<p>RNA is the molecular workhorse of nature, capable of doing many cellular tasks, from genetic data storage and regulation, to enzymatic synthesis--even to the point of self-catalyzing its own replication. While RNA can act as a catalyst on its own, as in the hammerhead ribozyme, the added efficiency of proteins is often a necessity; the ribosome--the large ribozyme responsible for peptide chain formation, is aided by proteins which ensure correct assembly and structural stability. These complexes of RNA and proteins feature in many essential cellular processes, including the RISC silencing complex and in the spliceosome. Despite its enormous utility, structural determination of RNA is notoriously difficult--particularly in the backbone, since a nucleotide standardly has 12 torsion angles (including &#967;) and 12 non-hydrogen atoms, compared to 4 torsions (including &#967;1) and 4 non-H atoms in a typical amino acid. The abundance of backbone atoms, their conformational flexibility, and experimental resolution limitations often result in systematic errors that can have a significant impact on the interpretation. False trails due to structural errors can lead to significant loss of time and effort, especially with such high-profile complexes as the ribosome and the RISC complex. </p><p>My research has focused on harnessing the recently discovered ribosome structures and the Richardsons' RNA dataset to find trends in RNA backbone conformations and motifs that were then used to develop structural validation techniques and provide improved diagnosis and correction techniques for RNA backbone. Methods for fixing RNA structure have been developed for both NMR and X-ray crystallography. For NMR structures, a method for assigning RNA backbone structure based on NOE data was developed, leading to improved identification and building of RNA backbone conformation in NMR ensembles. For crystallography, our method of diagnosing the correct ribose pucker from clear observables allows reliable assessment of pucker in validation or refinement. Observed differences in bond-lengths, bond-angles, and dihedrals have been categorized by sugar pucker in the PHENIX refinement package. I have shown that this improves the refinement behavior of both pucker and geometry. </p><p>There have also been improvements in identifying structural motifs. Many previously identified structural motifs have now been defined in terms of backbone suitestrings, a series of 2-character code divisions of RNA backbone that show the best clustering of dihedral angle correlations. Combined with a BLAST-like alignment program called SuiteAlign, these suitestrings were quickly and easily identified in a number of structures, eventually leading to the discovery of multiple instances of T&#968;C-loop structures in the ribosome.</p><p>To facilitate error diagnosis and corrections in RNA-protein complexes, as well as to expand the knowledge base of the scientific community as a whole, a database of RNA-protein interaction motifs has been developed. This database is rooted in the quality-filtering, visualization, and analysis techniques of the Richardson lab, particularly those developed by Laura Murray specifically for RNA structures.</p><p>The consensus backbone conformers, pucker diagnosis, and all-atom contacts have been combined to develop first manual and then automated tools for RNA structure correction. I have applied all these techniques to improve the accuracy of a number of important RNA and RNA/protein complex structures.</p> / Dissertation

RNA Binding Motif (RBM) domain proteins RBM5 and RBM10 have been shown to influence apoptosis, cell cycle arrest and splicing in transformed cells. In this study, RBM5 and RBM10 were examined in non-transformed cells in order to gain a wider range of knowledge regarding their function. Expression of Rbm5 and Rbm10, as well as select splice variants, was examined at the mRNA and protein level throughout H9c2 skeletal and cardiac myoblast differentiation. Results suggest that Rbm5 and Rbm10 may (a) be involved in regulating cell cycle arrest and apoptosis during skeletal myoblast differentiation and (b) undergo post-transcriptional or translational regulation throughout myoblast differentiation. All in all, the expression profiles obtained in the course of this study will help to suggest a role for Rbm5 and Rbm10 in differentiation, as well as possible differentiation-specific target genes with which they may interact.

DNA mismatch repair (MMR) is a highly conserved pathway that maintains genomic stability primarily by correcting mismatches generated during DNA replication. MMR deficiency leads to microsatellite instability (MSI), which is a hallmark of HNPCC (Hereditary Nonpolyposis Colorectal Cancer). Human mismatch repair is initiated by MutSα, a heterodimer of MSH2 and MSH6 subunits. Mismatch binding by MutSα triggers a series of downstream MMR events including interacting and communicating with other MMR proteins. The ATPase domain of MutSα is situated in the C-termini of its both subunits, and ATP binding is required for dissociation of MutSα from a mismatch. In eukaryotic cells, a strand break, which resides either 3’ or 5’ to the mismatch up to several hundred base pair away, determines the strand specificity of MMR. However, in spite of extensive studies, the mechanism by which MutSα locates and senses a nick from the mismatch, and coordinates the subsequent steps of MMR remains poorly understood. Two controversial models have been proposed to explain how the mismatch and the strand break communicate each other. Sliding model proposes that MutSα slides along the DNA helix from the mismatch to the strand break in an ATP binding-dependent but not ATP hydrolysis-dependent manner. Stationary model postulates that MutSα remains bound at the mismatch, and a protein-mediated DNA loop forms, physically bringing the mismatch and the nick in contact. Here, we tested these models in vitro, using a circular plasmid DNA substrate with a single GT mismatch and two Lac repressor (Lac I) binding sites as conditional physical 'roadblocks', one on either side of the mismatch, which when present, prevent MutSα from sliding bi-directionally along the DNA. The results showed that DNA excision initiates under conditions that block MutSα sliding, suggesting that initiation of excision is independent of whether MutSα slides from the mismatch to the nick. This result implies that the communication between the mismatch and the nick is likely through interactions between the mismatch-bound MutSα and other MMR components at the strand break, supporting the stationary model. Therefore, these studies provide significant insight into the mechanisms of mismatch correction in human cells.

Text-to-speech synthesis enables the speech-impeded user of an augmentative and alternative communication system to partake in any conversation on any topic, because it can produce dynamic content. Current synthetic voices do not sound very natural, however, lacking in the areas of emphasis and emotion. These qualities are furthermore important to convey meaning and intent beyond that which can be achieved by the vocabulary of words only. Put differently, speech synthesis requires a more comprehensive analysis of its text input beyond the word level to infer the meaning and intent that elicit emphasis and emotion. The synthesised speech then needs to imitate the effects that these textual factors have on the acoustics of human speech. This research addresses these challenges by commencing with a literature study on the state of the art in the fields of natural language processing, text-to-speech synthesis and speech prosody. It is noted that the higher linguistic levels of discourse, information structure and affect are necessary for the text analysis to shape the prosody appropriately for more natural synthesised speech. Discourse and information structure account for meaning, intent and emphasis, and affect formalises the modelling of emotion. The OCC model is shown to be a suitable point of departure for a new model of affect that can leverage the higher linguistic levels. The audiobook is presented as a text and speech resource for the modelling of discourse, information structure and affect because its narrative structure is prosodically richer than the random constitution of a traditional text-to-speech corpus. A set of audiobooks are selected and phonetically aligned for subsequent investigation. The new model of discourse, information structure and affect, called e-motif, is developed to take advantage of the audiobook text. It is a subjective model that does not specify any particular belief system in order to appraise its emotions, but defines only anonymous affect states. Its cognitive and social features rely heavily on the coreference resolution of the text, but this process is found not to be accurate enough to produce usable features values. The research concludes with an experimental investigation of the influence of the e-motif features on human speech and synthesised speech. The aligned audiobook speech is inspected for prosodic correlates of the cognitive and social features, revealing that some activity occurs in the into national domain. However, when the aligned audiobook speech is used in the training of a synthetic voice, the e-motif effects are overshadowed by those of structural features that come standard in the voice building framework. / PhD (Information Technology), North-West University, Vaal Triangle Campus, 2014

RNA is amazing. We found that without changing the backbone connectivity, RNA can maintain structural conservation in 3D via topology switches, at a single residue level. I developed a method of representing RNA structure in multiresolution, called the PBR approach (P stands for Phosphate; B stands for Base; R stands for Ribose). In this method, structural data is viewed through a series of resolutions from finest to coarsest. At a single nucleotide resolution (fine resolution), RNA is abstruse and elaborate with structural insertions/deletions, strand clips, and 3,2-switches. The compilation of structural deviations of RNA, called DevLS (Deviations of Local Structure), provides a new descriptive language of RNA structure, allowing one to systematize and investigate RNA structure. Using PBR analysis, a total of 103 tetraloops within the crystal structures of the 23s rRNA of H. marismortui and the 70s rRNA of T. thermophilus are found and classified. Combining them, I constructed a 'tetraloop family tree', using a tree formalism, to unify and re-define the tetraloop motif and to represent relationships between tetraloops, as grouped by DevLS. To date, structural alignment of very large RNAs remains challenge due to the large size, intricate backbone choreography, and tertiary interactions. To overcome these obstacles, I developed a concept of structural anchors along with a 'Divide and Conquer' strategy for performing superimposition of 23s rRNAs. The successful alignment and superimpositions of the 23s rRNAs of T. thermophilus and H. marismortui gives an overall RMSD of atomic positions of 1.2 Å, as utilized 73% of RNA backbone atoms (~ 2129 residues). By using principles of inorganic chemistry along with structural alignment technique as described above, a recurrent magnesium-binding motif in large RNAs is revealed. These magnesium-binding motifs play a critical role in the framework of the ribosomal PTC by their locations, topologies, and coordination geometries. Common features of Mg2+-mc's include direct phosphate chelation of two magnesium ions in the form of Mg2+(i)-(O1P-P-O2P)-Mg2+(j), phosphate groups of adjacent RNA residues as ligands of a given Mg2+, and undulated RNA surfaces with unpaired and unstacked bases.

The diploma thesis is focused on analysis, comparison and interpretation of Thomas Hardy?s literary work, especially on the attitude of characters towards the environment in the chosen ?Wessex novels? by Thomas Hardy. The principle of this work is to contextualize and characterize Hardy?s writings in general, together with his historical background and philosophical inspiration, which influenced his concept of space. The main aim is to point out the importance of the environment in Hardy?s literary work, problems of his protagonists? existence and their tragic fatality stemming for their connection with the place they dwell in. Furthermore, this thesis underlines other, mainly social circumstances that influence characters? lives. Another aim is to concentrate on the development of themes and motifs and to find out how Hardy?s work develops in particular, chronologically ordered novels. Answers to these questions can be found in the fundamental part of the diploma thesis, which is based on literary analysis of particular novels, in which the environment plays the very crucial role.

Este é um memorial descritivo e dissertativo da investigação artística feita através da utilização da Motif Writing (escrita por motivos) como propulsora de tarefas de movimento para a construção de dramaturgia em dança. A coreografia que resultou deste processo, A Cadeira _/ Uma Ilha, foi criada a partir da Motif Writing de um trecho do solo Experimento da Cadeira, visando a recriação e a multiplicação das qualidades expressivas dos movimentos do motif em outros corpos/sujeitos e relações dinâmicas entre estes, objetos, espaço e tempo. A pesquisa-criação conta com a participação das bailarinas Juliana Vicari e Luciana Hoppe, transformando o solo original em um trio. A escrita do motif relativo ao trecho selecionado da obra de referência utiliza o instrumental da Análise Laban de Movimento como ferramenta metodológica utilizada também para a condução do processo de criação. Inserido num contexto de pesquisa pós-positivista, com perspectiva artística, este memorial contém aspectos dissertativos sobre a Análise Laban em Movimento e a dramaturgia da dança como aportes para o relato do processo de criação. Ambos ajudam a compreender algumas das opções de trabalho buscadas e alcançadas, ou não, nesta investigação. Ao final deste texto comprovo a eficácia da motif writing tanto como ferramenta para reposição como para recriação da dramaturgia da obra de referência e que uma construção através deste procedimento requer a auto-revelação dos artistas. Descubro a 'reduplicação do Outro em mim‘ como potência criadora e afirmativa da minha ação como intérprete. / This thesis describes the creative and analytical process I used to develop choreography for three dancers called The Chair_/ An Island. I developed this trio choreography from one fragment of an earlier solo choreography called Chair Experiment, using an aspect of Laban Movement Analysis called "motif writing". I used this method as a score to multiply the original motifs into other bodies/subjects, objects, spaces, and time. My work was done in Brazil in collaboration with two other dancers, Juliana Vicari and Luciana Hoppe. From a post-positivist approach and artistic perspective, this thesis describes features of Laban Movement Analysis and Dance Dramaturgy which enhance the creative process. Both help us to understand possible choices and directions which are available to us when creating a choreography. I conclude that motif writing is efficient as a movement score for dramaturgy composition or reposition and that the use of this procedure encourages the artists for a self revelation. I found the 'reduplication of the Other in myself‘ as a creative potential and affirmative way as a performer.

Este é um memorial descritivo e dissertativo da investigação artística feita através da utilização da Motif Writing (escrita por motivos) como propulsora de tarefas de movimento para a construção de dramaturgia em dança. A coreografia que resultou deste processo, A Cadeira _/ Uma Ilha, foi criada a partir da Motif Writing de um trecho do solo Experimento da Cadeira, visando a recriação e a multiplicação das qualidades expressivas dos movimentos do motif em outros corpos/sujeitos e relações dinâmicas entre estes, objetos, espaço e tempo. A pesquisa-criação conta com a participação das bailarinas Juliana Vicari e Luciana Hoppe, transformando o solo original em um trio. A escrita do motif relativo ao trecho selecionado da obra de referência utiliza o instrumental da Análise Laban de Movimento como ferramenta metodológica utilizada também para a condução do processo de criação. Inserido num contexto de pesquisa pós-positivista, com perspectiva artística, este memorial contém aspectos dissertativos sobre a Análise Laban em Movimento e a dramaturgia da dança como aportes para o relato do processo de criação. Ambos ajudam a compreender algumas das opções de trabalho buscadas e alcançadas, ou não, nesta investigação. Ao final deste texto comprovo a eficácia da motif writing tanto como ferramenta para reposição como para recriação da dramaturgia da obra de referência e que uma construção através deste procedimento requer a auto-revelação dos artistas. Descubro a 'reduplicação do Outro em mim‘ como potência criadora e afirmativa da minha ação como intérprete. / This thesis describes the creative and analytical process I used to develop choreography for three dancers called The Chair_/ An Island. I developed this trio choreography from one fragment of an earlier solo choreography called Chair Experiment, using an aspect of Laban Movement Analysis called "motif writing". I used this method as a score to multiply the original motifs into other bodies/subjects, objects, spaces, and time. My work was done in Brazil in collaboration with two other dancers, Juliana Vicari and Luciana Hoppe. From a post-positivist approach and artistic perspective, this thesis describes features of Laban Movement Analysis and Dance Dramaturgy which enhance the creative process. Both help us to understand possible choices and directions which are available to us when creating a choreography. I conclude that motif writing is efficient as a movement score for dramaturgy composition or reposition and that the use of this procedure encourages the artists for a self revelation. I found the 'reduplication of the Other in myself‘ as a creative potential and affirmative way as a performer.

abstract: The communication of genetic material with biomolecules has been a major interest in cancer biology research for decades. Among its different levels of involvement, DNA is known to be a target of several antitumor agents. Additionally, tissue specific interaction between macromolecules such as proteins and structurally important regions of DNA has been reported to define the onset of certain types of cancers. Illustrated in Chapter 1 is the general history of research on the interaction of DNA and anticancer drugs, most importantly different congener of bleomycin (BLM). Additionally, several synthetic analogues of bleomycin, including the structural components and functionalities, are discussed. Chapter 2 describes a new approach to study the double-strand DNA lesion caused by antitumor drug bleomycin. The hairpin DNA library used in this study displays numerous cleavage sites demonstrating the versatility of bleomycin interaction with DNA. Interestingly, some of those cleavage sites suggest a novel mechanism of bleomycin interaction, which has not been reported before. Cytidine methylation has generally been found to decrease site-specific cleavage of DNA by BLM, possibly due to structural change and subsequent reduced bleomycin-mediated recognition of DNA. As illustrated in Chapter 3, three hairpin DNAs known to be strongly bound by bleomycin, and their methylated counterparts, were used to study the dynamics of bleomycin-induced degradation of DNAs in cancer cells. Interestingly, cytidine methylation on one of the DNAs has also shown a major shift in the intensity of bleomycin induced double-strand DNA cleavage pattern, which is known to be a more potent form of bleomycin induced cleavages. DNA secondary structures are known to play important roles in gene regulation. Chapter 4 demonstrates a structural change of the BCL2 promoter element as a result of its dynamic interaction with the individual domains of hnRNP LL, which is essential to facilitate the transcription of BCL2. Furthermore, an in vitro protein synthesis technique has been employed to study the dynamic interaction between protein domains and the i-motif DNA within the promoter element. Several constructs were made involving replacement of a single amino acid with a fluorescent analogue, and these were used to study FRET between domain 1 and the i-motif, the later of which harbored a fluorescent acceptor nucleotide analogue. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2014

Este é um memorial descritivo e dissertativo da investigação artística feita através da utilização da Motif Writing (escrita por motivos) como propulsora de tarefas de movimento para a construção de dramaturgia em dança. A coreografia que resultou deste processo, A Cadeira _/ Uma Ilha, foi criada a partir da Motif Writing de um trecho do solo Experimento da Cadeira, visando a recriação e a multiplicação das qualidades expressivas dos movimentos do motif em outros corpos/sujeitos e relações dinâmicas entre estes, objetos, espaço e tempo. A pesquisa-criação conta com a participação das bailarinas Juliana Vicari e Luciana Hoppe, transformando o solo original em um trio. A escrita do motif relativo ao trecho selecionado da obra de referência utiliza o instrumental da Análise Laban de Movimento como ferramenta metodológica utilizada também para a condução do processo de criação. Inserido num contexto de pesquisa pós-positivista, com perspectiva artística, este memorial contém aspectos dissertativos sobre a Análise Laban em Movimento e a dramaturgia da dança como aportes para o relato do processo de criação. Ambos ajudam a compreender algumas das opções de trabalho buscadas e alcançadas, ou não, nesta investigação. Ao final deste texto comprovo a eficácia da motif writing tanto como ferramenta para reposição como para recriação da dramaturgia da obra de referência e que uma construção através deste procedimento requer a auto-revelação dos artistas. Descubro a 'reduplicação do Outro em mim‘ como potência criadora e afirmativa da minha ação como intérprete. / This thesis describes the creative and analytical process I used to develop choreography for three dancers called The Chair_/ An Island. I developed this trio choreography from one fragment of an earlier solo choreography called Chair Experiment, using an aspect of Laban Movement Analysis called "motif writing". I used this method as a score to multiply the original motifs into other bodies/subjects, objects, spaces, and time. My work was done in Brazil in collaboration with two other dancers, Juliana Vicari and Luciana Hoppe. From a post-positivist approach and artistic perspective, this thesis describes features of Laban Movement Analysis and Dance Dramaturgy which enhance the creative process. Both help us to understand possible choices and directions which are available to us when creating a choreography. I conclude that motif writing is efficient as a movement score for dramaturgy composition or reposition and that the use of this procedure encourages the artists for a self revelation. I found the 'reduplication of the Other in myself‘ as a creative potential and affirmative way as a performer.