble, mono-dispersed, biocompatible, and easily adaptable for multifunctional bioconjugation of probes and ligands. We have successfully delivered magnetic nanoparticle bioconjugates into live cells and demonstrated their effect on relaxivity. We have further studied the role of coating thickness for optimization of contrast and further enhance the fundamental understanding of contrast mechanisms.

Mobile technology is developing quickly and becomming a part of daily life and communication. Bluetooth Low Energy is presented as a new way of mobile interaction. However there are not enough studies in this field on the interaction between mobile devices and the physical world. In order to understand user needs, and to encourage users to interact, we simulated an interaction process through designing and testing a prototype in a specific indoor environment. Prototype effectiveness and main characteristics for future mobile application was evaluated through a qualitative user study with 20 participants who interacted with our prototype. / Den mobila tekniken växer snabbt och blir en del av våra liv och vår kommunikation. BLE presenteras på ett nytt sätt inom mobilinteraktion. Det förekommer inte tillräckligt med studier inom området för interaktionen mellan mobila enheter och den fysiska världen. För att förstå användarbehov och få användare att interagera har vi simulerat en interaktiv process genom att designa och testa en prototyp i en specifik inomhusmiljö. Utvärdering av prototypens effektivitet och heuristiken har evaluerats genom en kvalitativ studie med 20 användare som har deltagit i interaktionen med vår prototyp.

Cette thèse s’inscrit dans le cadre d’un projet visant à développer un robot mobile autonome capable de réaliser des tâches spécifiques dans une zone préalablement définie par l’utilisateur. Afin de faciliter la mise en œuvre du système, des balises radiofréquences fournissant des mesures de distance par rapport au robot sont disposées au préalable autour du terrain. Le déploiement du robot s’effectue en deux phases, une première d’apprentissage du terrain, puis une seconde, où le robot effectue ses tâches de façon autonome. Ces deux étapes nécessitent de résoudre les problèmes de localisation et de localisation et cartographie simultanées pour lesquels différentes solutions sont proposées et testées en simulation et sur des jeux de données réelles. De plus, afin de faciliter l’installation et d’améliorer les performances du système, un algorithme de placement des balises est présenté puis testé en simulation afin de valider notamment l’amélioration des performances de localisation. / This thesis is part of a project which aims at developing an autonomous mobile robot able to perform specific tasks in a preset area. To ease the setup of the system, radio-frequency beacons providing range measurements with respect to the robot are set up beforehand on the borders of the robot’s workspace. The system deployment consists in two steps, one for learning the environment, then a second, where the robot executes its tasks autonomously. These two steps require to solve the localization and simultaneous localization and mapping problems for which several solutions are proposed and tested in simulation and on real datasets. Moreover, to ease the setup and improve the system performances, a beacon placement algorithm is presented and tested in simulation in order to validate in particular the improvement of the localization performances.

La survie d’un État dépend de la capacité de ses dirigeants à être informés et à communiquer leurs décisions, puis de contrôler leur exécution. L’information dans l’Antiquité circulait par courrier mais aussi par transmissions de signaux sonores et/ou lumineux. Après une étude documentaire des récits attestant la transmission d’informations par signaux lumineux et sonores au Levant au premier millénaire av. J.-C., nous avons démontré que les logiciels utilisés pour la conception des réseaux actuels de télécommunications peuvent suggérer des sites relais utilisés pour cette transmission. Les limites techniques des moyens de l’époque sont des paramètres déterminants. Nous en avons déduit des modèles de réseaux. Enfin ces hypothèses ont été validées sur le terrain. / The survival of a State depends on the capacity of its leaders to be informed and on their successes to communicate their decisions, then to control their executions. The information in the ancient times circulated by mail, but also by transmissions of sound and/or light signals.After a documentary survey of stories attesting the transmission of informations by light and sound signals in the Near East during the first millennium BCE, we tried to see if the software used for telecommunications networks design and planning can hint repeater sites used for this transmission. The limitations of the technology at that time will determine parameters. Models of networks have been deducted. Finally these hypotheses have been validated on the ground.

Force est de constater aujourd’hui que la localisation d’un bien ou d’une personne est devenue une nécessité. Plusieurs solutions existent en extérieur, largement dominées par le système GPS. Pour la localisation en intérieur, la précision se dégrade en raison des trajets multiples et de l’atténuation des signaux traversant les murs. Cette thèse se focalise sur la problématique de localisation à l’intérieur d’un bâtiment en utilisant les technologies présentes dans des smartphones et des tablettes fonctionnant sous le système d’exploitation Android disponible dans divers marques. Les systèmes de localisation en intérieur exploitent différents supports tels que les ondes radio-fréquence (RF) ou les capteurs inertiels embarqués dans un terminal. Dans le cas RF, ils utilisent des points références dont la répartition sur la zone couverte influe sur la performance en localisation. Une première contribution est un développement d’algorithme d’optimisation d’emplacement des balises basé sur le recuit simulé. Les signaux extraits des capteurs inertiels sont utilisés par la navigation pédestre à l’estime (NPE) pour déterminer le trajet effectué depuis une position connue. Ils dépendent de la sensibilité des paramètres intrinsèques de ces capteurs et ils sont corrompus par des bruits. Dans le cas NPE, une calibration permet d’obtenir des données exploitables pour l’estimation de l’orientation de déplacement et pour la détection des pas. Cette orientation est supposée identique à celle du terminal mais il y a un intérêt à prendre en compte le biais d’orientation entre les deux. Une autre contribution est une proposition d’algorithme de détection des pas exploitant la logique floue. / Nowadays, the localization of a device or person has become mandatory. If many solutions exist for outdoor environment, as the GPS one, any fails to provide an expected accuracy for indoor environment because of the multipath phenomena and the attenuation of signals crossing walls. This thesis focuses on the localization problem in buildings by using existed technologies in smartphones and tablets managed by Android OS - which is available in several brands. The indoor localization systems are using different technologies like radio-frequency (RF) waves or inertial sensors embedded in handsets. In the RF case, they use anchors or beacons, whose position impacts the localization performance for the covered zone. Our first contribution was the placement optimization of beacons using simulated annealing algorithm. Next to improve the localization performance, the inertial sensors, embedded in smartphones, have been used. The pedestrian dead reckoning (PDR) algorithm employs the extracted signals from the inertial sensors and determines the path done since a known position. These extracted signals are affected by the intrinsic parameters of sensors and they are corrupted by noises. The calibration of the sensors is compulsory to obtain data that could be used to estimate the walking orientation and the number of done steps by the user. It is often supposed that the walking orientation is the same as the smartphone orientation; however it might be interesting to consider the bias between these two orientations. A last contribution, in this thesis, consists on a proposed algorithm for step detection using fuzzy logic.