Avena sativa L. (2n = 6x = 42, AACCDD genome composition) or common oat is the cereal grain possessing the highest levels of water-soluble seed (1-3,1-4)-β-D-glucan (β-glucan), a hemicellulose important to human health due to its ability to lower serum LDL cholesterol levels. Understanding the mechanisms of β-glucan accumulation in oat endosperm is, consequently, of great interest. We report a genome-wide association study (GWAS) to identify quantitative trait loci (QTLs) controlling β-glucan production in oat, identifying 58 significantly associated markers. Synteny with the barley (Hordeum vulgare L.) genome identified four major regions of interest, the CslF and CslH gene families along with UGPase and AGPase as candidate genes. Subgenome-specific expression of the A, C, and D homoeologs of major β-glucan synthase AsCslF6 revealed that AsCslF6_C is the least expressed in all tissue types and time points, with low-β-glucan varieties recording the highest proportion of AsCslF6_C expression. In order to further investigate the candidate genes identified in our GWAS study and gain a greater understanding of the other cell wall polysaccharides that comprise the total fiber content in oat we sought to characterize five additional genes. Accordingly, we cloned and sequenced the three homoeologs of AsUGP and AsAGPS1. AsAGPS1 is the small subunit 1 gene of the enzyme ADP-glucose pyrophosphorylase (AGPase), which is responsible for catalyzing the first committed step in the starch biosynthesis pathway through the production of ADP-glucose. AsUGP is the gene the codes for UDP-glucose pyrophosphorylase (UGPase) an enzyme responsible for the reversible production of UDP-glucose (UDPG). UDPG is used directly or indirectly as a precursor for the biosynthesis of cell wall polysaccharides. In high β-glucan mutant line ‘OT3044’ we observed increased expression of AsUGP with a corresponding reduction of AsAGPS1 expression. Similarly, we observed an inverse expression pattern in low-fiber mutant line ‘OT3018’, wherein AsUGP expression was decreased in favor of AsAGPS1 expression. Further, we also found evidence that these changes in both AsUGP and AsAGPS1 expression are due primarily to up- or down-regulation in the A-genome homoeoalleles. Additionally, we characterized genes in the CslC family (CslC4, CslC9) and CslA family (CslA7) responsible for xyloglucan and glucomannan synthesis, respectively. High-fiber line ‘HiFi’ showed the least amount of overall expression of these three genes, raising the possibility that the increased β-glucan is due to a reduction in other hemicelluloses. After analyzing homoeolog-specific expression in multiple genes we observed that the A genome consistently had the most highly expressed homoeoallele, hinting at a universal preference for expression of this subgenome. We present hypotheses regarding multiple points in carbohydrate metabolism having the potential to alter β-glucan content in oat.

No description available.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2007. / Sugarcane is cultivated because of the high levels of sucrose it stores in its internodes. Starch metabolism has been a neglected aspect of sugarcane research despite the problems caused by it during sugarcane processing. Currently there is no information available on the starch content in different South African commercial sugarcane varieties. This project had two main aims of which the first was to determine the starch content in the internodal tissues of six commercial sugarcane varieties. The activities of ADP-Glucose Pyrophosphorylase (AGPase) and β- amylase were also determined. The second aim of the project was to manipulate starch metabolism in sugarcane using transgenesis. To achieve this, transformation vectors for the down-regulation of AGPase activity and over-expression of β-amylase activity were designed. These vectors were then used to transform sugarcane calli and the results were analysed in suspension cultures. Starch levels in sugarcane internodal tissue increased more than 4 times from young to mature internodes. There were also large differences between varieties. When mature tissues of different varieties were compared, their starch concentration varied between 0.18 and 0.51 mg g-1 FW, with the majority of the varieties having a starch concentration between 0.26 and 0.32 mg g-1 FW. NCo376’s starch concentration was much lower than the rest at 0.18 mg g-1 FW and N19’s was much higher at 0.51 mg. g-1 FW. There was also a very strong correlation between starch and sucrose concentration (R2 = 0.53, p ≤ 0.01) which could be due to the fact that these metabolites are synthesized from the same hexose-phosphate pool. No correlation was evident between starch concentration and AGPase activity. This was true for correlations based on either tissue maturity or variety. β-amylase activity expressed on a protein basis was almost 5 times higher in the young internodes compared to mature internodes, suggesting that carbon might be cycled through starch in these internodes. AGPase activity in the transgenic suspension cultures was reduced by between 0.14 and 0.54 of the activity of the wild type control. This reduction led to a reduction in starch concentration of between 0.38 and 0.47 times that of the wild type control. There was a significant correlation between the reduction in AGPase activity and the reduction in starch (R2 = 0.58, p ≤ 0.05). β-amylase activity in the transgenic suspension cultures was increased to 1.5-2 times that of the wild type control. This led to a reduction in starch concentration of between 0.1 and 0.4 times that of the wild type control. Once again the increase in β-amylase activity could be correlated to the reduction in starch concentration of the transgenic suspension cultures (R2 = 0.68, p ≤ 0.01). In both experiments there was no significant effect on sucrose concentration.

Jaburetox (Jbtx) é um peptídeo de 10 kDa derivado de uma das isoformas de urease de Canavalia ensiformis. Em um estudo anterior realizado com o triatomíneo vetor da doença de Chagas Triatoma infestans, esse peptídeo foi encontrado interagindo com a proteína UDP-N-acetilglicosamina pirofosforilase (UAP), alterando também sua atividade enzimática no sistema nervoso central, in vivo e in vitro. A UAP já foi encontrada em eucariotos, bactérias e vírus, estando relacionada com as rotas de produção de quitina, N-glicosilação e síntese de glicoinositolfosfolipídeos. Assim, o presente trabalho tem três objetivos: i) investigar o efeito de Jbtx sobre a atividade enzimática e a expressão gênica da UAP do inseto modelo Rhodnius prolixus, ii) clonar e expressar a UAP e iii) estudar a UAP filogeneticamente. Para a primeira parte, foram avaliados, no triatomíneo R. prolixus, a atividade enzimática da UAP e o perfil de expressão dessa enzima e da quitina sintase em insetos controles e alimentados com Jbtx. Para a segunda, o cDNA da enzima de R. prolixus foi clonado em vetor pET-15b e expressado em Escherichia coli Rosetta 2. A purificação da enzima recombinante foi feita por cromatografia de afinidade a níquel. Para a terceira parte, foram buscadas sequências de aminoácidos homólogas às da UAP de R. prolixus no servidor pHmmer e foi construída uma árvore filogenética com o método de Máxima Verossimilhança. Os resultados obtidos indicam que o Jbtx aumenta a atividade enzimática da UAP em glândulas salivares, corpo gorduroso e epiderme, enquanto diminui a expressão da UAP em intestino médio anterior, túbulos de Malpighi, glândulas salivares, corpo gorduroso, epiderme e sistema nervoso central, assim como a expressão da quitina sintase nos mesmos órgãos e no intestino médio posterior. Foi obtida uma UAP recombinante de 56 kDa, compatível com peso molecular previsto in silico. A árvore filogenética construída contém 40 sequências, sendo 38 de insetos e 2 sequências de grupo externo. A árvore segue o padrão de evolução dos insetos e foi identificado um novo organismo com potenciais dois genes codificantes de UAP. Esse trabalho apresenta a primeira evidência de que Jbtx altera a expressão gênica em R. prolixus. O resultado obtido pela análise filogenética indica que a UAP é uma enzima ancestral à diversificação em Insecta. / Jaburetox (Jbtx) is a 10 kDa peptide derived from a urease isoform of Canavalia ensiformis. In a previous work with the triatomine vector of Chagas’ disease Triatoma infestans, this peptide was found interacting with the protein UDP-N-acetylglucosamine pyrophosphorylase (UAP), also increasing the UAP enzymatic activity in the central nervous system in vivo and in vitro. UAP has been described in eukaryotes, bacteria and virus, and is involved in chitin production, N-linked glycosylation and glyco inositol phospholipids synthesis pathway. Thus, the present work has three main aims: i) to understand the effect of Jbtx on this enzyme on the model insect Rhodnius prolixus, ii) to clone and express UAP and iii) to study UAP from a phylogenetic point of view. Firstly, UAP enzymatic activity and its expression profile, as well as the chitin synthase expression, were analysed in the triatomine R. prolixus in saline- or Jbtx-fed insects. Secondly, the cDNA from R. prolixus’ UAP was cloned into the pET-15b vector and expressed in Escherichia coli Rosetta 2. The recombinant enzyme was purified through a nickel affinity chromatography. Thirdly, homolog sequences to R. prolixus’ UAP were searched in pHmmer database and a phylogenetic tree was built using the Maximmum Likelihood method. The results obtained indicate that Jbtx increases UAP enzymatic activity in salivary glands, fat body and epidermis, while decreasing UAP’s expression in the anterior and posterior midgut, Malpighian tubules, salivary glands, fat body, epidermis and central nervous system, as well as the chitin synthase expression in the same organs and the posterior midgut. A 56 kDa recombinant UAP was obtained, in agreement with the in silico estimated size. The phylogenetic tree built has 40 sequences, from which 38 are from insects and 2 are from mammals (external group). The tree follows the insect evolution patterns and a new organism containing two potential UAP coding genes was identified. This work presents the first evidence that Jbtx is able to interfere in the gene expression in R. prolixus. The results obtained through phylogenetic analysis shows that UAP is an enzyme ancestral to the diversification in Insecta.

Jaburetox (Jbtx) é um peptídeo de 10 kDa derivado de uma das isoformas de urease de Canavalia ensiformis. Em um estudo anterior realizado com o triatomíneo vetor da doença de Chagas Triatoma infestans, esse peptídeo foi encontrado interagindo com a proteína UDP-N-acetilglicosamina pirofosforilase (UAP), alterando também sua atividade enzimática no sistema nervoso central, in vivo e in vitro. A UAP já foi encontrada em eucariotos, bactérias e vírus, estando relacionada com as rotas de produção de quitina, N-glicosilação e síntese de glicoinositolfosfolipídeos. Assim, o presente trabalho tem três objetivos: i) investigar o efeito de Jbtx sobre a atividade enzimática e a expressão gênica da UAP do inseto modelo Rhodnius prolixus, ii) clonar e expressar a UAP e iii) estudar a UAP filogeneticamente. Para a primeira parte, foram avaliados, no triatomíneo R. prolixus, a atividade enzimática da UAP e o perfil de expressão dessa enzima e da quitina sintase em insetos controles e alimentados com Jbtx. Para a segunda, o cDNA da enzima de R. prolixus foi clonado em vetor pET-15b e expressado em Escherichia coli Rosetta 2. A purificação da enzima recombinante foi feita por cromatografia de afinidade a níquel. Para a terceira parte, foram buscadas sequências de aminoácidos homólogas às da UAP de R. prolixus no servidor pHmmer e foi construída uma árvore filogenética com o método de Máxima Verossimilhança. Os resultados obtidos indicam que o Jbtx aumenta a atividade enzimática da UAP em glândulas salivares, corpo gorduroso e epiderme, enquanto diminui a expressão da UAP em intestino médio anterior, túbulos de Malpighi, glândulas salivares, corpo gorduroso, epiderme e sistema nervoso central, assim como a expressão da quitina sintase nos mesmos órgãos e no intestino médio posterior. Foi obtida uma UAP recombinante de 56 kDa, compatível com peso molecular previsto in silico. A árvore filogenética construída contém 40 sequências, sendo 38 de insetos e 2 sequências de grupo externo. A árvore segue o padrão de evolução dos insetos e foi identificado um novo organismo com potenciais dois genes codificantes de UAP. Esse trabalho apresenta a primeira evidência de que Jbtx altera a expressão gênica em R. prolixus. O resultado obtido pela análise filogenética indica que a UAP é uma enzima ancestral à diversificação em Insecta. / Jaburetox (Jbtx) is a 10 kDa peptide derived from a urease isoform of Canavalia ensiformis. In a previous work with the triatomine vector of Chagas’ disease Triatoma infestans, this peptide was found interacting with the protein UDP-N-acetylglucosamine pyrophosphorylase (UAP), also increasing the UAP enzymatic activity in the central nervous system in vivo and in vitro. UAP has been described in eukaryotes, bacteria and virus, and is involved in chitin production, N-linked glycosylation and glyco inositol phospholipids synthesis pathway. Thus, the present work has three main aims: i) to understand the effect of Jbtx on this enzyme on the model insect Rhodnius prolixus, ii) to clone and express UAP and iii) to study UAP from a phylogenetic point of view. Firstly, UAP enzymatic activity and its expression profile, as well as the chitin synthase expression, were analysed in the triatomine R. prolixus in saline- or Jbtx-fed insects. Secondly, the cDNA from R. prolixus’ UAP was cloned into the pET-15b vector and expressed in Escherichia coli Rosetta 2. The recombinant enzyme was purified through a nickel affinity chromatography. Thirdly, homolog sequences to R. prolixus’ UAP were searched in pHmmer database and a phylogenetic tree was built using the Maximmum Likelihood method. The results obtained indicate that Jbtx increases UAP enzymatic activity in salivary glands, fat body and epidermis, while decreasing UAP’s expression in the anterior and posterior midgut, Malpighian tubules, salivary glands, fat body, epidermis and central nervous system, as well as the chitin synthase expression in the same organs and the posterior midgut. A 56 kDa recombinant UAP was obtained, in agreement with the in silico estimated size. The phylogenetic tree built has 40 sequences, from which 38 are from insects and 2 are from mammals (external group). The tree follows the insect evolution patterns and a new organism containing two potential UAP coding genes was identified. This work presents the first evidence that Jbtx is able to interfere in the gene expression in R. prolixus. The results obtained through phylogenetic analysis shows that UAP is an enzyme ancestral to the diversification in Insecta.

Jaburetox (Jbtx) é um peptídeo de 10 kDa derivado de uma das isoformas de urease de Canavalia ensiformis. Em um estudo anterior realizado com o triatomíneo vetor da doença de Chagas Triatoma infestans, esse peptídeo foi encontrado interagindo com a proteína UDP-N-acetilglicosamina pirofosforilase (UAP), alterando também sua atividade enzimática no sistema nervoso central, in vivo e in vitro. A UAP já foi encontrada em eucariotos, bactérias e vírus, estando relacionada com as rotas de produção de quitina, N-glicosilação e síntese de glicoinositolfosfolipídeos. Assim, o presente trabalho tem três objetivos: i) investigar o efeito de Jbtx sobre a atividade enzimática e a expressão gênica da UAP do inseto modelo Rhodnius prolixus, ii) clonar e expressar a UAP e iii) estudar a UAP filogeneticamente. Para a primeira parte, foram avaliados, no triatomíneo R. prolixus, a atividade enzimática da UAP e o perfil de expressão dessa enzima e da quitina sintase em insetos controles e alimentados com Jbtx. Para a segunda, o cDNA da enzima de R. prolixus foi clonado em vetor pET-15b e expressado em Escherichia coli Rosetta 2. A purificação da enzima recombinante foi feita por cromatografia de afinidade a níquel. Para a terceira parte, foram buscadas sequências de aminoácidos homólogas às da UAP de R. prolixus no servidor pHmmer e foi construída uma árvore filogenética com o método de Máxima Verossimilhança. Os resultados obtidos indicam que o Jbtx aumenta a atividade enzimática da UAP em glândulas salivares, corpo gorduroso e epiderme, enquanto diminui a expressão da UAP em intestino médio anterior, túbulos de Malpighi, glândulas salivares, corpo gorduroso, epiderme e sistema nervoso central, assim como a expressão da quitina sintase nos mesmos órgãos e no intestino médio posterior. Foi obtida uma UAP recombinante de 56 kDa, compatível com peso molecular previsto in silico. A árvore filogenética construída contém 40 sequências, sendo 38 de insetos e 2 sequências de grupo externo. A árvore segue o padrão de evolução dos insetos e foi identificado um novo organismo com potenciais dois genes codificantes de UAP. Esse trabalho apresenta a primeira evidência de que Jbtx altera a expressão gênica em R. prolixus. O resultado obtido pela análise filogenética indica que a UAP é uma enzima ancestral à diversificação em Insecta. / Jaburetox (Jbtx) is a 10 kDa peptide derived from a urease isoform of Canavalia ensiformis. In a previous work with the triatomine vector of Chagas’ disease Triatoma infestans, this peptide was found interacting with the protein UDP-N-acetylglucosamine pyrophosphorylase (UAP), also increasing the UAP enzymatic activity in the central nervous system in vivo and in vitro. UAP has been described in eukaryotes, bacteria and virus, and is involved in chitin production, N-linked glycosylation and glyco inositol phospholipids synthesis pathway. Thus, the present work has three main aims: i) to understand the effect of Jbtx on this enzyme on the model insect Rhodnius prolixus, ii) to clone and express UAP and iii) to study UAP from a phylogenetic point of view. Firstly, UAP enzymatic activity and its expression profile, as well as the chitin synthase expression, were analysed in the triatomine R. prolixus in saline- or Jbtx-fed insects. Secondly, the cDNA from R. prolixus’ UAP was cloned into the pET-15b vector and expressed in Escherichia coli Rosetta 2. The recombinant enzyme was purified through a nickel affinity chromatography. Thirdly, homolog sequences to R. prolixus’ UAP were searched in pHmmer database and a phylogenetic tree was built using the Maximmum Likelihood method. The results obtained indicate that Jbtx increases UAP enzymatic activity in salivary glands, fat body and epidermis, while decreasing UAP’s expression in the anterior and posterior midgut, Malpighian tubules, salivary glands, fat body, epidermis and central nervous system, as well as the chitin synthase expression in the same organs and the posterior midgut. A 56 kDa recombinant UAP was obtained, in agreement with the in silico estimated size. The phylogenetic tree built has 40 sequences, from which 38 are from insects and 2 are from mammals (external group). The tree follows the insect evolution patterns and a new organism containing two potential UAP coding genes was identified. This work presents the first evidence that Jbtx is able to interfere in the gene expression in R. prolixus. The results obtained through phylogenetic analysis shows that UAP is an enzyme ancestral to the diversification in Insecta.

The catalytic necessity of tyrosine residues in uridine diphospho- glucose pyrophosphorylase [E.C. 2.7.7.9] was investigated. Chemical modification of the pyrophosphorylase by N-acetylimidazole indicated that tyrosine residues were essential for activity. Approximately 23 of 112 tyrosines per molecule of 475,000 Daltons could be 0-acetylated. Solvent perturbation difference spectroscopy supported this number of exposed tyrosine side chains and in conjunction with chemical modification indicated that at least 11 to 12 tyrosyl residues per protein molecule are fully exposed. it her subst rate, uridine t riphosphate or uridine diphosphoglucose, afforded significant protection against inactivation by N-acetylimidazole. The significance of these tyrosine residues is discussed in terms of a quaternary subunit model for uridine diphosphoglucose pyrophosphorylase.

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme of carbohydrate metabolism in all living organisms. The main aim of this thesis was to investigate the function and regulation of plant UGP genes as well as the UGPase proteins. Both in vivo and in vitro approaches were used, including the use of transgenic plants deficient in UGPase activity, and using purified proteins and their mutants to elucidate the structure/ function properties of UGPase. In both Arabidopsis and aspen, there are two highly similar UGP genes being actively transcribed, but not to the same extent. For both species, the UGP genes could be classified into two categories: a “house-keeping” gene and a subsidiary gene, with the former functioning universally in all the tissues to support the normal growth, whereas the latter usually expressed at a lower level in most of the organs/tissues tested. Besides, the two UGP genes were also found being differentially regulated under abiotic stress conditions, e.g. low temperature. By investigating the Arabidopsis T-DNA insertion mutants, which respectively have one or both of the UGP genes knocked out, we noticed that as little as 10% of the remaining UGPase activity could still support normal growth and development under controlled conditions, with little or no changes in carbohydrate contents, including soluble sugars (e.g. sucrose), starch and cell wall polysaccharides. Those plants, however, had a significantly decreased fitness under field conditions, i.e. the plants most deficient in UGPase activity produced up to 50% less seeds than in wt. Therefore, we concluded that UGPase is not a rate-limiting enzyme in carbohydrate synthesis pathways, but still is essential in viability of Arabidopsis plants. In order to characterize two Arabidopsis UGPase isozymes, both proteins were heterologously overexpressed in prokaryotic cells and purified by affinity chromatography. The two isozymes showed little differences in physical and biochemical properties, including substrate specificity, Km values with substrates in both directions of the reaction, molecular masses, isoelectric point (pI), and equilibrium constant. On the other hand, possibilities of distinct post-translational regulatory mechanisms were indicated, based on amino acid (aa) motif analyses, and on 3D analyses of derived crystal structures of the two proteins. We used the heterologous bacterial system also to overexpress barley UGPase and several of its mutants, both single mutants and those with whole domains/ exons deleted. As a result, we have identified several aa residues/ protein domains that may be essential for structural integrity and catalytic/ substrate-binding properties of the protein. For instance, we found that the last exon of UGPase (8 aa at the end of C-terminus) was important for the protein ability to oligomerize and that Lys-260 and the second-to-last exon were essential for pyrophosphate (but not UDP-glucose) binding. The data emphasized the critical role of central part of the active site (so called NB-loop) in catalysis, but also pointed out to the role of N-terminus in catalysis and oligomerization, but not substrate binding, and that of C-terminus in both catalysis/substrate binding and oligomerization.

Submitted by Ricardo Cedraz Duque Moliterno (ricardo.moliterno@uefs.br) on 2015-07-27T21:18:33Z No. of bitstreams: 1 Dissertac?o Jos? Jorge Silva Junior.pdf: 3038189 bytes, checksum: be43dcbd4f764e08ab18fd95a388e950 (MD5) / Made available in DSpace on 2015-07-27T21:18:33Z (GMT). No. of bitstreams: 1 Dissertac?o Jos? Jorge Silva Junior.pdf: 3038189 bytes, checksum: be43dcbd4f764e08ab18fd95a388e950 (MD5) Previous issue date: 2014-07-31 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Pests are responsible for high losses in cocoa production in Brazil and other countries, among them the witches' broom (WB) is one of the most important and destructive to the cocoa, even causing losses of up to 95% of production. This plague has spread very easily in the state of Bahia due to environmental conditions that provided the spread of WB, caused by the fungus Moniliophthora pernicious. Several chemical compounds have been tested in order to prevent or eradicate WB, however it has not showed good results, so the present study aimed to perform in silico assays were obtained in order to identify inhibitors of UDP-N-acetylglucosamine pyrophosphorylase (UNAcP) of M. perniciosa. For achieve this goal, computational methods have been employed in the search for new inhibitors for UNAcP where different stages of research and evaluation were performed. The initial stage of virtual screening consisted of the choice of the scoring function, thus the following scoring functions were evaluated: Broyden Fletcher Goldfarb Shanno (BFGS) present in AutoDock VINA 1.1.2; Grid and Grid Score Score+Hawkins GB/SA both present in DOCK 6.5 and calculate the consensus score. The results were analyzed by calculating Enrichment Factor (EF) analysis of the ROC curve and its respective Area under an ROC curve (AUC). The Grid Score presented EF(5)=7.85. The ROC curve analysis allowed us to observe that the Grid Score function can identify almost 40% of active molecules with less than 10% of the database (false positive and active molecules), AUC analysis demonstrated that the Grid Score has greater accuracy (AUC=0.87). Thus, these results showed what the Grid Score was the best scoring function for this system. A database composed of molecules derived from natural sources was also used. The top ten results of virtual screening of the DOCK6.5 underwent online platform ChemGPS-NP, for the calculation of chemical descriptors. Thus, the molecules were re-categorized, based on the values of the Grid Score DOCK6.5 and chemical descriptors ChemGPS-NP. The results indicate the ZINC68592326 molecule his the best score and the analysis indicates that this has hydrophobic interactions with Ala380, Gln113, Gly112, Gly381, Ser168, Arg383, Pro221 and hydrogen bond interaction (3.32?) with Asn224. The virtual screening database of molecules derived from natural products research allowed with a universe of structures with very different characteristics. It was possible to obtain molecules with great structural diversity between the top ranking, however, also found very similar to the reference molecules. The use of chemometric methods is considered very useful and allows a systematic and consistent choice of structures, mainly by taking into account chemical descriptors and molecular characteristics, allowing for a more detailed evaluation. / Pragas s?o respons?veis por elevadas perdas na produ??o de cacau no Brasil e no mundo, dentre elas a vassoura-de-bruxa (VB) ? uma das mais importantes e destrutivas para o cacaueiro, chegando a causar perdas de at? 95% da produ??o. Essa praga disseminou-se muito facilmente no estado da Bahia devido a condi??es ambientais que proporcionaram a propaga??o da VB, causada pelo fungo Moniliophthora perniciosa. Diversos compostos qu?micos v?m sendo testados com o objetivo de prevenir ou erradicar a VB, por?m n?o foram obtidos bons resultados, portanto, o presente trabalho teve como principal objetivo realizar ensaios in silico, a fim de identificar inibidores da UDP-N-acetilglicosamina pirofosforilase (UNAcP) do M. perniciosa. Para tanto, foram empregados m?todos computacionais na busca de novos inibidores para a UNAcP, onde foram realizadas diferentes etapas de busca e avalia??o. A etapa inicial da triagem virtual consistiu na escolha da fun??o de pontua??o, assim, foram avaliadas as seguintes fun??es de pontua??o: Broyden?Fletcher?Goldfarb?Shanno (BFGS) presente no AUTODOCK VINA 1.1.2; Grid Score e Grid Score+Hawkins GB/SA ambos presentes no DOCK 6.5 e o c?lculo do escore de consenso. Os resultados foram analisados atrav?s do c?lculo de Fator de Enriquecimento (FE), analise da curva ROC e sua respectiva ?rea Sobre a Curva (AUC). O Grid Score apresentou FE(5)=7,85. A an?lise da curva ROC permitiu observar que a fun??o Grid Score consegue identificar quase 40% das mol?culas ativas com menos de 10% do banco de dados (mol?culas ativas e falso positivos), a an?lise da AUC demonstrou que o Grid Score tem maior exatid?o (AUC=0,87). Assim os resultados da avalia??o apontaram o Grid Score como melhor fun??o de pontua??o para esse sistema. Foi utilizado um banco de dados composto por mol?culas oriundas de fontes naturais. Os dez melhores resultados da triagem virtual feita no DOCK6.5 foram submetidos ? plataforma on line ChemGPS-NP, para o c?lculo dos descritores qu?micos. Assim, as mol?culas foram recategorizadas, baseando-se nos valores do Grid Score do DOCK6.5 e descritores qu?micos do ChemGPS-NP. Os resultados apontaram a mol?cula ZINC68592326 com a melhor pontua??o, a an?lise das intera??es intermoleculares indica que est? mol?cula apresenta intera??es hidrof?bicas com os res?duos Ala380, Gln113, Gli112, Gli381, Ser168, Arg383, Pro221 e liga??o de hidrog?nio do tipo aceptora com dist?ncia de 3,32? com o res?duo Asn224. A triagem virtual em banco de dados de mol?culas oriundas de produtos naturais permitiu a investiga??o com um universo de estruturas com caracter?sticas muito diversas. Foi poss?vel obter mol?culas com grande diversidade estrutural entre os primeiros do ranking, por?m, foram encontradas tamb?m mol?culas muito similares ?s mol?culas de refer?ncia. A utiliza??o de m?todos quimiom?tricos, ? considerada muito ?til e permitem uma escolha sistem?tica e consistente das estruturas, principalmente por levar em considera??o, descritores qu?micos e caracter?sticas moleculares, permitindo uma avalia??o mais criteriosa.

<p>Photosynthesis captures light from the sun and converts it into carbohydrates, which are utilised by almost all living organisms. The conversion between the different forms of carbohydrates is the basis to form almost all biological molecules.</p><p>The main intention of this thesis has been to study the role of UDP-glucose in carbohydrate synthesis and metabolism, and in particular the genes that encode UDP-glucose pyrophosphorylase (UGPase) and UDP-glucose dehydrogenase (UGDH) in plants and their regulation. UGPase converts glucose-1-phosphate to UDP-glucose, which can be utilised for sucrose synthesis, or cell wall polysaccharides among others. UGDH converts UDP-glucose to UDP-glucuronate, which is a precursor for hemicellulose and pectin. As model species I have been working with both Arabidopsis thaliana and poplar.</p><p>Sequences for two full-length EST clones of Ugp were obtained from both Arabidopsis and poplar, the cDNAs in Arabidopsis correlate with two genes in the Arabidopsis genomic database.</p><p>The derived protein sequences are 90-93% identical within each plants species and 80-83% identical between the two species.</p><p>Studies on Ugp showed that the expression is up-regulated by Pi-deficiency, sucrose-feeding and by light exposure in Arabidopsis. Studies with Arabidopsis plants with mutations in sugar/ starch- and Pi-content suggested that the Ugp expression is modulated by an interaction of signals derived from Pi-deficiency, sugar content and light/ dark conditions, where the signals act independently or inhibiting each other, depending on conditions. Okadaic acid, a known inhibitor of certain classes of protein phosphatases, prevented the up-regulation of Ugp by Pi-deficiency and sucrose-feeding. In poplar, sucrose also up-regulated the expression of Ugp. When poplar and Arabidopsis were exposed to cold, an increase of Ugp transcript content was detected as well as an increase in UGPase protein and activity. In poplar, Ugp was found to be expressed in all tissues that were examined (differentiating xylem, phloem, apical leaves and young and mature leaves).</p><p>By using antisense strategy, Arabidopsis plants that had a decrease in UGPase activity of up to 30% were obtained. In the antisense plants, the soluble carbohydrate content was reduced in the leaves by at least 50%; in addition the starch content decreased. Despite the changes in carbohydrate content, the growth rate of the antisense plants was not changed compared to wild type plants under normal growth conditions. However, in the antisense lines the UGPase activity and protein content in sliliques and roots increased, perhaps reflecting compensatory up-regulation of second Ugp gene. This correlates with a slightly larger molecular mass of UGPase protein in roots and siliques when compared to that in leaves. Maximal photosynthesis rates were similar for both wild type and antisense plants, but the latter had up to 40% lower dark respiration and slightly lower quantum yield than wild type plants.</p><p>Two Ugdh cDNAs from poplar and one from Arabidopsis were sequenced. The highest Ugdh expression was found in xylem and younger leaves. Expression data from sugar and osmoticum feeding experiment in poplar suggested that the Ugdh expression is regulated via an osmoticumdependent pathway.</p>