All twenty amino acids did not appear simultaneously in nature. Instead some of them appeared early, while others were added into the genetic code later. The amino acids that were formed by Miller (1953) are suggested to have appeared early in evolutionary history, and the amino acids associated with codon capture developed late in the course of evolution. The chronological order of appearance of the amino acids proposed by Trifonov (2000) was G/A, V/D, P, S, E/L, T, R, N, K, Q, I, C, H, F, M, Y, W. According to Romero et al. (1997) amino acids G, D, E, P and S are disorder-promoting residues and C, F, W and Y are order-promoting residues this means that the early or the ancient amino acids were disorder promoting and the order promoting residues came late into the genetic code. These observations led to the hypothesis that the first proteins, which were comprised of the early amino acids only, were disordered, and, furthermore, that the appearance of the late amino acids and the appearance of the structural proteins were concurrent. Software developed by Brooks et al. (2004) to find the amino acid composition of the LUA (Last Universal Ancestor) was used to test this hypothesis. For this work, the Clusters of Orhtologous Groups of proteins (65 COGs) were split into enzymes and non-enzymes. It was found that intrinsic disorder was abundant in both the groups of proteins, with non enzymes being much more disorder than enzymes. Further analysis was done to check for the frequency of the modern amino acids C, F, W, and Y in the Protein data bank (PDB) and Swissprot.

The work undertaken within this thesis is towards the development of a representative set of sequence driven features for the prediction of structural classes of proteins. Proteins are biological molecules that make living things function, to determine the function of a protein the structure must be known because the structure dictates its physical capabilities. A protein is generally classified into one of the four main structural classes, namely all-α, all-β, α + β or α / β, which are based on the arrangements and gross content of the secondary structure elements. Current methods manually assign the structural classes to the protein by manual inspection, which is a slow process. In order to address the problem, this thesis is concerned with the development of automated prediction of structural classes of proteins and extraction of a small but robust set of sequence driven features by using the amino acid indices. The first main study undertook a comprehensive analysis of the largest collection of sequence driven features, which includes an existing set of 1479 descriptor values grouped by ten different feature groups. The results show that composition based feature groups are the most representative towards the four main structural classes, achieving a predictive accuracy of 63.87%. This finding led to the second main study, development of the generalised amino acid composition method (GAAC), where amino acid index values are used to weigh corresponding amino acids. GAAC method results in a higher accuracy of 68.02%. The third study was to refine the amino acid indices database, which resulted in the highest accuracy of 75.52%. The main contributions from this thesis are the development of four computationally extracted sequence driven feature-sets based on the underused amino acid indices. Two of these methods, GAAC and the hybrid method have shown improvement over the usage of traditional sequence driven features in the context of smaller and refined feature sizes and classification accuracy. The development of six non-redundant novel sets of the amino acid indices dataset, of which each are more representative than the original database. Finally, the construction of two large 25% and 40% homology datasets consisting over 5000 and 7000 protein samples, respectively. A public webserver has been developed located at http://www.generalised-protein-sequence-features.com, which allows biologists and bioinformaticians to extract GAAC sequence driven features from any inputted protein sequence.

To examine the response of wheat to different temperatures and photoperiods at the proteomic level, a series of experiments was performed at the University of Saskatchewan, College of Agriculture and Bioresources, Department of Plant Science. Tandem-mass spectrometry (MS/MS) was used for protein identification. The iTRAQ approach was used to generate raw data for protein quantification. The Pro Group protein identification software was used for protein identification and quantification of differentially expressed proteins. Despite the input samples being from a plant,the software reported non-plant proteins. The traditional approach used by scientists to deal with this problem is to use sequence alignment software to find close green-plant homologs of the non-plant proteins from a plant-only database. Such a technique is problematic since homology-based sequence similarity does not generally equate to similarity of mass spectra. In this work a more radical approach was investigated and implemented. A bioinformatics pipeline was designed and implemented to report plant proteins misidentified by the Pro Group software. The approach drew its idea from the fact that MS/MS-based protein identification uses peptide fragments/ions bearing unique m/z values in the mass spectra. From the reported non-plant proteins and associated peptides, putative m/z values of the peptides are generated and then used to find alternate hits from a green plant-only database. The pipeline uses three different heuristics, each generating a list of candidate proteins. The proteins reported consistently across the three reported lists have the highest likelihood to be present in the original sample. To evaluate the performance of the pipeline, three separate experiments were performed. A set of known plant peptides, a combination of known plant and non-plant peptides and a set of known non-plant peptides were used as input to the pipeline. For each experiment a stringency value (threshold value) was set by the user. Better results were observed by specifying a tighter stringency; that is, more plant proteins were reported consistently across the three reported lists. The research presented in this thesis shows that m/z values, consideration of unique peptides and accounting for proteins with shorter sequences can be used to identify proteins. These characteristics can be used to identify proteins when limited information is available, in this case a list of non-plant proteins reported as being present in a plant-derived sample. The information available was limited because the original input data was already processed by the Pro Group software. The approach presented here is an alternative to a wet lab scientist using sequence alignment tools, sequence databases, and homology-based search. The pipeline can be enhanced by adding various other modules. The results presented here could be used as a foundation for a further study.

To examine the response of wheat to different temperatures and photoperiods at the proteomic level, a series of experiments was performed at the University of Saskatchewan, College of Agriculture and Bioresources, Department of Plant Science. Tandem-mass spectrometry (MS/MS) was used for protein identification. The iTRAQ approach was used to generate raw data for protein quantification. The Pro Group protein identification software was used for protein identification and quantification of differentially expressed proteins. Despite the input samples being from a plant,the software reported non-plant proteins. The traditional approach used by scientists to deal with this problem is to use sequence alignment software to find close green-plant homologs of the non-plant proteins from a plant-only database. Such a technique is problematic since homology-based sequence similarity does not generally equate to similarity of mass spectra. In this work a more radical approach was investigated and implemented. A bioinformatics pipeline was designed and implemented to report plant proteins misidentified by the Pro Group software. The approach drew its idea from the fact that MS/MS-based protein identification uses peptide fragments/ions bearing unique m/z values in the mass spectra. From the reported non-plant proteins and associated peptides, putative m/z values of the peptides are generated and then used to find alternate hits from a green plant-only database. The pipeline uses three different heuristics, each generating a list of candidate proteins. The proteins reported consistently across the three reported lists have the highest likelihood to be present in the original sample. To evaluate the performance of the pipeline, three separate experiments were performed. A set of known plant peptides, a combination of known plant and non-plant peptides and a set of known non-plant peptides were used as input to the pipeline. For each experiment a stringency value (threshold value) was set by the user. Better results were observed by specifying a tighter stringency; that is, more plant proteins were reported consistently across the three reported lists. The research presented in this thesis shows that m/z values, consideration of unique peptides and accounting for proteins with shorter sequences can be used to identify proteins. These characteristics can be used to identify proteins when limited information is available, in this case a list of non-plant proteins reported as being present in a plant-derived sample. The information available was limited because the original input data was already processed by the Pro Group software. The approach presented here is an alternative to a wet lab scientist using sequence alignment tools, sequence databases, and homology-based search. The pipeline can be enhanced by adding various other modules. The results presented here could be used as a foundation for a further study.

Recently, several spectrometers for small volume measurements in order of microliter have been introduced. They are primarily intended for protein determination (or determination of proteins and nucleic acids in one measurement) by direct spectrophotometry or other spectral methods. One of such instruments is the NanoVueTM Plus (GE Healthcare). In this work, we first tried to characterize the instrument in general terms (stability) and to optimize measurement condititions (sample volume). Proteins have been determined by direct spectrophotometry using internal programs of the instrument, data were controlled by an independent computation. We studied also influence of differences in composition of various proteins on the results. According to the results of this Thesis, the most accurate values could be obtained using the internal program E 1%, using the E 1% value from an experiment. On the other hand, the program Protein UV is producing often inaccurate values, strongly infleunced by the protein amino acid composition. Keywords: protein determination, spectrophotometer NanoVueTM Plus, influence of amino acid composition

O enxofre é um dos elementos essenciais para as plantas e as exigências nutricionais nesse nutriente variam com a espécie e a taxa de crescimento das plantas. Com o objetivo geral de avaliar o efeito da nutrição em enxofre no crescimento e no metabolismo do capimtanzânia (Panicum maximum cv. Tanzânia) e do estilosante (Stylosanthes guianensis cv. Mineirão) desenvolveu-se a presente pesquisa com os objetivos específicos de avaliar os efeitos do fornecimento de enxofre em: i) modificações morfológicas, produtivas e nutricionais ocorridas na parte aérea e nas raízes; ii) metabolismo do nitrogênio e as consequentes alterações na composição e concentrações de aminoácidos; iii) concentrações de enxofre total, enxofre-sulfato e glutationa e na atividade das enzimas glutationa redutase e glutationa sulfo-transferase nas folhas recém-expandidas e raízes; iv) crescimento, metabolismo da glutationa e atividade das enzimas envolvidas no ciclo ascorbato-glutationa e v) absorção de sulfato e a expressão de genes de transportadores de sulfato. Os experimentos foram conduzidos em casa de vegetação e camara de crescimento, empregando-se soluções nutritivas. As doses de enxofre aplicadas foram ajustadas de modo a permitir nutrição baixa, intermediária e alta em enxofre para cada espécie. O enxofre afetou diretamente na emissão de folhas e de perfilhos, área foliar, comprimento e superfície radicular do capim-tanzânia e do estilosante Mineirão, aumentando a produção de massa seca da parte aérea e das raízes. A baixa disponibilidade de enxofre ocasionou o desequilíbrio nutricional com o nitrogênio nas plantas, evidenciado por alta relação nitrogênio:enxofre e altas concentrações de nitrato e aminoácidos livres no tecido vegetal. Sob limitação de enxofre, o capim apresentou predomínio de asparagina na composição aminoacídica, enquanto no estilosante ocorreu a predominância de arginina. A aplicação de enxofre aumentou as concentrações de enxofre total, enxofre-sulfato e glutationa nas folhas diagnósticas e raízes para ambas as espécies forrageiras. As plantas crescidas sob limitação de enxofre apresentaram alta atividade da enzima glutationa redutase visando regenerar a glutationa reduzida, que atua protegendo as células contra danos oxidativos decorrentes do estresse da deficiência nutricional. O fornecimento de enxofre aumentou a atividade da glutationa sulfo-transferase incrementando a capacidade do vegetal de suportar estresses ambientais. A baixa disponibilidade de enxofre induziu o aumento da atividade de enzimas antioxidantes que atuam na regeneração da glutationa e do ascorbato na forma reduzida. As plantas crescidas em baixa disponibilidade de enxofre apresentaram aumento da concentração de glutationa e maior alocação desse composto nas raízes. A distribuição de glutationa das folhas para as raízes em condição de limitação de enxofre regula a absorção de sulfato no capim e no estilosante de modo diferenciado. Para o capim com alta concentração de glutationa nas raízes decresce o influxo total de 34S, enquanto para o estilosante não ocasiona a redução da absorção de sulfato. / Sulfur is an essential element required by plants and the nutritional requirements in this nutrient vary according to species and plant growth rate. This research had the main objective of evaluating the effect of sulfur nutrition on growth and metabolism of Guinea grass (Panicum maximum cv. Tanzânia) and stylo (Stylosanthes guianensis cv. Mineirão) and was developed with the specific objectives to determine the effects on i) morphological, productive and nutritional changes in plant shoots and roots, ii) nitrogen metabolism and the changes in the composition and concentrations of amino acids, iii) concentrations of total sulfur, sulfur-sulfate and glutathione and the activity of the enzymes glutathione reductase and glutathione sulfo-transferase in recently expanded leaves and roots, iv) growth, glutathione metabolism and activity of enzymes involved in ascorbate-glutathione cycle and v) sulfate uptake and expression of sulfur transporters genes. The experiments were carried out in greenhouse and growth chamber, by using nutrient solutions. Sulfur supply were adjusted to low, intermediate and high S nutrition for each species. Sulfur supply influences the emission of leaves, tillering, leaf area, root length and surface of Guinea grass and stylo increasing production of dry mass of aboveground and roots. Sulfur limitation alters the distribution of photosynthates between aboveground and roots of Guinea grass and stylo providing reduction in dry matter production of roots. The plants of Guinea grass increase root surface as a mechanism for adaptation to limited S in the culture medium. The relative chlorophyll index (RCI) in the recently expanded leaves relates to the production of dry mass of aboveground and can be used to assess S nutritional status in Guinea grass and stylo. The application of S proves necessary to increase production of dry mass in Guinea grass and stylo. Low S availability caused nutritional imbalance with N in Guinea grass and stylo plants, as shown by a high N:S ratio and high concentrations of N-nitrate and free amino acids in plant tissues. Among amino acids, asparagine predominated in S-limited guineagrass and arginine in Slimited stylo. Increased S supply regulates N:S ratio at values close to 20:1, which provides N and S concentrations that are more suitable for protein synthesis and forage production in plants of both species. Adding S increased concentrations of total S, S-sulfate, and glutathione in diagnostic leaves and roots of both species collected at the two harvests. Plants grown under S limitation showed high levels of GR activity, related to the regeneration of GSH, which acts to protect cells against oxidative damage caused by the stress of nutritional deficiency. S supply increased GST activity, and consequently plants\' capacity to withstand environmental stresses. Low S availability increased activity of the antioxidant enzymes that act in the regeneration of GSH and AsA. Plants grown with low S availability showed higher concentration of glutathione and greater allocation of glutathione to roots. For Guinea grass, high glutathione concentrations in roots decrease the 34S uptake. For stylo not cause reduction of 34S uptake.

It has been discovered during the past few years that demosponges of the order Verongida such as Aplysina cavernicola exhibit chitin-based skeletons. Verongida sponges are well known to produce bioactive brominated tyrosine derivatives. We could recently demonstrate that brominated compounds do not exclusively occur in the cellular matrix but also in the skeletons of the marine sponges Aplysina cavernicola and Ianthella basta. Our measurements imply that these yet unknown compounds are strongly, possibly covalently bound to the sponge skeletons. In the present work, we determined the skeletal amino acid composition of the demosponge A. cavernicola especially with respect to the presence of halogenated amino acids. The investigations of the skeletons before and after MeOH extraction confirmed that only a small amount of the brominated skeleton-bound compounds dissolves in MeOH. The main part of the brominated compounds is strongly attached to the skeletons but can be extracted for example by using Ba(OH)2. Various halogenated tyrosine derivatives were identified by GC-MS and LC-MS in these Ba(OH)2 extracts of the skeletons.

O enxofre é um dos elementos essenciais para as plantas e as exigências nutricionais nesse nutriente variam com a espécie e a taxa de crescimento das plantas. Com o objetivo geral de avaliar o efeito da nutrição em enxofre no crescimento e no metabolismo do capimtanzânia (Panicum maximum cv. Tanzânia) e do estilosante (Stylosanthes guianensis cv. Mineirão) desenvolveu-se a presente pesquisa com os objetivos específicos de avaliar os efeitos do fornecimento de enxofre em: i) modificações morfológicas, produtivas e nutricionais ocorridas na parte aérea e nas raízes; ii) metabolismo do nitrogênio e as consequentes alterações na composição e concentrações de aminoácidos; iii) concentrações de enxofre total, enxofre-sulfato e glutationa e na atividade das enzimas glutationa redutase e glutationa sulfo-transferase nas folhas recém-expandidas e raízes; iv) crescimento, metabolismo da glutationa e atividade das enzimas envolvidas no ciclo ascorbato-glutationa e v) absorção de sulfato e a expressão de genes de transportadores de sulfato. Os experimentos foram conduzidos em casa de vegetação e camara de crescimento, empregando-se soluções nutritivas. As doses de enxofre aplicadas foram ajustadas de modo a permitir nutrição baixa, intermediária e alta em enxofre para cada espécie. O enxofre afetou diretamente na emissão de folhas e de perfilhos, área foliar, comprimento e superfície radicular do capim-tanzânia e do estilosante Mineirão, aumentando a produção de massa seca da parte aérea e das raízes. A baixa disponibilidade de enxofre ocasionou o desequilíbrio nutricional com o nitrogênio nas plantas, evidenciado por alta relação nitrogênio:enxofre e altas concentrações de nitrato e aminoácidos livres no tecido vegetal. Sob limitação de enxofre, o capim apresentou predomínio de asparagina na composição aminoacídica, enquanto no estilosante ocorreu a predominância de arginina. A aplicação de enxofre aumentou as concentrações de enxofre total, enxofre-sulfato e glutationa nas folhas diagnósticas e raízes para ambas as espécies forrageiras. As plantas crescidas sob limitação de enxofre apresentaram alta atividade da enzima glutationa redutase visando regenerar a glutationa reduzida, que atua protegendo as células contra danos oxidativos decorrentes do estresse da deficiência nutricional. O fornecimento de enxofre aumentou a atividade da glutationa sulfo-transferase incrementando a capacidade do vegetal de suportar estresses ambientais. A baixa disponibilidade de enxofre induziu o aumento da atividade de enzimas antioxidantes que atuam na regeneração da glutationa e do ascorbato na forma reduzida. As plantas crescidas em baixa disponibilidade de enxofre apresentaram aumento da concentração de glutationa e maior alocação desse composto nas raízes. A distribuição de glutationa das folhas para as raízes em condição de limitação de enxofre regula a absorção de sulfato no capim e no estilosante de modo diferenciado. Para o capim com alta concentração de glutationa nas raízes decresce o influxo total de 34S, enquanto para o estilosante não ocasiona a redução da absorção de sulfato. / Sulfur is an essential element required by plants and the nutritional requirements in this nutrient vary according to species and plant growth rate. This research had the main objective of evaluating the effect of sulfur nutrition on growth and metabolism of Guinea grass (Panicum maximum cv. Tanzânia) and stylo (Stylosanthes guianensis cv. Mineirão) and was developed with the specific objectives to determine the effects on i) morphological, productive and nutritional changes in plant shoots and roots, ii) nitrogen metabolism and the changes in the composition and concentrations of amino acids, iii) concentrations of total sulfur, sulfur-sulfate and glutathione and the activity of the enzymes glutathione reductase and glutathione sulfo-transferase in recently expanded leaves and roots, iv) growth, glutathione metabolism and activity of enzymes involved in ascorbate-glutathione cycle and v) sulfate uptake and expression of sulfur transporters genes. The experiments were carried out in greenhouse and growth chamber, by using nutrient solutions. Sulfur supply were adjusted to low, intermediate and high S nutrition for each species. Sulfur supply influences the emission of leaves, tillering, leaf area, root length and surface of Guinea grass and stylo increasing production of dry mass of aboveground and roots. Sulfur limitation alters the distribution of photosynthates between aboveground and roots of Guinea grass and stylo providing reduction in dry matter production of roots. The plants of Guinea grass increase root surface as a mechanism for adaptation to limited S in the culture medium. The relative chlorophyll index (RCI) in the recently expanded leaves relates to the production of dry mass of aboveground and can be used to assess S nutritional status in Guinea grass and stylo. The application of S proves necessary to increase production of dry mass in Guinea grass and stylo. Low S availability caused nutritional imbalance with N in Guinea grass and stylo plants, as shown by a high N:S ratio and high concentrations of N-nitrate and free amino acids in plant tissues. Among amino acids, asparagine predominated in S-limited guineagrass and arginine in Slimited stylo. Increased S supply regulates N:S ratio at values close to 20:1, which provides N and S concentrations that are more suitable for protein synthesis and forage production in plants of both species. Adding S increased concentrations of total S, S-sulfate, and glutathione in diagnostic leaves and roots of both species collected at the two harvests. Plants grown under S limitation showed high levels of GR activity, related to the regeneration of GSH, which acts to protect cells against oxidative damage caused by the stress of nutritional deficiency. S supply increased GST activity, and consequently plants\' capacity to withstand environmental stresses. Low S availability increased activity of the antioxidant enzymes that act in the regeneration of GSH and AsA. Plants grown with low S availability showed higher concentration of glutathione and greater allocation of glutathione to roots. For Guinea grass, high glutathione concentrations in roots decrease the 34S uptake. For stylo not cause reduction of 34S uptake.

Demosponges of the order Verongida such as Ianthella basta exhibit skeletons containing spongin, a collagenous protein, and chitin. Moreover, Verongida sponges are well known to produce bioactive brominated tyrosine derivatives. We recently demonstrated that brominated compounds do not only occur in the cellular matrix but also in the skeletons of the marine sponges Aplysina cavernicola and I. basta. Further investigations revealed the amino acid composition of the skeletons of A. cavernicola including the presence of several halogenated amino acids. In the present work, we investigated the skeletal amino acid composition of the demosponge I. basta, which belongs to the Ianthellidae family, and compared it with that of A. cavernicola from the Aplysinidae family. Seventeen proteinogenic and five non-proteinogenic amino acids were detected in I. basta. Abundantly occurring amino acids like glycine and hydroxyproline show the similarity of I. basta and A. cavernicola and confirm the collagenous nature of their sponging fibers. We also detected nine halogenated tyrosines as an integral part of I. basta skeletons. Since both sponges contain a broad variety of halogenated amino acids, this seems to be characteristic for Verongida sponges. The observed differences of the amino acid composition confirm that spongin exhibits a certain degree of variability even among the members of the order Verongida.

Les objectifs de cette thèse étaient d'apporter une percée conceptuelle pour une compréhension en profondeur des mécanismes moléculaires des protéines intrinsèquement désordonnées (IDPs) et de leurs rôles dans la physiologie cellulaire de Chlamydomonas reinhardtii. La combinaison d’approches expérimentale et bioinformatique m’a permis d’identifier 682 protéines thermorésistantes chez C. reinhardtii. Parmi celles-ci, 299 protéines sont systématiquement prédites comme potentielles IDP par quatre algorithmes de prédiction de désordre. Nos résultats indiquent que le pourcentage désordonné moyen de ces protéines prédites comme étant des IDPs est d'environ 20%, et la plupart d'entre elles (~70%) sont adressées à d'autres compartiments que la mitochondrie et le chloroplaste. Leur composition en acides aminés est biaisée par rapport à d'autres IDPs de la base de données de protéines désordonnées (DisProt). Ces IDPs potentielles jouent des fonctions moléculaires diverses, et 54% d'entre elles sont des cibles de phosphorylation.Notre travail a également augmenté l’état des connaissances sur l'adénylate kinase 3 (ADK3), une enzyme contenant une région intrinsèquement désordonnée (IDR). Cette enzyme a été isolée par notre approche globale pour caractériser les IDPs de l’algue verte. L’extension C-terminale désordonnée (CTE) de cette enzyme lui confère de nouvelles fonctions comme par exemple, la formation d’un complexe bi-enzymatique avec la glycéraldéhyde-3-phosphate déshydrogénase (GAPDH), la régulation (négative) de l'activité GAPDH avec le NADPH comme cofacteur, et le rôle de chaperon pour la GAPDH en la protégeant de la dénaturation par traitement thermique et de l’agrégation. / The objectives of this work were to bring a conceptual breakthrough for an in-depth understanding of the molecular mechanisms of intrinsically disordered proteins (IDPs) and their roles in the cellular physiology of Chlamydomonas reinhardtii. Using experimental approaches, 682 heat-resistant proteins were identified as putative IDPs. Among them, 299 proteins were consistently predicted as IDPs by all four disordered predictors. The mean percentage of disordered residues content of these IDPs is about 20%, and most of them (~70%) are addressed to other compartments than mitochondrion and chloroplast. These newly identified IDPs from C. reinhardtii have a biased amino acid composition as regard to other IDPs from the Database of protein disorder (DisProt). Furthermore, they play diverse molecular functions, and 54% of them are the targets for phosphorylation. Our work also revealed more knowledge of the IDR-containing protein adenylate kinase 3 (ADK3) that was extracted by heat-treatment. Its disordered C-terminal extension (CTE) brought new functions to this protein. For instance, via its CTE, ADK3 can form a bi-enzyme complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), down-regulates the NADPH-dependent GAPDH activity, and behaves as a chaperone for GAPDH against its aggregation and inactivation under heat-treatment.