Zur Detektion neuer IgE- reaktiver Proteine wurde in dieser Arbeit ein zweidimensionales Proteintrennverfahren verwendet. Resultierende Proteinfraktionen wurden mithilfe von 18 tomatensensibiliesierten Patientenseren im Immunoblot getestet. Detektierte Proteine in der SDS-PAGE wurden mittels LC-MS/MS identifiziert. Dadurch konnten 2 Tomatensamenproteine, die im Immunoblot ein IgE- reaktives Signal zeigten eindeutig mittels Massenspektrometrie identifiziert werden. Diese Proteine sind Legumin und Vicilin. Durch Sequenzabgleich und Proteinstrukturmodellierung im Vergleich zu bereits bekannten Allergenen (Erdnuss und Cashewnuss), konnte eine hohe Homologie gezeigt werden. / For the detection of tomato allergens a multidimensional protein fractionation strategy and LC-MS/MS was used. Putative allergens were detected by IgE immunoblotting using sera from 18 adult tomato sensitised patients selected based on a positive history skin prick test and specific Immunglobulin (Ig) E levels. Two legumin- and vicilin- proteins were purified and showed strong IgE-reactivity in immunoblots. Individual patient sera exhibited varying IgE-sensitivity against the purified proteins. In silico structural modelling indicates high homology between epitopes of known peanut and cashewnut allergens and the detected IgE-crossreactive tomato proteins.

The use of various types of gel electrophoresis for characterization and identifying plant varieties is well estabilished. This diploma work deals with the possibility characterize potato cultivar (Solanum tuberosum L.) by the help of electrophoretic technique SDS {--} PAGE tuber protein profiles. Soluble potato tuber proteins can be classified according to their molecular weight (kDa) into three groups: First, the major tuber protein patatin (37 {--} 44 kDa); second, a group constituted of protease inhibitors (6 {--} 24 kDa); and third group containing all other proteins. First two group manifest genetic variability, which is necessary evalute for its potential using of potato cultivar characterization.

Orientador: Marcos Silveira Buckeridge / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-12T17:19:07Z (GMT). No. of bitstreams: 1 Tonini_PatriciaPinho_D.pdf: 2946398 bytes, checksum: 0c45bddc87c835268cd2cfd05f89d364 (MD5) Previous issue date: 2008 / Resumo: Sementes de Sesbania virgata (Cav.) Pers. acumulam suas reservas de carbono no endosperma na forma de um polissacarídeo de parede celular, o galactomanano. A mobilização deste ocorre após a germinação e envolve três enzimas hidrolíticas, dentre elas a a-galactosidase. Além da reserva de carbono, há uma grande quantidade de corpos protéicos, no citoplasma das células endospérmicas, que constituem a principal reserva de nitrogênio nestas sementes. Para que ocorra a correta distribuição dos produtos de degradação das reservas deve haver sincronização entre os processos de degradação das reservas de carbono e nitrogênio, porém para compreender tais mecanismos, é necessário estudar aspectos do controle da produção e ação das enzimas responsáveis pela hidrólise das reservas. Buscando determinar em que ponto do metabolismo a semente de S. virgata se encontra em relação à produção destas enzimas hidrolíticas, durante e após a germinação, e supostamente os tecidos envolvidos nesta produção, sementes desta espécie foram embebidas em actinomicina-D (inibidor de transcrição) e cicloheximida (inibidor de tradução) e os efeitos destes inibidores verificados através da atividade e detecção da a-galactosidase no tegumento e endosperma destas sementes. Além disso, buscando verificar e relacionar o efeito do ácido abscísico, do etileno e dos açúcares na degradação das reservas após a germinação de S. virgata, sementes desta espécie foram embebidas em ABA, etileno, glucose e sacarose, e os efeitos destes foram verificados através dos teores protéicos, da atividade da a-galactosidase e da produção endógena de ABA e etileno nestas sementes. Como a presença de actinomicina-D e cicloheximida não inibiram a produção e a atividade da a-galactosidase no endosperma durante e após a germinação, sugere-se que a produção desta enzima ocorra principalmente durante a maturação da semente. Aparentemente, a partir do período pós-germinativo, a enzima pré-formada seria processada e ativada, para conseqüente degradação dos galactomananos, através de um processo proteolítico. Em contrapartida, como a presença deactinomicina-D e cicloheximida inibiram a degradação das proteínas de reserva no tegumento e endosperma e, inclusive, induziram a atividade da a-galactosidase nestes tecidos no final do processo de degradação dos galactomananos, sugerese a síntese de novo de proteases durante e após a germinação e a relação íntima destas enzimas com a degradação das proteínas de reserva e da a-galactosidase no final do processo de degradação dos galactomananos. Quanto aos hormônios, a presença de ABA exógeno retardou o início da degradação das proteínas de reserva no endosperma, assim como diminuiu a atividade da a-galactosidase no mesmo tecido no final do processo de degradação do galactomanano, sugerindo um efeito modulador deste hormônio durante a degradação das reservas, reprimindo a ação das enzimas hidrolíticas. A presença de etileno exógeno, entretanto, aumentou a atividade da a-galactosidase no endosperma e, inclusive, no tegumento no final do processo de degradação do galactomanano, sugerindo um efeito indutor deste hormônio na ativação e ação das enzimas hidrolíticas. De fato, analisando a produção endógena de ABA e etileno, observou-se um aumento brusco dos hormônios no período de mobilização das reservas, que pode estar relacionado à influência destes hormônios na atividade das enzimas hidrolíticas em sementes de S. virgata. Ainda, como ocorreram mudanças na produção de ABA e etileno endógeno na presença de glucose e sacarose, sugere-se uma relação íntima entre a via de sinalização destes hormônios e a dos açúcares, a fim de controlar o processo de degradação das reservas. Desta forma, estas evidências sugerem que o ABA e o etileno controlariam antagonicamente a mobilização de reservas em sementes de S. virgata, juntamente com os açúcares, através da ativação e ação das enzimas hidrolíticas, a fim de controlar o processo de degradação das proteínas e dos galactomananos, evitando a produção dos açúcares redutores e de sacarose em excesso durante o período pós-germinativo e garantindo o afluxo eficiente de carbono e nitrogênio para o desenvolvimento da plântula. / Abstract: Seeds of Sesbania virgata (Cav.) Pers. have an endosperm which accumulates galactomannan as a storage polysaccharide in the cell walls that is hydrolysed after germination by three enzymes (a-galactosidase, endo-ß-mannanase and exo-ß-mannosidase). Besides the storage of carbon, there is a great amount of protein bodies in the cytoplasm of endospermic cells, which play the major role as a nitrogen reserve in this seed. It is likely that a synchronization between the process of galactomannan and protein degradation occurs for a more efficient distribution and use of the storage degradation products. However, to understand these mechanisms, it is necessary to study aspects of the production and action control of the hydrolytic enzymes responsible for the mobilisation of these reserves. Aiming to determine in which point of the metabolism the seed of S. virgata is in relation to the production of the hydrolytic enzymes, during and after germination, and the supposed tissues involved in this production, seeds of this specie were imbibed in actinomycin-D (transcription inhibitor) and cycloheximide (translation inhibitor), and the effects of these inhibitors verified thought the a-galactosidase activity and detection in the testa and endosperm of these seeds. Besides of this, for to verify and relate the effects of abscisic acid, ethylene and sugars on the reserves degradation after germination of the S. virgata, seeds of this specie were imbibed in ABA, ethylene, glucose and sucrose, and the effects of these were verified through the protein content, a-galactosidase activity and endogenous production of ABA and ethylene in this seeds. In the presence of actinomycin-D and cycloheximide, the production and activity of the a- galactosidase were not inhibited during and after germination, suggesting that the production of this enzyme occurs principally during the maturation of the seed. Apparently, after the germination, this enzyme is processed by proteolysis, so that it becomes activated to perform galactomannan degradation. On the other hand, the presence of actinomycin-D and cycloheximide inhibited the protein degradation in the testa and endosperm and at the same time induced the a-galactosidaseactivity in the same tissues at the final steps of the galactomannan degradation process. This suggests the existence of synthesis de novo of the proteases during and after germination and a possible relationship between the storage protein and galactomannan mobilisation. Regarding the hormones, the presence of exogenous ABA retarded the beginning of the storage protein degradation in the endosperm and also decreased a-galactosidase activity in the same tissue at the end of galactomannan degradation. This suggests that there is a regulator effect of this hormone during the storage degradation, repressing the enzymes action. In the presence of exogenous ethylene an increase in the a-galactosidase activity in the endosperm and in the testa were observed at the end of galactomannan degradation, suggesting an inducing effect of this hormone on the hydrolytic enzymes. The finding of endogenous ABA and ethylene production, observed during the period of the storage mobilisation, give support to the above raised hypothesis that the two hormones participate in the control of the hydrolytic enzyme activities in seeds of S. virgata. Furthermore, the changes observed in the endogenous ABA and ethylene production in the presence of glucose and sucrose, suggested a relation between the signaling pathway of these hormones and the sugars signaling, controlling the process of storage degradation. Thus, our results add evidence to suggest that ABA and ethylene antagonistically control the storage mobilisation in seeds of S. virgata, together with the sugars, through the hydrolytic enzymes activation, controlling the process of storage protein and galactomannan degradation, in other to avoiding the excess production of reductor sugars and sucrose during the post-germinative period and assuring the efficient afflux of the carbon and nitrogen to the seedling development. / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Orientador: Paulo Arruda / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-08T07:58:05Z (GMT). No. of bitstreams: 1 Figueira_ThaisRezendeeSilva_D.pdf: 6195048 bytes, checksum: a6968f696fa4837277a389b9699a272b (MD5) Previous issue date: 2007 / Resumo: O seqüenciamento de ESTs e a sua organização em bancos de dados constituem poderosas ferramentas para a identificação de genes expressos em determinados tecidos e/ou tipos celulares. Buscando obter informações sobre o metabolismo e o desenvolvimento da semente do milho, nós criamos um banco de ESTs denominado MAIZEST. Esse banco de dados contem ESTs de um grande número de tecidos do milho e é especialmente rico em seqüências provenientes do endosperma em desenvolvimento. O MAIZEST contém 227.431 ESTs oriundas de mais de 30 órgãos e tecidos de milho. Desses, 64.537 são provenientes de endosperma em desenvolvimento sendo que 30.531 foram seqüenciados em nosso laboratório. Utilizando ferramentas de bioinformatica, identificamos no MAIZEST um conjunto de genes preferencialmente expressos no endosperma, dentre os quais destacam-se fatores de transcrição posivelmente envolvidos em processos chave do metabolismo, diferenciação e desenvolvimento do endosperma. Em seguida, identificamos no MAIZEST os genes que codificam as proteínas de reserva de milho, as zeínas, e fizemos um estudo comparativo entre os genes que coficam as prolaminas do milho, do sorgo e da cana-de-açúcar. Estudos evolutivos sugerem que o milho seja o produto de uma alotetraploidização ocorrida pela hibridização interespecífica de dois ancestrais n=5. O milho deve ter sido originado através de um evento de alotetraploidização segmental, ocorrido a aproximadamente 11 milhões de anos, ou através de um evento único de duplicação, ocorrido acerca de 5 milhões de anos. Todavia, até o momento, os progenitores do milho ainda não haviam sido identificados. Neste trabalho, demonstramos que um destes progenitores pertence à linhagem Saccharum, que deu origem à moderna cana-de-açúcar. Comparando os padrões das prolaminas do milho, do sorgo e da cana-de-açúcar, observamos uma grande similaridade entre milho e cana. As prolaminas, que se acumulam no endosperma dos cereais, podem ser agrupadas em classes estruturalmente distintas, denominadas de a-, ß-, ?- e d-prolaminas. Quase a totalidade dessas classes de prolaminas são encontradas nas três espécies, mas em milho existem duas classes de a-zeinas, as de 22KD e as de 19KD. Sorgo possui apenas a-kafirinas de 22 KD, enquanto a cana, como o milho, possui as a-prolaminas de 22 e 19KD, as quais denominamos caneinas. O alinhamento das seqüências de aminoácido das a-zeínas, a-caneinas e a-kafirinas revelou que em cana e milho, o sexto domínio, de uma estrutura composta por 10 domínios a-helices que caracterizam as a-prolaminas de 22 KD, está ausente nas a-prolaminas de 19KD. Uma vez que as a-prolaminas de 22KD estão presentes em sorgo, e em outras espécies da tribo Andropogoneae, como o Coix, nós postulamos que as a-prolaminas de 19KD foram origindas pela deleção do sexto domínio das a-prolaminas de 22KD, ocorrida em uma linhagem acestral de Saccharum. Saccharum e Sorgo divergiram entre 8-9 milhões de anos, quando apenas as a-prolaminas de 22KD existiam. As caneínas de 19KD foram originadas após esta divergência o que sugere que o milho herdou a a-zeina de 19KD de Saccharum, após a hibridização interespecífica entre Saccharum e outro progenitor Androponeae n=5 / Abstract: The sequencing of ESTs (expressed sequence tags) and its organization in databases constitute powerful tools to identify genes of interest in certain tissues and/or cell types. In this work we have created a database of ESTs expressed in diverse maize tissues called MAIZEST. MAIZEST contains 227,431 ESTs coming from over 30 different maize tissues, 64,357 of which coming from developing endosperm. The analysis of MAIZEST database leaded to the identification of 4,032 transcripts preferentially expressed in endosperm, and its annotation revealed a great variety of new genes involved in endosperm metabolism and development. We used the information of the genes encoding the maize storage protein, zein, to comparre with the storage protein genes from sorghum and sugarcane. Genetic and molecular evolution studies have suggested that maize is the product of a tetraploid event which occurred by the interspecific hybridisation of two n = 5 ancestors. It has been proposed that maize may have originated through a segmental allotetraploid event occurred at 11.5 Mya or by a single duplication event occurred about 4.8 Mya. However, the progenitors of the allotetraploid have not as yet been identified. Here we show that one of the progenitors belongs to the saccharum lineage from which modern sugarcane originates. Comparing patterns of the seed storage protein, prolamins, of maize, sorghum and sugarcane, a striking similarity was found between maize and sugarcane. Prolamins, which accumulate in the endosperm of cereal seeds, can be grouped into structurally distinct classes named a-, ß-, ?- and d-prolamins. Almost all prolamin classes are present in maize, sorghum and sugarcane, but in maize there are two molecular weight distinct classes of a-prolamins, the 22 KD and the 19 KD a-zeins. Sorghum possesses only the 22 KD a-kafirin while sugarcane possesses both the 22KD and the 19 KD a-prolamins, which we called caneins. Alignment of the 22 and the 19 KD amino acid sequences revealed that both the 19 KD a-zein and the 19 KD a-canein lack the 6th a-helices domain from the 10 a-helices domains found in the 22 KD a-prolamins. Since the 22 KD a-prolamins are present in sorghum and in the more ancient Andropogoneae species Coix, we postulate that the 19 KD a-prolamins originated, by a deletion of the 6th a-helices domain of the 22 KD a-prolamins, in the saccharum lineage. Saccharum and sorghum diverged about 5-9 Mya, when only the 22 KD a-prolamins existed. The 19KD a-canein originated after this divergence. Therefore, maize inherited the 19KD a-zein from saccharum after the interspecific hybridisation between saccharum and another n = 5 Andropogoneae progenitor / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

Os cereais representam importantes fontes de proteína para alimentação humana e animal. Entretanto, são caracterizados pela baixa qualidade nutricional de suas proteínas devido à composição desbalanceada de aminoácidos, causada pelo excesso dos aminoácidos prolina e glutamina e deficiência de lisina, treonina e triptofano. As proteínas de reserva prolaminas constituem 50% do conteúdo total de proteínas no endosperma e são as principais responsáveis por tais características nos cereais. As informações sobre o metabolismo de lisina e o acúmulo de proteínas de reserva no endosperma vêm sendo utilizadas para desenvolver e aplicar estratégias em programas de melhoramento de plantas que visam suprir a deficiência de lisina encontrada nos cereais. Lange e colaboradores (2007) relataram a produção de linhagens transgênicas de cevada com padrão de proteínas de reserva alterado e que apresentaram incremento no teor de lisina e outros aminoácidos essenciais. O presente trabalho teve como objetivo identificar os mecanismos responsáveis pelas alterações observadas. Para tanto, avaliou-se a atividade das enzimas envolvidas na síntese e degradação de lisina, além da caracterização das proteínas de reserva e sua composição de aminoácidos. Observou-se redução na fração protéica das prolaminas (5,91 a 18,34%) e incrementos compensatórios na fração protéica das glutelinas (2,16 a 6,52%). As demais frações apresentaram respostas variáveis dependendo do evento avaliado. Além disso, a composição de aminoácidos foi alterada nas diferentes frações protéicas. As prolaminas exibiram incrementos nos teores de lisina (1,79 a 49,13%), treonina (5,04 a 22,60%) e metionina (13,57 a 45,38%), enquanto que as globulinas aumentaram principalmente o conteúdo de metionina (32,30 a 142,56%). Para os aminoácidos solúveis, foram observados incrementos na ordem de duas a três vezes de histidina, lisina, fenilalanina e metionina. A análise das enzimas envolvidas no metabolismo de lisina revelou que ocorreram alterações nas três principais enzimas da via do ácido aspártico. A enzima aspartato quinase (AK) apresentou aumentos na atividade (4,44 a 47,27%), entretanto, foi mais sensível a inibição causada por lisina. A enzima dihidrodipicolinato sintase (DHDPS) também apresentou incremento na atividade (1,50 a 66,32%), mas diferente da AK, foi menos sensível à inibição causada por lisina. A enzima homoserina desidrogenase (HSDH), a qual compete o substrato ASA com a enzima DHDPS, exibiu redução na atividade (3,36% a 28,80%) (exceto um evento de transformação) e foi menos sensível a inibição causada por treonina. Embora as enzimas envolvidas na degradação de lisina também foram alteradas, os resultados foram variáveis para os diferentes eventos. Para aqueles que foram observados redução na atividade da enzima lisina cetoglutarato redutase (LOR), foi também verificado para enzima sacaropina desidrogenase (SDH), mas na ordem de duas vezes, sendo válido para aqueles que apresentaram incremento. Este trabalho mostrou que a alteração no padrão de proteínas de reserva ocasionou mudanças no metabolismo de aminoácidos, neste caso a lisina, para suprir a demanda necessária para incorporação em proteínas de reserva / Cereals represent an important source of protein to human food and animal feed. However, they are characterized by low nutritional quality of proteins due to the unbalanced composition of amino acids, caused by the excess of the amino acids proline and glutamine and deficiency of lysine, threonine and tryptophan. The prolamin storage proteins constitute 50% of the total protein content in the endosperm and is primarily responsible for these characteristics in cereals. Information on the metabolism of lysine and accumulation of storage proteins in endosperm have been used to develop and implement strategies in plant breeding programs that aim to address the deficiencies found in cereals. Lange and coworkers (2007) reported the production of transgenic lines of barley with a pattern of storage proteins that showed altered and increase in the levels of lysine and other amino acids. This study aimed to identify what were the mechanisms responsible for observed changes. For this, we evaluated the activity of enzymes involved in synthesis and degradation of lysine, besides the characterization of storage proteins and their amino acid composition. There was a reduction in the prolamin protein fraction (5.91 to 18.34%) and compensatory increases in the glutelin fractions (2.16 to 6.52%). The other fractions had variable responses depending on the event evaluated. Moreover, the amino acid composition was changed in the different protein fractions. Prolamins exhibited increases in levels of lysine (1.79 to 49.13%), threonine (5.04 to 22.60%) and methionine (13.57 to 45.38%), whereas increases were mainly globulins content of methionine (32.30 to 142.56%). With respect to soluble amino acids, increases were observed in the order of 2-3 fold of histidine, lysine, phenylalanine and methionine. Analysis of enzymes involved in lysine metabolism showed that changes in three key enzymes of the pathway of aspartic acid. The enzyme aspartate kinase (AK) showed increase in activity (4.44 to 47.27%), however, was more sensitive to inhibition by lysine. The enzyme dihydrodipicolinate synthase (DHDPS) also showed increased activity (from 1.50 to 66.32%), but unlike the AK, was less sensitive to inhibition by lysine. The enzyme homoserine dehydrogenase (HSDH) that competes for the substrate ASA with the DHDPS, exhibited reduced activity (3.36% to 28.80%) (an exception one transgenic line) and was less sensitive to inhibition by threonine. The enzymes involved in degradation of lysine were also changed, though the results varied for different events. Those who observed decreased activity of the enzyme lysine ketoglutarate reductase (LOR) was also found for enzyme saccharopine dehydrogenase (SDH), but the order of twice, which was valid for those who had increased. This study showed that the change in the pattern of storage proteins produced changes in amino acid metabolism, in this case lysine, to supply the demand needed for incorporation into storage proteins.

L’autophagie est un processus vésiculaire des organismes eucaryotes permettant de véhiculer au sein d’autophagosomes des protéines dysfonctionnelles et/ou des organites défectueux qui sont apportés à la vacuole pour y être dégradés. Les acides aminés et les squelettes carbonés ainsi générés pourront ensuite être exportés vers le cytosol et recyclés. Ce travail de thèse a consisté à identifier les rôles que joue l’autophagie au cours du développement et de la germination de la graine d’Arabidopsis thaliana. Dans une première partie, les graines du mutant d’autophagie atg5 ont été caractérisées d’un point de vue morphologique et leur remplissage en molécules de réserve a été étudié. Il a notamment été montré que la graine mutante présente une maturation accélérée et accumule plus de protéines que la graine sauvage. Dans une seconde partie, l’expression de gènes ATG8 a été mise en évidence au cours du développement de l’embryon, dans le phloème de la silique et du funicule, dans les téguments externes et internes, ainsi que dans l’endosperme de la graine. L’activité autophagique a été visualisée par l’observation en microscopie de structures autophagiques dans l’embryon en développement. Enfin, dans une dernière partie, les rôles de l’autophagie au cours de la germination ont été étudiés via le suivi de la mobilisation des molécules de réserve chez le mutant atg5, comparativement à des graines sauvages. Il a ainsi été montré que la graine mutante présente un défaut de mobilisation des protéines. Les résultats obtenus montrent donc que l’autophagie jouerait différents rôles dans la graine, notamment dans sa maturation et son vieillissement, dans l’apport des nutriments depuis la plante mère jusqu’à l’embryon, et encore dans la constitution des réserves au cours du développement, puis leur mobilisation après la germination. / Autophagy is a vesicular process of eukaryotic organisms, which consists of the transport of dysfunctional proteins and/or defective organelles within auto phagosomes toward the vacuole in order to be degraded. The generated amino acids and carbon skeletons are transported to the cytosol and recycled. The aim of this thesis work was to identify the roles of autophagy during seed development and germination in Arabidopsis thaliana. In the first part, seeds of the atg5 autophagy mutant have been morphologically characterized in order to study the accumulation of storage molecules. We demonstrated that atg5 mutant seeds are affected by an accelerated maturation and accumulate more proteins than wild type seeds. In a second part, the expression of ATG8 genes has been exhibited during the embryo development, into the phloem of silique and funiculus, in the outer and the inner integument, and in the seed endosperm. Autophagic activity has been visualized by microscopy observation of autophagic structures in the developing embryo. Finally, in the last part, the roles of autophagy during germination have been studied by monitoring the mobilization of storage molecules in the atg5 mutant seeds and compared with the wild type. We thus established that mutant seeds are affected by a defect in protein mobilization. These results show that autophagy may play several roles in seeds, for instance in the ageing and maturation processes, in the transport of nutrients from the mother plant to the embryo, or in the constitution of storage compounds during seed development and their mobilization after germination.

Although work on the prolamins of pearl millet (Pennisetum glaucum) has revealed partial amino acid sequences for several alcohol-soluble storage proteins (Marcellino et al. 2002) the genes encoding them have not yet been isolated. We constructed a cDNA library from developing P. glaucum seed tissue and screened it using maize zein gene probes to isolate several α-prolamin-like gene sequences. The proteins encoded by these genes generally fall into two size classes: 20.6kD and 27.1kD, which we call the 21kD and 27kD pennisetins. Both proteins are similar in composition and sequence to α-prolamins from maize, sorghum and Coix. Protein bodies that appear as occlusions within the rough ER of P. glaucum endosperm cells are also very similar in size and shape to maize and sorghum protein bodies. The SDS-PAGE gel of the alcohol soluble protein fraction shows two distinct bands in the region corresponding to the 19kD and 22kD of maize α-zein. Both classes of pennisetins appear to be more similar to the 19kD α-zein of maize than to the 22-kD α-zein judging from sequence homology and maize antibody binding. Phylogenetic reconstruction suggests that P. glaucum may have branched from maize prior to the gene duplication which created the 19kD and 22kD α-zein families.

Le blé dur, du fait de sa vitrosité et de sa richesse en protéines est particulièrement adapté àla fabrication des pâtes alimentaires. Cette céréale, largement cultivée dans le bassinméditerranéen, est fréquemment soumise à des stress hydriques et thermiques. Les objectifsde cette thèse ont été d'approfondir les connaissances sur les modalités d'accumulation etd'assemblage des protéines de réserve au cours du développement du grain. Un suivi fin duremplissage du grain, de la morphologie des corps protéiques, des changements d'état redox etde la distribution en taille des polymères de gluténines a été réalisé. L'influence, sur cesdifférents paramètres, de températures élevées appliquées à différents stades dedéveloppement du grain a été étudiée.En privilégiant l'accumulation des protéines au détriment de l'amidon, le stress thermiquejoue un rôle non négligeable dans l'obtention de grains vitreux et à fortes teneurs en protéines.L'arrêt de la croissance du grain, observé à 45% de teneur en eau, est précédé de la mise enplace de la matrice protéique. L'accumulation de la matière sèche apparaît étroitement liée à ladynamique de l'eau, confirmant le lien entre teneur en eau et poids final du grain. Au cours duremplissage du grain, les températures élevées exercent un effet significatif sur la formationdes polymères de gluténines insolubles dans le SDS. Leur formation n'intervient qu'en fin dedessiccation et en deçà de 30% de teneur en eau. La durée de cette phase ultime d'oxydation etd'assemblage des polymères de gluténines a été reliée à la teneur en protéines.L'évolution du statut redox du grain montre qu'une accumulation massive de polymères degluténines intervient juste avant l'entrée en dessiccation du grain qui coïncide avecl'effondrement de l'ascorbate dans le grain. Coïncidant avec cette oxydation manifeste, unphénomène de glutathionylation des protéines a été détecté avec une rupture de l'activité de laCAT. Au cours de la dessiccation, les activités de la SOD et de la GR augmentent de façonsignificative. Cette synthèse tardive pourrait se produire au niveau de la couche à aleurone etdu germe en réponse à l'oxydation massive de l'albumen.Enfin, nous avons analysé le couplage entre oxydation des thiols protéiques et croissance entaille des polymères de gluténines. Très précocement, les sous-unités de gluténiness'assemblent sous forme d'oligomères partiellement réduits. Durant le remplissage du grain,une oxydation massive des thiols intervient avec la formation de polymères majoritairementcomposés de SG-HPM sur lesquels viendraient se fixer les SG-FPM. Au cours de la dessiccation,la taille des polymères augmente. Les structures polymériques formées au cours del'élaboration du grain possèdent un nombre de cystéines réduites élevé et incompatible avecun modèle d'assemblage linéaire. Les résultats obtenus nous permettent de proposer unrepliement tardif des SG-FPM, postérieur à leur insertion dans les assemblages oligomériques.Ils nous amènent à mettre en avant le rôle du glutathion comme co-facteur de la genèse despolymères, à l'opposé des thèses classiques qui en font plutôt un inhibiteur de la croissance entaille des polymères de gluténines. / Durum wheat is particularly recognized as the most suitable raw material for pasta makingdue to its vitreousness and its high protein content. This cereal is commonly grown inMediterranean environment and then frequently submitted to high temperature and waterstress. The aim of this thesis was to better understand the accumulation and assembly ofstorage proteins during the grain development. Evaluation of the grain filling, the morphologyof protein bodies, the redox status and the size distribution of glutenin polymers were carriedout. The effect of high temperatures applied at different stages of the grain development onthese parameters was studied.By favouring protein accumulation at the expense of starch, heat stress appeared essentialto obtain vitreous grains and high protein content. The arrest of grain growth observed at 45%grain water content is preceded by the formation of a continuous protein matrix. The dry massaccumulation is closely related to water dynamics, confirming the link between water contentand final grain weight. During grain filling, high temperatures have a significant effect on theformation of SDS-insoluble glutenin polymers. It occurred when grain water concentrationdropped below 30%. The duration of this final stage of oxidation and assembly of gluteninpolymers was related to protein content.Changes in the grain redox status showed an accumulation of glutenin polymers mainlyoccurring before the onset of grain desiccation phase and coinciding with the ascorbateoxidation. This clear oxidation coincided also with the glutathionylation of proteins and thedrop of the CAT activity. During desiccation, the activities of SOD and GR increased significantly.This late synthesis could occur in the aleurone layer and germ in response to massive oxidationof the endosperm cells.Finally, we evaluated the coupling between protein thiol oxidation and size increase ofglutenin polymers. In the early stages, glutenin subunits assembled as oligomers partiallyreduced. During grain filling, a strong thiol oxidation took place with the formation of polymersmainly composed of HMW-GS associated with LMW-GS branching. The polymer size increasedduring desiccation. The polymeric structures obtained during the grain development presenteda high number of reduced cysteines inconsistent with a linear concatenation model ofassembly. It allowed us to propose that the folding of LMW-GS is a late event, subsequent totheir insertion into oligomeric assemblies. These results highlighted the role of glutathione asco-factor in the polymers formation, contrasting with the common assumption that considers itas an inhibitor of the size increase of glutenin polymers.

O arroz está entre os cereais mais consumidos do mundo. A desnutrição por micronutrientes como zinco (Zn) e ferro (Fe) é generalizada, especialmente em populações pobres, cujas calorias diárias são obtidas principalmente pelo consumo de cereais. Apesar de o arroz ser diferenciado por possuir maior quantidade de lisina em seus grãos em comparação ao trigo, milho ou cevada, a sua quantidade de proteínas representa somente de 8-9% do grão. Sendo o Zn é um micronutriente correlacionado com o funcionamento vital de plantas e animais, e estando relacionado direta e indiretamente com a síntese e funcionamento de várias proteínas, a proposta deste trabalho foi analisar a possível correlação da quantidade de Zn encontrado no grão com o perfil das proteínas de reserva, aminoácidos solúveis, enzimas envolvidas no metabolismo do ácido aspártico e do fator antinutricional ácido fítico, nos grãos de diferentes genótipos de arroz, avaliando primordialmente as diferenças inerentes de cada material genético a fim de agregar conhecimentos que possam ser utilizados como ferramenta para o melhoramento dessa espécie, visando ganho nutricional em seu grão. Para tanto, foram executados dois experimentos distintos. No primeiro foram avaliados três genótipos, os quais foram expostos à adubação foliar com sulfato de zinco, nas concentrações 0, 2 e 4 kg.ha-1 de ZnSO4.7H2O, buscando aumentar-se o nível de Zn nos grãos. No entanto não se verificou incremento significativo do metal na porção de interesse alimentar, sendo a variação encontrada nas concentrações de Zn em grãos integrais e polidos foram inerentes aos genótipos. Ainda assim, foram verificados os outros aspectos nutricionais, para os quais os resultados apontaram diferenças significativas entre os tipos de grãos analisados, sendo indicado o consumo de grão integral ao polido, destacando-se o genótipo Primavera, que apresentou maior concentração de proteínas e menor quantidade de fitato. Esse experimento ainda confirmou que o catabolismo da lisina é a principal via de modulação para o acréscimo desse aminoácido no grão. Para a execução do segundo experimento foram utilizados 16 genótipos cultivados no Brasil, buscando caracterizar a diversidade genética em relação às principais propriedades do grão relacionadas à qualidade nutricional humana. Os dados mostraram que apesar do Zn estar correlacionado positivamente com fitato, não há relação direta entre a concentração desse elemento com a síntese e concentração de aminoácidos e nem com a concentração total de proteínas de reserva. No entanto, com os resultados obtidos para grão integral em relação às características nutricionais, permite apresentar os genótipos Carolino e Goiano como os de melhor desempenho, além de ressaltar a importância da camada da aleurona do grão, já que está relacionada com todas as características nutricionais importantes para um projeto de bioforticação, indicando que estudos devem ser intensificados para entender o transporte de nutrientes até o interior do endosperma, ou ainda, que o consumo de arroz integral deve ser mais reforçado. / Human malnutrition due to low intake of the micronutrients zinc (Zn) and iron (Fe) is relatively common in poor population, who uses cereals as main sources of calories. Currently, rice is one of the most consumed cereals worldwide but, despite its high lysine concentration in comparison to other cereal crops, rice grains have low protein contents (8-9%). Therefore, the current work aimed to evaluate the influence of additional Zn supply on amino acid profile, storage proteins, macro- and micronutrients, as well as on phytate concentration, and on activity of enzymes from lysine metabolism in grains of different rice genotypes, primarily evaluating the inherent differences of each genetic material in order to aggregate knowledge like a tool for the improvement of this species, aiming nutritional gain in its grain. For this purpose, two different experiments were performed. First of all, three genotypes were evaluated and exposed to foliar fertilization with zinc sulfate, in the concentrations 0, 2 and 4 kg.ha-1 of ZnSO4.7H2O, seeking to increase the level of Zn in the grains. However, there was no significant increase of the metal in the grain, and the variation found in Zn concentrations in brown and polished grains was according to the genotypes. Nevertheless, the other nutritional aspects were verified, for which the results indicated significant differences between the types of grains analyzed, being indicated the consumption of brown grain to instead polished one, and the genotype \'Primavera\' stood out with higher concentration of proteins and smaller amount of phytate. This experiment also confirmed that lysine catabolism is the main modulation pathway to increase this amino acid in the grain. For the second experiment 16 genotypes cultivated in Brazil were used to characterize the genetic diversity in relation to the major grain properties related to human nutritional quality. Zinc concentration was positively correlated to phytate, but no direct association between Zn and synthesis and concentration of amino acids or storage proteins was detected. Based on results, \'Carolino\' and \'Goiano\' rice cultivars presented the best performance. Moreover, due to the relevance of aleurone layer in retaining important nutritional features in rice grains, biofortification programs should investigate nutrient transportation from this region to endosperm in order to achieve better results.

Lectins and hemagglutinins are carbohydrate binding proteins present in a diversity of organisms including humans, vertebrate and invertebrate animals, plants, fungi, and bacteria. They are usually the abundant storage proteins in leguminous plants. They display a host of biological activities such as antitumor, antifungal, antiviral, insecticidal, and antibacterial activities. / The biological properties of isolated proteins, including hemagglutinating, antifungal, anti-tumor and HIV-1 reverse transcriptase inhibitory activities, were examined. Their biochemical and biological properties were compared with other purified proteins. / The seeds contain an abundance of proteins, some of which are storage proteins but may play a role of protection from pathogenic microbes and phytophagous insects. Antifungal peptides/proteins, antiviral proteins, ribosome-inactivating proteins, proteinase inhibitors, chitinases, proteinases, and defensins, are some examples of the myriad of seed proteins. The aforementioned proteins are collectively called plant defense proteins in view of their antipathogenic activities. These antifungal proteins exhibit a wide range of molecular masses and amino acid sequences. / Two lectins with potentially exploitable activities were purified from Capparis spinosa seeds and Hibiscus mutabilis seeds, respectively. A hemagglutinin was isolated from Phaselous vulgaris , cultivar "French bean 35", and detailed apoptotic pathway in breast cancer cells, MCF-7 cells, was investigated. A novel dimeric beta-lactoglobulin-like antifungal protein and an antifungal amidase were purified from Passiflora edilus seeds and Peltophorum pterocarpum, respectively. / Lam, Sze Kwan. / Adviser: Tsi Bun Ng. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 188-204). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.