Regensburg, Univ., Diss., 2008.

Regensburg, Univ., Diss., 2008.

L'étude de la ramification chez le pois, à partir des mutants hyper-ramifiés ramosus (rms) a permis de mettre en évidence l'existence d'une nouvelle famille d'hormones végétales : les strigolactones, inhibant la ramification des plantes à graines. La découverte de cette hormone végétale ouvre de nouvelles pistes de recherche sur la biosynthèse et la perception de cette nouvelle hormone. Nous avons montré le rôle du gène PsBRC1, codant un facteur de transcription de type TCP et homologue du gène TEOSINTE BRANCHED1 du maïs, dans la voie de signalisation des strigolactones. L’étude de ce gène nous a permis d'avoir une meilleure compréhension de l’interaction entre strigolactones et cytokinines dans le contrôle de la ramification, de la dynamique de la levée de dormance des bourgeons axillaires, et d'effectuer les premières études de relations structure-activité des strigolactones sur l’inhibition de la ramification chez le pois.Nous avons étudié et caractérisé d'autres éléments dans la voie de signalisation. Chez le pois, deux mutants, autres que Psbrc1, ne répondent pas à l’application de strigolactones, rms3 et rms4. Le gène RMS4 code pour une protéine à boîte F. Nous nous sommes focalisés ici sur le mutant hyper-ramifié rms3. Nous avons montré que RMS3 est l'homologue du gène D14 du riz, codant pour une protéine de la superfamille des α-β/hydrolases. Ces protéines peuvent avoir une activité enzymatique et ainsi pourraient modifier les strigolactones en un composé actif. Le récepteur des gibbérellines GID1 appartient aussi à cette famille, RMS3 est donc un bon candidat pour être le récepteur des strigolactones. Nous avons utilisé une strigolactone radiomarquée afin d’étudier le métabolisme de l'hormone. Nous avons découvert que la strigolactone synthétique, 3H-GR24 est clivée en un composé inconnu au contact des racines, indépendamment de l'activité de la protéine RMS3. Ce composé de structure inconnue se retrouve aussi dans la sève du xylème alors que 3H-GR24 y est absent.Outre un phénotype hyperbranché les mutants rms présentent une diminution de la taille de leurs entre-nœuds, qui n'est pas due à l’augmentation de la ramification. Nous avons étudié l'origine du nanisme des mutants déficients en strigolactones et affectés dans la réponse à l’hormone. Des approches génétiques et moléculaires ont été utilisées pour tester une interaction possible entre les strigolactones et les gibbérellines. Nous avons montré que les strigolactones régulaient l’élongation des entre-nœuds indépendamment des gibbérellines.Le pois est un excellent modèle en génétique et en physiologie. Avec le développement de nouvelles techniques à l'INRA (TILLING; UNIGENE : ensemble de plus de 40000 séquences exprimées de pois), nous avons pu identifier de nouveaux gènes de biosynthèse des strigolactones chez le pois et obtenir plusieurs nouveaux mutants de pois. Ces mutants seront essentiels pour les futures études du laboratoire et pourront permettre d'identifier de nouveaux intermédiaires dans la biosynthèse et le métabolisme des strigolactones. / The study of shoot branching in pea, using the high branching ramosus (rms) mutants has highlighted the existence of a new family of plant hormones: the strigolactones, inhibiting shoot branching in seed plants. The discovery of this novel plant hormone opens novel research areas in the deciphering of strigolactone biosynthesis and strigolactone perception. We have shown the role of the pea TCP transcription factor, PsBRC1, the homolog of the maize TEOSINTE BRANCHED (TB1) in strigolactone signaling. The PsBRC1 gene was shown to have a role in integrating strigolactone and cytokinin pathways, and allowed to have a better understanding of the dynamics of bud outgrowth, and to perform the first strigolactone Structure-Activity Relationship studies for branching inhibition in pea. We investigated and characterized other elements in the signaling pathway, including the strigolactone receptor. In pea, two mutants, other than Psbrc1, do not respond to the application of strigolactones, rms3 and rms4. The RMS4 gene encodes an F-BOX protein and here we focused on the high branching rms3 pea mutant. We have shown that RMS3 is the homolog of the rice D14 gene encoding a protein of the α-β/hydrolase superfamily. Consequently RMS3 may have an enzymatic activity to modify strigolactone into an active compound. The gibberellin receptor GID1 also belongs to this family, therefore RMS3 is also a good candidate for the strigolactone receptor. We used a radiolabeled synthetic strigolactone, 3H-GR24, to investigate the metabolism of the hormone. We discovered that the synthetic strigolactone, 3H-GR24 is cleaved in an unknown compound in the root media independently of RMS3 activity, compound which is also found in the xylem sap in contrast to 3H-GR24. The rms mutants exhibit not only a high branching phenotype but also a reduced height which is not due to this high branching. We investigated the origin of the dwarfism of strigolactone-deficient and response mutants in pea. Genetic and molecular approaches have been used to test a possible interaction between strigolactones and gibberellins. We have shown that strigolactones regulate stem elongation independently of gibberellin. Pea is a powerful model plant for genetics and physiology. With the development of new facilities at INRA (TILLING; UNIGENE set of more than 40000 expressed sequences), we were able to identify new biosynthesis genes in pea and to obtain several novel pea mutants. These mutants will be essential for future studies of the laboratory in particular to identify new intermediates in strigolactone biosynthesis and metabolism.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 100-117).

The tautomerase superfamily is composed of a group of proteins characterized by two key features: the N-terminal proline and a beta-alpha-beta-motif. This superfamily has been divided into five families represented by 4-oxalocrotonate tautomerase (4-OT), 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI), cis-3-chloroacrylic acid dehalogenase (cis-CaaD), malonate semialdehyde decarboxylase (MSAD), and macrophage migration inhibitory factor (MIF). Cg10062 is a homologue of cis-CaaD, but has several distinct biochemical properties from cis-CaaD. For example, Cg10062 can be irreversibly inhibited by (R)- or (S)-oxirane-2-carboxylate, whereas cis-CaaD can only be irreversibly inhibited by (R)-oxirane-2-carboxylate. FG41MSAD is a homologue of MSAD, with comparable decarboxylase activity but missing Arg-73 known to be crucial for the MSAD activity. In order to understand the unique biochemical characteristics of Cg10062 and FG41MSAD, we have solved five crystal structures. These crystal structures have established a solid structural basis for understanding the mechanisms of their activities. The eukaryotic protein phosphatases are composed of a group of proteins that are responsible for reversible phosphorylation. The eukaryotic protein phosphatases have been divided into three families, the phosphoprotein phosphatase (PPP) family, the protein phosphatase Mg2+- or Mn2+-dependent (PPM) family and the protein Tyr phosphatase (PTP) family. PDP1 is a member of PPM family. PDP1 is also an important component of the large pyruvate dehydrogenase complex (PDC) which catalyzes the decarboxylation of pyruvate to yield acetyl-CoA with the accompanying reduction of NAD+. In order to understand the mechanism in which it dephosphorylates its target protein we have solved the structure of the catalytic domain of PDP1. Analysis of these structures in the light of their evolutionary contexts enables us to appreciate the unity and diversity of the biological systems at the chemical level and help us solve interesting problems, such as the possible physiological functions for some members within the tautomerase superfamily. / text

Introdução: A rede lisossômica é um complexo de vias metabólicas que influenciam processos como degradação de organelas danificadas ou senescentes, processamento de antígenos, reutilização de aminoácidos essenciais e, em última instância, um sistema de fundamental importância para a fisiologia celular normal. Nesse contexto, os lisossomos possuem uma relevância muito grande, uma vez que é nessa organela que ocorre a destruição das moléculas envolvidas em todos os processos citados acima. Entre as unidades operacionais nos lisossomos estão as hidrolases lisossômicas, que são mais de 50 enzimas com capacidade, em ambiente ácido, de realizar a quebra de substratos específicos. A GlcNAc-fosfotransferase é um complexo hexamérico (J2K2L2) residente na porção cis do complexo de Golgi que realiza a adição de resíduos de manose-6- fosfato nas cadeias de oligossacarídeos das hidrolases lisossômicas. Com ação subsequente, a enzima descobridora realiza a remoção da manose, expõe os resíduos de fosfato e possibilita que as hidrolases sejam reconhecidas pelos receptores de manose-6- fosfato e direcionadas aos compartimentos lisossomais. As subunidades J e K da GlcNAc-fosfotransferase são codificadas pelo gene GNPTAB, localizado no cromossomo 12, constituído de 21 éxons, e a subunidade L pelo gene GNPTG, localizado no cromossomo 16, constituído de 11 éxons. Alterações patogênicas em GNPTAB podem causar as doenças Mucolipidose II ou III alfa/beta e alterações em GNPTG causam a doença Mucolipidose III gama. O defeito genético leva à atividade residual, ou nula, da enzima que acaba por gerar o extravasamento das hidrolases lisossômicas ao meio extracelular e o acúmulo de substratos nos lisossomos. Objetivos: (1) caracterizar as alterações patogênicas em GNPTAB em um grupo de pacientes brasileiros não relacionados com Mucolipidose II ou III alfa/beta, e (2) definir um protocolo de pesquisa molecular para os pacientes brasileiros. Metodologia: É um estudo transversal, com amostragem por conveniência, e inclui pacientes com diagnóstico clínico e bioquímico de Mucolipidose II ou III. Foi extraído DNA genômico dos pacientes a partir de sangue obtido por punção venosa periférica. O gene GNPTAB foi sequenciado através da técnica de Sanger. As alterações do tipo troca de sentido foram analisadas pelos programas de Bioinformática Polyphen2, Sift e Consurf, e as preditas como patogênicas foram pesquisadas em alelos controles brasileiros e analisadas por estudos funcionais, quando possível. A geração de construtos das alterações p.Ser385Leu e c.3503_3504delTC foi realizada por mutagênese sítio-dirigida e a atividade residual destes foi avaliada 24 horas após expressão em células HEK. Para a análise financeira, valores atuais de equipamentos, reagente de biologia molecular e materiais plásticos foram utilizados para estimar o custo de uma extração de DNA, reação de PCR, purificação com PEG8000 e sequenciamento. Resultados: Foram incluídos 13 pacientes (ML II= 8; ML III= 5) e, adicionalmente, de uma mãe de paciente com diagnóstico clínico e bioquímico de ML II. A análise molecular identificou seis alterações patogênicas novas, as c.831delT, c.1763insA, c.1927delAATT, p.Ser385Leu, p.(Asp76Gly) e p.Try1111*. A análise de bioinformática das alterações do tipo troca de sentido as caracterizaram como prejudiciais para a função da proteína e os resíduos 76 e 385 como estrutural e funcional, respectivamente, além de ambos como altamente conservados entre as espécies. A análise funcional dos mutantes p.Ser385Leu e c.3503_3504delTC identificaram atividades residuais de 1,5% e nula, respectivamente. Também foram identificadas outras seis alterações patogênicas previamente descritas. A alteração c.3503_3504delTC foi a que apresentou a maior frequência (40%, n= 10/25 alelos), seguido pela p.Ile403The (12%, n=3/25 alelos). Quanto às relações genótipo-fenótipo, sete pacientes com ML II possuem genótipos combinados de alterações do tipo mudança de fase de leitura e sem sentido, enquanto que os cincos pacientes com ML III alfa/beta apresentam pelo menos uma alteração do tipo troca de sentido, o que evidencia a relação entre alterações que impactam a funcionalidade da proteína e fenótipos mais graves. A análise retrospectiva definiu o protocolo 1.0 que finalizaria o diagnóstico com um custo médio de R$ 338,45, em uma amostra de 25 pacientes. A análise prospectiva do protocolo 2.0, sobre a mesma amostra de pacientes, indicou que o mesmo finalizaria o diagnóstico com o custo médio de R$ 299,80, uma economia de 25%. Discussão/Conclusão: As novas alterações patogênicas descritas nesse trabalho confirmam a alta heterogeneidade alélica do gene GNPTAB. A análise funcional da alteração p.Ser385Leu confirma sua patogenicidade, que está de acordo com o fenótipo ML II do paciente, e evidencia a necessidade de mais estudos a fim de constatar o motivo desse resíduo ser importante para a proteína. A síntese dos protocolos demonstrou ser uma estratégia interessante e economicamente importante, uma vez que diminui os gastos envolvidos para finalizar o diagnóstico molecular. / Introduction: The lysosomal network is a complex of metabolic ways that influence processes like damaged or senescent organelles degradation, antigen processing, essential amino acid reutilization, and, in the last instance, it is important for normal cell physiology. In this context, lysosomes have a great relevance since it is in this organelle that the destruction of the molecules involved in all the processes mentioned above occurs. The operational units in the lysosomes are the lysosomal hydrolases that are more than 50 enzymes with capacity, in an acid environment, to breakdown specific substrates. GlcNAc-phosphotransferase is a hexameric complex (J2K2L2) located in the cis portion of the Golgi complex that performs the addition of mannose-6-phosphate residues in oligosaccharides chains on lysosomal hydrolases. In a subsequent way, the uncovering enzyme removes the mannose residues, exposes the phosphate residues, and enables the recognition of hydrolases by mannose-6-phosphate receptors. The J and K subunits are codified by GNPTAB gene, which is located in chromosome 12 and consists of 21 exons, and the L subunit, encoded by GNPTG gene, that is located in chromosome 16 and consists of 11 exons. The consequences of pathogenic alterations in GNPTAB are Mucolipidosis II or III alpha/beta diseases and alterations in the GNPTG are Mucolipidosis III gamma disease. The genetic defect leads to residual or absent activity of enzyme which ultimately generates an overflow of lysosomal hydrolases to the extracellular environment and accumulation of substrates in lysosomes. Objectives: (1) to characterize, by sequencing of the GNPTAB gene, the pathogenic alterations in a group of unrelated Brazilian patients with Mucolipidosis II or III alpha/beta, and (2) to define a molecular research protocol for Brazilian patients. Methodology: It is a crosssectional study with convenience sampling, and it includes patients with biochemical and clinical diagnosis of Mucolipidosis II or III. The DNA was amplified by PCR technique and sequencing by Sanger technique. All patients in the present study had all exons amplified. The missense alterations were analyzed by Polyphen2, Sift, and ConSurf softwares, and the alterations predicted as pathogenic were studied through research in Brazilian control alleles. The p.Ser385Leu and c.3503_3504delTC were evaluated by site-direct mutagenesis and the residual activity was evaluated 24 hours after expression in HEK cells, through radioactive assays. For cost-price analysis, current values for equipment, molecular biology reagents, and plastic materials were utilized to estimate the cost of DNA extraction, PCR reaction, PEG8000 purification, and sequencing. Results: Of the 13 patients, 8 were clinically diagnosed with Mucolipidosis II and 5 with Mucolipidosis III alpha/beta and, additionally, a mother of one patient with biochemical and clinical diagnosis of Mucolipidosis II was also analyzed. The DNA analysis identified six novel pathogenic alterations in GNPTAB: c.831delT, c.1763insA, c.1927delAATT, p.Ser385Leu, p.(Asp76Gly), and p.Tyr1111*. The bioinformatics analysis of missense alterations were characterized as damaging for protein function, and residues 76 and 385 as structural and functional, respectively, and both as highly conserved among the species. The functional analysis of mutants p.Ser385Leu and c.3503_3504delTC showed the residual activity of GlcNAcphosphotransferase of 1.5% and 0%, respectively. Six others pathogenic alterations previously described were also identified. The alteration c.3503_3504delTC showed the highest frequency (40%, n=10/25 alleles) followed by p.Ile403The (12%, n=3/25 alleles). The retrospective analysis defined the 1.0 protocol that finalized the molecular diagnosis at the cost of R$ 338,45 per sample, in a group of 25 patients. The prospective analysis of 2.0 protocol, in the same patients, indicated that it would finalize diagnosis at the cost of R$ 299,80 per sample, a saving of 25%. Discussion/Conclusion: The novel pathogenic alterations described confirm the high allelic heterogeneity of GNPTAB gene. In the genotype-phenotype relationship, 7 patients with Mucolipidosis II have combined genotype of frameshift or nonsense alterations, or both, and 5 Mucolipidosis III alpha/beta patients have at least one missense alteration, that shows the correlation between alterations that cause impact on the protein function and severe phenotype. The functional analysis of alteration p.Ser385Leu confirms its pathogenicity and makes evident the need of more studies in order to determine the reason this residue is so important for protein function. Protocol synthesis proves to be an interesting and economically important strategy, once it decreases costs to conclude the molecular diagnosis.

A redução eletroquímica de 3,3\'-(1,3- e 1,4-fenileno)-bis-(2-propenoatos de etila), sistemas diolefínicos cujas duplas ligações estão separadas por um anel benzênico, foi investigada em N,N-dimetilformamida (DMF) e acetonitrila (MeCN) e metanol/acetonitrila (MeOH/MeCN) (7:3) (v/v). As eletrólises a potencial controlado em DMF e MeCN conduziram a hidrodímeros cíclicos derivados da ciclopentanona com elevada estereosseletividade, análogos aos produtos formados a partir de ésteres do ácido cinâmico nas mesmas condições experimentais. Ao lado dos hidrodímeros, quantidades variáveis de produtos de polimerização e de hidrogenação das duplas ligações dos grupos etoxicarbonilvinila eram formados. Foram examinados a influência do pH, concentração inicial dos substratos e presença de doadores de prótons (MeOH/MeCN) sobre o curso da redução eletroquímica. A maior complexidade dos produtos de eletrólise no substrato 1,4-substituído quando comparada as produtos do 1,3-substituído pode ser explicada, em parte, pela maior interação eletrônica dos grupos etoxicarbonilvinila através do anel aromático no primeiro. / The electrochemical reduction of ethyl 3,3\' -(1,3 and 1,4-phenylene)-bis-(2-propenoates), diolefinic systems whose double bonds are separated by a benzene ring, was investigated in N,N-dimethylformamide (DMF), acetonitrile (MeCN) and methanol/acetonitrile (MeOH/MeCN) (7:3) (v/v). Controlled potential electrolyses in DMF and MeCN afforded cyclic hydrodimers derivatives of cyclopentanone with high stereoselectivity as are the products formed from cinnamic acid esters under similar experimental conditions. Besides the hydrodimers variable amounts of polymerization and side chain carbon-carbon double bond hydrogenation products were formed. The influence of pH, initial concentration of substrates and the presence of proton donors (MeOH/MeCN) upon the electrochemical reduction was examined. The formation of more complex reaction mixtures in the case of the 1,4-substituted substrate when compared with the 1,3-substituted may be explained, at the least partially, due to a larger electronic interaction between the ethoxycarbonylvinyl groups through the aromatic ring in former.

AMP-activated protein kinase (AMPK) is an important metabolic stress-sensing protein kinase responsible for regulating metabolism in response to changing energy demand and nutrient supply. Mammalian AMPK is a stable aß? heterotrimer comprising a catalytic a subunit and two non-catalytic subunits, ß and ?. The ß subunit targets AMPK to membranes via an N-terminal myristoyl group and to glycogen via a mid-molecule glycogen-binding domain. Here I show that the conserved C-terminal 85-residue sequence of the ß subunit, ß1(186-270), is sufficient to form an active AMP-dependent heterotrimer a1ß1(186-270)?1, whereas the 25-residue ß1 C-terminal (246-270) sequence is sufficient to bind ?1, ?2, or ?3 but not the a subunit. Within this sequence (246-270), two residues were essential for ß? association based on Ala scanning mutagenesis. / Substitution of ß1 Tyr-267 for Ala precludes ß? but not aß association suggesting independent binding requirements. Substitution of Tyr-267 for Phe or His but not Ala or Ser can rescue ß? binding. Substitution of Thr-263 for Ala also resulted in decreased ß? but not aß association. Truncation of the a subunit reveals that ß1 binding requires the a1(313-473) sequence while the remainder of the a C-terminus is required for ? binding. The conserved C-terminal 85-residue sequence of the ß subunit (90% between ß1 and ß2) is the primary a? binding sequence responsible for the formation of the AMPK aß? heterotrimer. The ? subunits contain four repeat CBS sequences with variable N-terminal extensions and the ?1 isoform is N-terminally acetylated. The ?2 subunit can be multiply phosphorylated by protein kinase C (PKC) in vitro, with Ser-32 identified as a minor site. A detailed understanding of the structure and regulation of AMPK will enable rational drug design for treatment of such linked diseases as obesity, insulin resistance and type 2 diabetes.

This thesis presents the formulation and validation of tracking algorithms for vehicular motion for use in active collision prevention in V2V communications. The main objective is to estimate position and velocity of a vehicle based on update from vehicular wireless network. By using vehicular wireless network, the range of position estimation improves when compare to conventional radars and sensors. On the other hand, from a vehicular wireless network point of view regular measurement information update is more difficult to obtain because of packet losses due to interference between communicating vehicles. Our proposed algorithms are based on methods from position tracking termed alpha-beta trackers in aerospace applications with constant rate of information updates, with some modifications to better solve the problem. We present the main algorithms and provide numerical evidence of their accuracy based on simulation data. The modified filters are shown to be computationally efficient (lightweight) and provide sufficient accuracy for estimation of vehicle positions based on information update in a wireless V2V system.

This thesis presents the formulation and validation of tracking algorithms for vehicular motion for use in active collision prevention in V2V communications. The main objective is to estimate position and velocity of a vehicle based on update from vehicular wireless network. By using vehicular wireless network, the range of position estimation improves when compare to conventional radars and sensors. On the other hand, from a vehicular wireless network point of view regular measurement information update is more difficult to obtain because of packet losses due to interference between communicating vehicles. Our proposed algorithms are based on methods from position tracking termed alpha-beta trackers in aerospace applications with constant rate of information updates, with some modifications to better solve the problem. We present the main algorithms and provide numerical evidence of their accuracy based on simulation data. The modified filters are shown to be computationally efficient (lightweight) and provide sufficient accuracy for estimation of vehicle positions based on information update in a wireless V2V system.