One of the challenges in the structural biological community is processing the wealth of protein data being produced today; therefore, the use of computational tools has been incorporated to speed up and help understand the structures of proteins, hence the functions of proteins. In this thesis, protein structure investigations were made through the use of Multivariate Analysis (MVA), and Fourier Transformed Infrared (FTIR), a form of vibrational spectroscopy. FTIR has been shown to identify the chemical bonds in a protein in solution and it is rapid and easy to use; the spectra produced from FTIR are then analysed qualitatively and quantitatively by using MVA methods, and this produces non-redundant but important information from the FTIR spectra. High resolution techniques such as X-ray crystallography and NMR are not always applicable and Fourier Transform Infrared (FTIR) spectroscopy, a widely applicable analytical technique, has great potential to assist structure analysis for a wide range of proteins. FTIR spectral shape and band positions in the Amide I (which contains the most intense absorption region), Amide II, and Amide III regions, can be analysed computationally, using multivariate regression, to extract structural information. In this thesis Partial least squares (PLS), a form of MVA, was used to correlate a matrix of FTIR spectra and their known secondary structure motifs, in order to determine their structures (in terms of "helix", "sheet", “310-helix”, “turns” and "other" contents) for a selection of 84 non-redundant proteins. Analysis of the spectral wavelength range between 1480 and 1900 cm-1 (Amide I and Amide II regions) results in high accuracies of prediction, as high as R2 = 0.96 for α-helix, 0.95 for β-sheet, 0.92 for 310-helix, 0.94 for turns and 0.90 for other; their Root Mean Square Error for Calibration (RMSEC) values are between 0.01 to 0.05, and their Root Mean Square Error for Prediction (RMSEP) values are between 0.02 to 0.12. The Amide II region also gave results comparable to that of Amide I, especially for predictions of helix content. We also used Principal Component Analysis (PCA) to classify FTIR protein spectra into their natural groupings as proteins of mainly α-helical structure, or protein of mainly β-sheet structure or proteins of some mixed variations of α-helix and β-sheet. We have also been able to differentiate between parallel and anti-parallel β-sheet. The developed methods were applied to characterize the secondary structure conformational changes of an unfolding protein as a function of pH and also to determine the limit of Quantitation (LoQ).Our structural analyses compare highly favourably to those in the literature using machine learning techniques. Our work proves that FTIR spectra in combination with multivariate regression analysis like PCA and PLS, can accurately identify and quantify protein secondary structure. The developed models in this research are especially important in the pharmaceutical industry where the therapeutic effect of drugs strongly depends on the stability of the physical or chemical structure of their proteins targets; therefore, understanding the structure of proteins is very important in the biopharmaceutical world for drugs production and formulation. There is a new class of drugs that are proteins themselves used to treat infectious and autoimmune diseases. The use of spectroscopy and multivariate regression analysis in the medical industry to identify biomarkers in diseases has also brought new challenges to the bioinformatics field. These methods may be applicable in food science and academia in general, for the investigation and elucidation of protein structure.

La liaison hydrogène est une interaction stabilisante très importante, qui est présente dans de nombreux systèmes moléculaires, des petits clusters d'eau à la molécule d'ADN. L'étude du cas de la liaison hydrogène intramoléculaire (LHI) est d'un intérêt particulier en raison du rôle important de ce type d'interaction dans les processus de transfert d'hydrogène interne, dans la photodynamique et la conformation structurelle. La famille des molécules β-dicarbonyles est un système modèle de LHI unique car il possède relativement peu de degrés de liberté et tous les processus mentionnés précédemment sont clairement présents. L'objectif principal de ce travail est d'étudier le lien entre structure isotopique et électronique des molécules β-dicarbonyles, force de la liaison hydrogène intramoléculaire, sélectivité sur le processus de photoisomérisation et couplage du transfert d'hydrogène avec d'autres mouvements de grande amplitude. Les expériences sont complétées par des calculs de chimie quantique. Quatre molécules de la famille des β-dicétones (acétylacétone doublement deutérée, 3-chloroacétylacétone, hexafluoroacétylacétone et trifluoroacétylacétone) et une β-dialdéhyde (2-chloromalonaldéhyde) sont étudiées dans des environnements inertes à basse température par spectroscopie électronique et vibrationnelle (FT- IR et Raman). Le néon et le para hydrogène ont été utilisés principalement comme matrices hôtes permettant une analyse spectroscopique claire. Les β-dicarbonyles se présentent sous deux formes tautomères: le céto et l'énol, mais ce dernier prédomine en grande partie en phase gazeuse, et par conséquent, dans les échantillons déposés étudiés. Huit différents conformères énoliques peuvent exister, mais celui avec LHI (énol chélaté) est le plus stable. Ces conformères peuvent être divisés en quatre paires dans lesquelles chaque couple partage la même structure squelettique et ne diffère que dans la conformation hydroxyle. Le conformère énolique fermé a toujours été trouvé comme l´espèce la plus stable dans nos expériences. L'influence de l'environnement et de la force de la liaison hydrogène sur des variables spectroscopiques comme la largeur de bande, l'intensité et le déplacement spectral sont discutées. On a également trouvé des preuves expérimentales du processus de conversion de spin nucléaire dans la forme énolique fermée de l'acétylacétone doublement deutérée en matrice de para-hydrogène. Différents conformères énoliques ouverts ont été produits dans chaque système après excitation par laser UV. Les conformères énoliques ouverts présentent des ordres d'énergie différents pour chaque analogue halogéné en raison de l'existence d'interactions non covalentes spécifiques, comme le révèlent les calculs théoriques. Néanmoins, dans tous les cas, les conformères produits sont les conformères les plus stables de leurs paires énoliques. Ceci est expliqué par un processus régit par passage tunnel de l'hydrogène hydroxylique, comme observé expérimentalement dans les isotopologues deutérés. A partir des résultats expérimentaux, nous avons proposé un mécanisme général pour expliquer la photo-isomérisation dans ces systèmes. Par ailleurs, la technique des gouttelettes d'hélium a également été utilisée pour avoir accès à des informations spectroscopiques précieuses (spectres ro-vibrationnels) sur des complexes fortement ou faiblement liés en milieu inerte. Le rôle de l'eau comme espèce donneur ou accepteur de protons dans un complexe peut facilement être modifié par un déséquilibre des forces d'interaction en jeu. Les résultats préliminaires sur le système à liaison hydrogène intermoléculaire propyne-eau dans des gouttelettes d'hélium sont présentés. / The hydrogen bond interaction is an important stabilizing interaction present in many kinds of molecular systems, from small water clusters to the big DNA molecule. The study of the specific case of the intramolecular hydrogen bond (IHB) is of special interest because of the important role of this kind of interaction in internal hydrogen transfer processes, photodynamic behavior and structural conformation. The β-dicarbonyl family of molecules is a unique model system with relatively small amount of degrees of freedom and where all the processes just mentioned are clearly present. The main aim of this work is to study the link between the isotopic and electronic structure of β-dicarbonyl molecules (model IHB system) with the strength of intramolecular hydrogen bond, selectivity on the photoisomerization process and coupling of hydrogen transfer with other large amplitude motions. Experiments are supported with quantum chemical calculations. Four molecules from the β-diketone family (double deuterated acetylacetone, 3-chloroacetylacetone, hexafluoroacetylacetone and trifluoroacetylacetone) and one from the β-dialdehyde (2-chloromalonaldehyde) are studied in low temperature inert environments by means of electronic and vibrational spectroscopy (FT-IR and Raman). Neon and para-hydrogen were mainly used as host matrices allowing clear spectroscopic analysis. The β-dicarbonyl molecules can be present in two tautomeric forms: keto and enol, but the latter largely predominates in the gas phase, and as a consequence, in the deposited isolated samples. Eight different enol conformers can exist, but the one with intramolecular hydrogen bond (chelated enol) is the most stable. The enol conformers can be divided in four pairs in which each couple shares the same skeletal structure and differs only in the hydroxyl conformation.In the deposited sample of all the molecules under study, the closed enolic conformer was found as the most stable species. The influence of the environment and the hydrogen bond strength on spectroscopic variables like bandwidth, intensity and frequency position are discussed. Experimental evidence of Nuclear Spin Conversion process in para-hydrogen matrix of the closed enol form of double deuterated acetylacetone was also found. Different open enol conformers were produced in each system after UV laser excitation. The open enol conformers show different energy ordering for each halogenated analog because of the existence of specific non covalent interactions, as revealed by theoretical calculations. Nevertheless, in all cases, the produced conformers are the most stable conformers of their enolic pairs. This is explained by a tunneling driven process in the hydroxyl hydrogen, as observed experimentally in deuterated isotopologues. From the experimental results we proposed a general mechanism to explain the photoisomerization in these systems. On the other hand, helium droplets technique was also used to allow recording valuable spectroscopic information (ro-vibrational spectrum) about strong and weak complexes in inert media. The role of water as donor or acceptor proton species in a complex can easily change by an imbalance of the interaction forces at play. Preliminary results about the intermolecular hydrogen bonded system propyne-water in helium droplets are presented.

Therapies of neurodegenerative diseases are often very difficult and their success depend on an early diagnose. From that reason we have been developing new diagnostic method for multiple sclerosis and Alzheimer disease by drop coating deposition Raman (DCDR) spectroscopy of cerebrospinal fluid (CSF) in this work. We found out conditions of measurements, where spectra were reproducible and accepted for standard diagnostic practices. We discovered that CSF has fast degradation at a room temperature, which was detectable in spectra after 5 hours, and degradation due to refreezing. DCDR spectra of CSF from individual patients were analyzed by factor and cluster analysis. Multiple sclerosis was manifested by lower intensity of a Raman band at 1080 cm−1 , which is probably connected with more general pathologic state. Spectral changes caused by Alzeheimer disease were more complex and beside changes mentioned above also changes connected with composition and conformation of proteins were identified in regions 1200-1800 cm−1 and 2870-2950 cm−1 . Additionally, we succeeded in distinguishing of young healthy patients from older patients in DCDR spectra. In this work were checked up, that DCDR is good diagnostic method for clinical practices for determining neurodegenerative diseases through the complex...

The work concerns to structural characterization of two important proteins of 2'-5' oligoadenylate pathway participating in an immune response of organism to a viral infection. Studied proteins were ankyrin domain of mouse RNase L, the C-terminal part of human phosphodiesterase 12 and the complete human phosphodiesterase 12. The proteins were characterized by Raman spectroscopy, infrared spectroscopy, electronic circular dichroism, dynamic light scattering and in addition by two non-spectroscopic methods- differential calorimetry and electrophoresis. For each protein the secondary structures, thermal stability, weight of oligomers and generally a basic characterization by above mentioned methods were provided.

Title: Cellulose dissolution: Comparison of two non-derivatizing solvent systems and the effect of additives Author: Nikolay Kotov Institute: Institute of Macromolecular Chemistry, Czech Academy of Sciences Supervisor of the doctoral thesis: RNDr. Jiří Dybal, CSc. Consultant: Mgr. Adriana Šturcová, Ph.D. Institute of Macromolecular Chemistry, Czech Academy of Sciences, Department of Vibrational Spectroscopy Abstract: Cellulose is an abundant renewable material, which processing and applicability is limited owing to cellulose inability to dissolve in commonly used solvents. Only specific solvents or their combinations are able to dissolve cellulose and its dissolution processes remain unclear till nowadays. Aim of this thesis was to acquire new experimental information on the changes which cellulose exhibits upon dissolution in two exemplary solvents: an ionic liquid 1-butyl-3-methylimidazolium chloride (bmimCl) and in an organic solvent N,N-dimethylacetamide (DMAc) with lithium chloride (LiCl). The main tool for that investigation is vibrational spectroscopy which provides valuable information about the polymer structure. Similarities and differences found in the spectra of cellulose in those two solvents and influence of cellulose on the solvents are analyzed. Furthermore, influence of additives on the...

Alzheimer disease (AD) is the most frequent dementia. The prevalence is approximately 10% in 65 years old people. The current treatment is only progression protective, therefore it is crucial to find a new diagnostic approach for diagnosing AD in early stage. We analysed a set of 55 patients by the drop coating deposition Raman spectroscopy with the goal to verify previously published high sensitivity of the AD spectroscopic diagnosis in cerebral spinal fluid (CSF) and to find a new diagnostic method for blood serum (BS). We optimized measurement conditions for BS. The results were evaluated by the cluster analysis and the principal component analysis. The small set of samples exhibited high sensitivity in both CSF and BS but that distinctly decreased in the whole set. The results for CSF were affected by the choice of the analysed spectral interval. The best for AD diagnose was the interval containing peaks at 980, 1080 and 1249 cm-1.The results for BS have been the most sensitive in the whole spectral range. They have low sensitivity but high specificity for AD (92%). The usage of neural networks has conversely high sensitivity and low specificity in both sets of samples of BS and CSF. Powered by TCPDF (www.tcpdf.org)

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