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Researchers use multiple sequence alignment algorithms to detect conserved regions in genetic sequences and to identify drug docking sites for drug development. In this dissertation, a novel algorithm is presented for using physicochemical properties to increase the accuracy of multiple sequence alignments. Secondary structures are also incorporated in the evaluation function. Additionally, the location of the secondary structures is assimilated into the function. Multiple properties are combined with weights, determined from prediction accuracies of protein secondary structures using artificial neural networks. A new metric, the PPD Score is developed, that captures the average change in physicochemical properties. Using the physicochemical properties and the secondary structures for multiple sequence alignment results in alignments that are more accurate, biologically relevant and useful for drug development and other medical uses. In addition to a novel multiple sequence alignment algorithm, we also propose a new protein-coding DNA reference alignment database. This database is a collection of multiple sequence alignment data sets derived from tertiary structural alignments. The primary purpose of the database is to benchmark new and existing multiple sequence alignment algorithms with DNA data. The first known comparative study of protein-coding DNA alignment accuracies is also included in this work.

Non-protein-coding RNAs play critical regulatory roles in cellular life. Many ncRNAs fold into specific structures in order to perform their biological functions. Some of the RNAs, such as riboswitches, can even fold into alternative structural conformations in order to participate in different biological processes. In addition, these RNAs can transit dynamically between different functional structures along folding pathways on their energy landscapes. These alternative functional structures are usually energetically favored and are stable in their local energy landscapes. Moreover, conformational transitions between any pair of alternate structures usually involve high energy barriers, such that RNAs can become kinetically trapped by these stable and local optimal structures. We have proposed a suite of computational approaches for analyzing and discovering regulatory RNAs through studying folding pathways, alternative structures and energy landscapes associated with conformational transitions of regulatory RNAs. First, we developed an approach, RNAEAPath, which can predict low-barrier folding pathways between two conformational structures of a single RNA molecule. Using RNAEAPath, we can analyze folding iii pathways between two functional RNA structures, and therefore study the mechanism behind RNA functional transitions from a thermodynamic perspective. Second, we introduced an approach, RNASLOpt, for finding all the stable and local optimal structures on the energy landscape of a single RNA molecule. We can use the generated stable and local optimal structures to represent the RNA energy landscape in a compact manner. In addition, we applied RNASLOpt to several known riboswitches and predicted their alternate functional structures accurately. Third, we integrated a comparative approach with RNASLOpt, and developed RNAConSLOpt, which can find all the consensus stable and local optimal structures that are conserved among a set of homologous regulatory RNAs. We can use RNAConSLOpt to predict alternate functional structures for regulatory RNA families. Finally, we have proposed a pipeline making use of RNAConSLOpt to computationally discover novel riboswitches in bacterial genomes. An application of the proposed pipeline to a set of bacteria in Bacillus genus results in the re-discovery of many known riboswitches, and the detection of several novel putative riboswitch elements.

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Non-coding RNAs are the most abundant class of RNAs found throughout genomes. These RNAs are key players of gene regulation and thus, the func- tion of whole organisms. Numerous methods have been developed so far for detecting novel classes of ncRNAs or finding homologs to the known ones. Because of their abundance, the sequence availability of these RNAs is rapidly increasing, as is the case for example for microRNAs. However, for classes of them, still only incomplete information is available, invertebrates 7SK snRNA for instance. Consequently, a lot of false positive outputs are produced in the former case, and more accurate annotation methods are needed for the latter cases to improve derivable knowledge. This makes the accuracy of gathering correct homologs a challenging task and it leads directly to a not less important problem, the curation of these data. Finding solutions for the aforementioned problems is more complex than one would expect as these RNAs are characterized not only by sequences informa- tion but also structure information, in addition to distinct biological features. In this work, data curation methods and sensitive homology search are shown as complementary methods to solve these problems. A careful curation and annotation method revealed new structural information in the invertebrates 7SK snRNA, which pushes the investigation in the area forward. This has been reflected by detecting new high potential 7SK RNA genes in different invertebrates groups. Moreover, the gaps between homology search and well- curated data on the one side, and between experimental and computational outputs on the other side, are closed. These gaps were bridged by a curation method applied to the microRNA data, which was then turned into a com- prehensive workflow implemented into an automated pipeline. MIRfix is a microRNA curation pipeline considering the detailed sequence and structure information of the metazoan microRNAs, together with biological features related to the microRNA biogenesis. Moreover, this pipeline can be integrated into existing methods and tools related to microRNA homology search and data curation. The application of this pipeline on the biggest open source microRNA database revealed its high capacity in detecting wrong annotated pre-miRNA, eventually improving alignment quality of the majority of the available data. Additionally, it was tested with artificial datasets highlighting the high accuracy in predicting the pre-miRNA components, miRNA and miRNA*.:Chapter 1: Introduction Chapter 2: Biological and Computational background 2.1 Biology 2.1.1 Non-coding RNAs 2.1.2 RNA secondary structure 2.1.3 Homology versus similarity 2.1.4 Evolution 2.2 The role of computational biology 2.2.1 Alignment 2.2.1.1 Pairwise alignment 2.2.1.2 Multiple sequence alignment (MSA) 2.2.2 Homology search 2.2.2.1 Sequence-based 2.2.2.2 Structure-based 2.2.3 RNA secondary structure prediction Chapter 3: Careful curation for snRNA 3.1 Biological background 3.2 Introduction to the problem 3.3 Methods 3.3.1 Initial seeds and models construction 3.3.2 Models anatomy then merging 3.4 Results 3.4.1 Refined model of arthropod 7SK RNA 3.4.1.1 5’ Stem 3.4.1.2 Extension of Stem A 3.4.1.3 Novel stem B in invertebrates 3.4.1.4 3’ Stem 3.4.2 Invertebrates model conserves the HEXIM1 binding site 3.4.3 Computationally high potential 7SK RNA candidate . 3.4.4 Sensitivity of the final proposed model 3.5 Conclusion Chapter 4: Behind the scenes of microRNA driven regulation 4.1 Biological background 4.2 Databases and problems 4.3 MicroRNA detection and curation approaches Chapter 5: Initial microRNA curation 5.1 Introduction 5.2 Methods 5.2.1 Data pre-processing 5.2.2 Initial seeds creation 5.2.3 Main course 5.3 Results and discussion 5.4 Conclusion Chapter 6: MIRfix pipeline 6.1 Introduction 6.2 Methods 6.2.1 Inputs and Outputs 6.2.2 Prediction of the mature sequences 6.2.3 The original precursor and its alternative 6.2.4 The validation of the precursor 6.2.5 Alignment processing 6.3 Results and statistics 6.4 Applications 6.4.1 Real life examples and artificial data tests 6.4.2 miRNA and miRNA* prediction 6.4.3 Covariance models 6.5 Conclusion Chapter 7: Discussion

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Um programa computacional foi desenvolvido para otimização de geometria e simulação de dinâmica molecular baseado em um campo de forças clássicas parametrizado. O solvente foi considerado como um contínuo eletrostático e a interface entre o meio aquoso e o interior de uma membrana biológica como uma superfície de descontinuidade dielétrica, tratada pelo \"método das imagens eletrostáticas\". Nesse método, o campo de polarização produzido na superfície de descontinuidade por uma carga pontual é representado por uma carga fictícia, colocada na fase oposta, cuja distância e sinal é definida pelas condições de contorno na superfície. Diversos sistemas foram estudados, tanto em solventes contínuos como na presença de superfícies de descontinuidade: a) Foram estudadas as distribuições populacionais dos rotâmeros do triptofano na forma zwitteriônica e no peptídeo Ala-Trp- Ala, em solvente polar e apolar. Foi demonstrado que a dinâmica do triptofano e as populações de rotâmeros são compatíveis com as observações experimentais de fluorescência resolvida no tempo e NMR; b) Em um estudo das conformações em polialanina, verificou-se que a estabilidade da estrutura secundária hélice- é um efeito cooperativo entre pontes de hidrogênio em solvente de baixa constante dielétrica. Na presença da interface água-membrana, a hélice- anfifílica de um modelo para a -endorfina estabiliza-se sobre a interface. Um comportamento anfifílico foi também observado na seqüência sinal para o receptor- da e. coli, a qual estabilizou-se perpendicularmente à interface, na conformação parcial hélice- proposta na literatura; c) Em um estudo sobre o hormônio -MSH observou-se que, em solvente polar, de uma conformação helicoidal ele passa para uma conformação estendida. Porém, ao atravessar para o interior hidrofóbico de uma membrana, o peptídeo estabiliza-se em dobra-. Observou-se ainda que a estabilidade dessa conformação no interior da membrana é reforçada por pontes salinas entre os resíduos carregados do peptídeo, os quais formam um \"caroço\" hidrofílico circundado por resíduos hidrofóbicos. Esse arranjo estrutural está em concordância com o proposto para a conformação biologicamente ativa. De um modo geral, o modelo para biomembrana proposto no presente trabalho reproduziu o comportamento hidrofóbico, hidrofílico ou anfifílico dos peptídeos estudados. / A software was developed for optimisation of geometry and molecular dynamics simulation, based on a parameterized classical force field. Solvent was assumed as an electrostatic continuum. The interface between the aqueous medium and the hydrophobic core of biological membranes was described by a surface of dielectric discontinuity, treated by the \"method of images\". In this method, the polarization field produced at the surface of discontinuity by a point charge was represented by a fictitious charge, placed in the opposite phase. The position and signal of this charge-image were defined by boundary conditions at the surface. Several systems were studied, either in continuous solvent, as in the presence of discontinuity surfaces: a) the population distribution of tryptophan rotamers was studied in the zwytterion and in the peptide Ala-Trp-Ala, in polar and apolar solvents; the results for the tryptophan dynamics and the rotamers populations agree with experimental observations using time resolved fluorescence and NMR spectroscopies. b) analysis of polyalanin conformations showed that the stabililty of the -helix is a cooperative effect between hydrogen bonds in low dielectric constant solvent; in the presence of the water-membrane interface, the amphyphilic -helix of a -endorphin model stabilizes on the interface; a similar behavior was observed in the signal sequence for the E. Coli -receptor, that stabilized perpendicular to the interface in a partial -helix conformation, as proposed in the literature. c) calculations on melanotropic hormone a.-MSH showed that in polar solvent it goes from helycoidal conformation to an extended one; in the presence of the interface water-membrane, the peptide goes into the interior of the membrane and stabilizes in a -turn; the stability ofthis conformation was reinforced by salt bridges between charged residues, forming a hydrophilic core surrounded by hydrophobic residues; this structural arrangement agrees with the one proposed for the biologically active conformation of the hormone. In general terms, the model proposed here for the biomembrane was able to mimic the hydrophobic, hydrophihlic or amphyphilic behavior of the peptides studied.

Um programa computacional foi desenvolvido para otimização de geometria e simulação de dinâmica molecular baseado em um campo de forças clássicas parametrizado. O solvente foi considerado como um contínuo eletrostático e a interface entre o meio aquoso e o interior de uma membrana biológica como uma superfície de descontinuidade dielétrica, tratada pelo \"método das imagens eletrostáticas\". Nesse método, o campo de polarização produzido na superfície de descontinuidade por uma carga pontual é representado por uma carga fictícia, colocada na fase oposta, cuja distância e sinal é definida pelas condições de contorno na superfície. Diversos sistemas foram estudados, tanto em solventes contínuos como na presença de superfícies de descontinuidade: a) Foram estudadas as distribuições populacionais dos rotâmeros do triptofano na forma zwitteriônica e no peptídeo Ala-Trp- Ala, em solvente polar e apolar. Foi demonstrado que a dinâmica do triptofano e as populações de rotâmeros são compatíveis com as observações experimentais de fluorescência resolvida no tempo e NMR; b) Em um estudo das conformações em polialanina, verificou-se que a estabilidade da estrutura secundária hélice- é um efeito cooperativo entre pontes de hidrogênio em solvente de baixa constante dielétrica. Na presença da interface água-membrana, a hélice- anfifílica de um modelo para a -endorfina estabiliza-se sobre a interface. Um comportamento anfifílico foi também observado na seqüência sinal para o receptor- da e. coli, a qual estabilizou-se perpendicularmente à interface, na conformação parcial hélice- proposta na literatura; c) Em um estudo sobre o hormônio -MSH observou-se que, em solvente polar, de uma conformação helicoidal ele passa para uma conformação estendida. Porém, ao atravessar para o interior hidrofóbico de uma membrana, o peptídeo estabiliza-se em dobra-. Observou-se ainda que a estabilidade dessa conformação no interior da membrana é reforçada por pontes salinas entre os resíduos carregados do peptídeo, os quais formam um \"caroço\" hidrofílico circundado por resíduos hidrofóbicos. Esse arranjo estrutural está em concordância com o proposto para a conformação biologicamente ativa. De um modo geral, o modelo para biomembrana proposto no presente trabalho reproduziu o comportamento hidrofóbico, hidrofílico ou anfifílico dos peptídeos estudados. / A software was developed for optimisation of geometry and molecular dynamics simulation, based on a parameterized classical force field. Solvent was assumed as an electrostatic continuum. The interface between the aqueous medium and the hydrophobic core of biological membranes was described by a surface of dielectric discontinuity, treated by the \"method of images\". In this method, the polarization field produced at the surface of discontinuity by a point charge was represented by a fictitious charge, placed in the opposite phase. The position and signal of this charge-image were defined by boundary conditions at the surface. Several systems were studied, either in continuous solvent, as in the presence of discontinuity surfaces: a) the population distribution of tryptophan rotamers was studied in the zwytterion and in the peptide Ala-Trp-Ala, in polar and apolar solvents; the results for the tryptophan dynamics and the rotamers populations agree with experimental observations using time resolved fluorescence and NMR spectroscopies. b) analysis of polyalanin conformations showed that the stabililty of the -helix is a cooperative effect between hydrogen bonds in low dielectric constant solvent; in the presence of the water-membrane interface, the amphyphilic -helix of a -endorphin model stabilizes on the interface; a similar behavior was observed in the signal sequence for the E. Coli -receptor, that stabilized perpendicular to the interface in a partial -helix conformation, as proposed in the literature. c) calculations on melanotropic hormone a.-MSH showed that in polar solvent it goes from helycoidal conformation to an extended one; in the presence of the interface water-membrane, the peptide goes into the interior of the membrane and stabilizes in a -turn; the stability ofthis conformation was reinforced by salt bridges between charged residues, forming a hydrophilic core surrounded by hydrophobic residues; this structural arrangement agrees with the one proposed for the biologically active conformation of the hormone. In general terms, the model proposed here for the biomembrane was able to mimic the hydrophobic, hydrophihlic or amphyphilic behavior of the peptides studied.

Understanding the complex dynamics that underpin the response of structures in the occurrence of earthquakes is of paramount importance in ensuring community resilience. The operational continuity of structures is influenced by the performance of nonstructural components, also known as secondary structures. Inherent vulnerability characteristics, nonlinearities and uncertainties in their properties or in the excitation pose challenges that render their response determination as a non-straightforward task. This dissertation settles in the context of mathematical modelling and response quantification of seismically driven secondary systems. The case of bilinear hysteretic, rigid-plastic and free-standing rocking oscillators is first considered, as a representative class of secondary systems of distinct behaviour excited at a single point in the primary structure. The equations governing their full dynamic interaction with linear primary oscillators are derived with the purpose of assessing the appropriateness of simplified analysis methods where the secondary-primary feedback action is not accounted for. Analyses carried out in presence of pulse-type excitation have shown that the cascade approximation can be considered satisfactory for bilinear systems provided the secondary-primary mass ratio is adequately low and the system does not approach resonance. For the case of sliding and rocking systems, much lighter secondary systems need to be considered if the cascade analysis is to be adopted, with the validity of the approximation dictated by the selection of the input parameters. Based on the premise that decoupling is permitted, new analytical solutions are derived for the pulse driven nonlinear oscillators considered, conveniently expressing the seismic response as a function of the input parameters and the relative effects are quantified. An efficient numerical scheme for a general-type of excitation is also presented and is used in conjunction with an existing nonstationary stochastic far-field ground motion model to determine the seismic response spectra for the secondary oscillators at given site and earthquake characteristics. Prompted by the presence of uncertainty in the primary structure, and in line with the classical modal analysis, a novel approach for directly characterising uncertainty in the modal shapes, frequencies and damping ratios of the primary structure is proposed. A procedure is then presented for the identification of the model parameters and demonstrated with an application to linear steel frames with uncertain semi-rigid connections. It is shown that the proposed approach reduces the number of the uncertain input parameters and the size of the dynamic problem, and is thus particularly appealing for the stochastic assessment of existing structural systems, where partial modal information is available e.g. through operational modal analysis testing. Through a numerical example, the relative effect of stochasticity in a bi-directional seismic input is found to have a more prominent role on the nonlinear response of secondary oscillators when compared to the uncertainty in the primary structure. Further extending the analyses to the case of multi-attached linear secondary systems driven by deterministic seismic excitation, a convenient variant of the component-mode synthesis method is presented, whereby the primary-secondary dynamic interaction is accounted for through the modes of vibration of the two components. The problem of selecting the vibrational modes to be retained in analysis is then addressed for the case of secondary structures, which may possess numerous low frequency modes with negligible mass, and a modal correction method is adopted in view of the application for seismic analysis. The influence of various approaches to build the viscous damping matrix of the primary-secondary assembly is also investigated, and a novel technique based on modal damping superposition is proposed. Numerical applications are demonstrated through a piping secondary system multi-connected on a primary frame exhibiting various irregularities in plan and elevation, as well as a multi-connected flexible secondary system. Overall, this PhD thesis delivers new insights into the determination and understanding of the response of seismically driven secondary structures. The research is deemed to be of academic and professional engineering interest spanning several areas including seismic engineering, extreme events, structural health monitoring, risk mitigation and reliability analysis.