This master's thesis focuses on search for sequences potentially forming quadruplexes in DNA sequences, their visualization and filtration. For this purpose a web application was created in cooperation with the Institute of biophysics of Academy of science of Czech republic. This application uses a newly created algorithm able to find all possible formations of quadruplex within given input sequence. Design and implementation of this algorithm are also part of this thesis.

Starting with the discovery of microRNAs and the advent of genome‐wide transcriptomics, non‐protein‐coding transcripts have moved from a fringe topic to a central field research in molecular biology. In this contribution we review the state of the art of “computational RNomics”, i.e., the bioinformatics approaches to genome‐wide RNA annotation. Instead of rehashing results from recently published surveys in detail, we focus here on the open problem in the field, namely (functional) annotation of the plethora of putative RNAs. A series of exploratory studies are used to provide non‐trivial examples for the discussion of some of the difficulties.

Dynamic Programming Algorithms solve many standard problems of RNA bioinformatics in polynomial time. In this contribution we discuss a series of variations on these standard methods that implement refined biophysical models, such as a restriction of RNA folding to canonical structures, and an extension of structural alignments to an explicit scoring of stacking propensities. Furthermore, we demonstrate that a local structural alignment can be employed for ncRNA gene finding. In this context we discuss scanning variants for folding and alignment algorithms.

In the field of protein research in general and the pharmaceutical industry in particular, it is now necessary to perform measurements of the secondary structure of proteins on many samples simultaneously, for instance to screen for molecules that stabilize proteins or to evaluate the action of multiple environmental conditions. In this context, we have proposed a new approach to evaluate the secondary structure of proteins on a very large scale (approximately 2000 to 4000 samples / cm2), by combining infrared imaging and 2D printing of protein microarrays. In view of the large amount of data, in a first step, methods for automating the extraction of spectra of interest from microarray infrared images and for automating the processing of the spectra were developed. Since the estimation of the secondary structure from infrared spectra is based on the construction of prediction models by chemometric methods, a relevant set of proteins for calibration was mandatory. A protein bank consisting of 92 commercially available proteins, the structure of which was well characterized by X-ray crystallography, was established for this purpose. After the development of predictive models for secondary structure determination and the validation of the protein microarray approach, we tried to optimize the models to improve the secondary structure prediction by different approaches as secondary structure definition, partial deuteration or subtraction of side chain contribution to the spectra. On the other hand, dealing with non-native structures not present in the reference protein library was a challenge. We took the opportunity to analyze the structural modifications of a subset of our protein library subjected to moderate denaturation conditions. Multivariate curve resolution-alternating least squares (MCR-ALS) was used to model a new spectral component appearing in the protein set subjected to denaturing conditions, which could represent a potential spectroscopic marker of aggregation and could allow a semi-quantitative evaluation of the aggregation. While the assessment of secondary structure was well established in the first part of this work, tertiary structure and stability are also critical. Hydrogen / deuterium exchange (HDX) is a potential approach for studying the structure and dynamics of proteins. In the last part of this work, we built a device which allowed to follow the HDX exchange kinetics simultaneously on the entire microarray. In conclusion, protein microarray FTIR imaging opens the door to high throughput analysis of protein secondary structure without any labelling and would allow better understanding of three-dimensional structure and dynamics of proteins through recording HDX curves. / Dans le domaine de la recherche sur les protéines et de l'industrie pharmaceutique, il s’avère désormais nécessaire d'effectuer des mesures de la structure secondaire des protéines sur de nombreux échantillons simultanément, de cribler des molécules qui stabilisent les protéines, ou d'évaluer l'action de multiples conditions environnementales. Dans ce contexte, nous avons proposé une nouvelle approche pour évaluer la structure secondaire des protéines à très grande échelle (environ 2 000 à 4 000 échantillons / cm2), en associant l'imagerie infrarouge et l'impression 2D de damiers de protéines. Dans un premier temps, des méthodes d'automatisation de l'extraction des spectres d'intérêt à partir des images infrarouges des damiers et d'automatisation des spectres ont été développées. L'estimation de la structure secondaire à partir des spectres infrarouges étant basée sur la construction de modèles de prédiction à partir de méthodes chimiométriques, un ensemble pertinent de protéines pour l'étape de calibration était obligatoire. Une banque de protéines constituée de 92 protéines disponibles dans le commerce, dont la structure était bien caractérisée par cristallographie aux rayons X, a été constituée dans ce but. Après élaboration des modèles prédictifs de la structure secondaire et la validation de l'approche des damiers de protéines, nous avons tenté d'optimiser les modèles pour améliorer les prédictions de structure secondaire par différentes approches. D'autre part, traiter des protéines présentant une structure jamais rencontrée dans les structures natives de notre bibliothèque de protéines de référence constituait un défi. Nous avons saisi l'opportunité d'analyser les modifications structurales d'un sous-ensemble de notre bibliothèque de protéines, caractérisé par un contenu structurel secondaire très différent en le soumettant à des conditions de dénaturation modérées La méthode de résolution de courbes multivariées des moindres carrés alternés (MCR-ALS) a été utilisée pour modéliser une nouvelle composante spectrale apparaissant dans l'ensemble protéique soumis à des conditions dénaturantes, et a permis de révéler un marqueur spectroscopique potentiel d'agrégation protéique permettant une évaluation semi-quantitative de son contenu. Alors que l’évaluation de la structure secondaire a été bien établie dans la première partie de ce travail, la structure tertiaire et la stabilité sont également critiques. L'échange hydrogène / deutérium (HDX) est une approche potentielle pour l’étude de la structure et de la dynamique des protéines. Dans la dernière partie de ce travail, nous avons construit un dispositif qui a permis de suivre la cinétique d’échange HDX simultanément sur l'ensemble d’un damier. En conclusion, l'imagerie FTIR de damiers de protéines ouvre la porte à une analyse à haut débit de la structure secondaire des protéines et permettrait de mieux comprendre la structure et la dynamique tridimensionnelles grâce à l'enregistrement des courbes HDX. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Protein solution structures were analyzed by horizontal attenuated total reflectance (ATR) FTIR spectroscopy. Secondary structure compositions determined from analyses of amide-I and II region and amide-III region difference spectra were compared. Data for proteins of known solution structure, cytochrome c, concanavalin A and lysozyme, were compared with those reported in the iiterature. Melittin, a peptide from bee venom whose secondary structural configuration varies depending upon solution conditions was also examined. Acyl-carrier protein (ACP) is a small protein of recognized dynamic structure that in its diverse physiologic roles interacts specifically with numerous different proteins. Horizontal ATR FTIR analysis of ACP's secondary structure indicated a predominately helical structure best defined as a combination of ordered and disordered helices. The FTIR-derived structural composition agreed with those determined for ACP by other techniques. Comparison of independent analyses of the amide-I and III regions to determine protein configuration compositions was a useful method of verifying the internal consistency of the calculated structural compositions of dynamically-structured proteins.

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Molecular structures of biomaterials have close relation to their functions. We are interested in how biological building blocks assemble into the structures of native biomaterials and the hierarchy of those structures. We tackled the problem mainly with X-ray diffraction experiments and developed a thorough analysis technique to assign the X-ray signals to protein secondary structures and chitin. Three different types of biomaterials were examined: vimentin fibres, squid pens, and human hair. In vimentin fibres, we found that the secondary protein structures play an important role in the strength of the fibres. In native squid pens, we found a self-similar, hierarchical structure from millimetres down to nanometres. In human hair, we compared the signals corresponding to keratin proteins, intermediate filaments, and lipids between different subjects, and found small deviations. The structures of these three biomaterials, which encompass different orders of length scales, were described both quantitatively and graphically. We hope that this work will eventually allow us to understand how and why nature builds biomaterials this way. / Thesis / Master of Science (MSc)

DNA中の二本鎖切断(DSB)に対するアスコルビン酸(AA)およびDMSOの保護効果を、蛍光顕微鏡による巨大DNA(T4 DNA; 166kbp)の単分子観察によって評価した。凍結／解凍の状態に対して3つの異なる形態の放射源、可視光、γ線、および超音波の環境下にさらした。1‐プロパノールと2‐プロパノールの間で異なる効果が表れた。ゲノムDNA分子の高次構造の変化は、1−プロパノールを用いると、長軸長が濃度60％で最小を示し、次にアルコール含有量の増加と共に増加する傾向があることを見出した。一方、2−プロパノールを用いると、長軸長はアルコール含有量の増加と共にほぼ単調な減少を示した。 / The protective effect of ascorbic acid (AA) and DMSO against double-strand breaks (DSBs) in DNA was evaluated by single-molecule observation of giant DNA (T4 DNA; 166kbp) through fluorescence microscopy. Samples were exposed to three different forms of radiation: visible light, γ-ray, and ultrasound or freeze/thawing. The change of the higher-order structure of genomic DNA molecules in the presence of alcohols by use of single DNA observation with fluorescence microscopy, by focusing our attention to unveil the different effect between 1-propanol and 2-propanol. / 博士(工学) / Doctor of Philosophy in Engineering / 同志社大学 / Doshisha University

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