Oldenburg, Universiẗat, Diss., 2003. / Dateiformat: zip, Dateien in unterschiedlichen Formaten.

Oldenburg, Universiẗat, Diss., 2002.

Dans l’espèce équine, les mécanismes de la fécondation sont mal connus. Afin de mieux les comprendre, nous avons développé une approche comparative entre les équins (faibles taux de fécondation in vitro : 0-60%) et les porcins (forts taux : 80–90%). Notre but était d’identifier les différences et les similitudes entre ces deux modèles opposés afin de mettre en évidence des éléments clés de la fécondation. Nous avons montré que 1) la zone pellucide de l’ovocyte est un élément clé dans l’interaction des gamètes, 2) sa composition et sa structure sont différentes entre les espèces équine et porcine, 3) chaque vertébré a son propre jeu de protéines permettant l’interaction des gamètes, 4) la ß-1,4-galactosyltransferase n’est pas nécessaire pour cette interaction dans les espèces équine et porcine, contrairement aux bovins, 5) les sécrétions de l’oviducte participent aux mécanismes de la fécondation équine, mais les protéines impliquées n’ont pas pu être identifiée. / In equine species, the mechanisms of fertilization remain largely enigmatic. In order to clarify these mechanisms, we have developped a comparative strategy between equine (low in vitro fertilization rates: 0- 60%) and porcine (high rates: 80-90%) species. Our objective was to identify differences and similarities between these two opposite models in order to highlight key components of fertilization. We showed that 1) the zona pellucida is a determining element in gamete interaction, 2) its composition and its structure are different between equine and porcine species, 3) each vertebrate has its own protein-set involved in gamete interaction, 4) the ß-1,4-galactosyltransferase is not necessary for gamete interaction in the horse and the pig, contrary to the bovine, 5) the secretions of oviduct cells take part in the mechanism of equine fertilization, but the proteins involved remain to be identified.

Les glycosyltransferases sont les enzymes responsables de la synthèse d'oligosaccharides, de polysaccharides et de glycoconjugués. Elles catalysent le transfert d'un saccharide à partir d'un substrat donneur, en général un nucléotide sucre, vers un substrat accepteur. Le mécanisme de réaction des glycosyltransférases peut avoir lieu avec une inversion ou une rétention de l'anomérie de liaison du sucre transféré. De nombreuses incertitudes subsistent au sujet du mécanisme des glycosyltransférases transférant avec rétention de l'anomérie. L'élucidation de ce mécanisme aiderait à la conception d'inhibiteurs ciblés afin de soigner des maladies allant des infections virales et bactériennes au cancer. De nombreuses protéines sont actives à l'état cristallin, ce qui fait de la cristallographie aus rayons X un outil de choix pour étudier le mécanisme d'enzymes. La « cristallographie cinétique » est un terme qui regroupe l'ensemble des techniques permettant d'initier une activité biologique in crystallo pour générer et piéger une quantité significative d'un état intermédiaire de réaction, afin de résoudre sa structure par cristallogrpahie aux rayons X. Le but de mon projet était d'étudier le mécanisme catalytique d'une glycosyltransférase transférant avec rétention de l'anomérie, par cristallographie cinétique. De cette façon, j'ai étudié une enzyme du groupe sanguin responsable du transfert d'un galactose à partir d'UDP-Gal vers l'antigène H. J'ai étudié les effets de la cryoprotection sur la structure de la protéine, et j'ai effectué les études préalables nécessaires à l'application de deux techniques issues de la cristallographie cinétique à l'étude de ces enzymes : « Déclencher-tremper »et « Tremper-déclencher ». / Glycosyltransferases are a large class of enzymes responsible for the synthesis of oligosaccharides, polysaccharides and glycoconjugates. They catalyze the transfer of a saccharide from a donor substrate, usually a nucleotide sugar, to an acceptor. Glycosyltransferase reactions can occur with either retention or inversion of the anomeric configuration of the transferred sugar. Many uncertainties remain concerning the catalytic mechanisms of retaining glycosyltransferases even though the elucidation of this mechanism would help in the rationale design of potent inhibitors to treat diseases ranging from viral and bacterial infections to cancer. Many proteins function in the crystalline state which makes X-ray crystallography a potential powerful tool for studying enzymatic mechanisms. ‘Kinetic crystallography' is a term coined to name the ensemble of techniques to initiate a biological turnover in crystallo in order to generate and trap a significant amount of a given intermediate reaction state, and then solve its X-ray structure. The aim of my project was to investigate the catalytic mechanism of a retaining glycosyltransferase, by kinetic crystallography methods. In this way, I studied a human blood group synthase responsible for the transfer of a galactose from UDP-Gal to the H antigen. I investigated the effects of the cryoprotectant on the structure of the protein, and I made preliminary studies to apply two kinetic crystallography techniques to the enzyme: freeze-trigger and trigger freeze experiments.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第25008号 / 医科博第155号 / 新制||医科||10(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 伊藤 貴浩, 教授 藤田 恭之, 教授 伊藤 能永 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Together with the advancements in animal cloning, the ability to efficiently target specific genes in somatic cells would greatly enhance several areas of research. While it has been possible for quite some time to target specific genes in the germ cells of mice, the advancements in somatic cell gene targeting has been slowed for two main reasons. First, the finite lifespan of somatic cells, due mainly to the inability of the somatic cells to regenerate or maintain their telomeres, poses a major problem given the lengthy selection process needed to identify a targeting event. The second problem is the overall inefficiency of homologous recombination. A double strand break or introduction of foreign DNA into a cell can be processed either through the homologous recombination or non-homologous end joining pathways. Of these two, non-homologous end joining is dominant in somatic cells. A two plasmid recombination system was used to study the effects of the manipulation of several non-homologous end joining and homologous recombination pathway molecules on the rates of homologous recombination in porcine fetal fibroblasts. In addition, the effect of telomerase expression, cell synchrony, and DNA nuclear delivery was examined. Results indicate a strong positive relationship between inactivation of p53, cell synchronization, and efficient DNA nuclear delivery in enhancing the rate of homologous recombination. These findings were then applied to an actual locus in the pig, the α1,3 galactosyltransferase gene. Results from these transfections are compared to published accounts of successful targeting at this locus and possibilities for the differences found are discussed.

Mutants showing developmental hyper-responsiveness to limited light were screened and designated as seedlings hyper-responsive to light (shl). These mutants showed an etiolated phenotype similar to wild type in the dark, yet had shorter hypocotyls, larger cotyledons, and more advanced development of true leaves than wild type in low light. The SHL genes act (genetically) as light-dependent negative regulators of photomorphogenesis, possibly in a downstream signaling or developmental pathway that is shared by the major photoreceptor genes (CRY1, PHYA, and PHYB) and other photoreceptors (CRY2, PHYC, PHYD, and PHYE). shl1 and shl2 were shown to be partially dependent on HY5 activity for their light-hyperresponsive phenotypes. shl1-1 showed a defect in responding to auxin in its root development in both white and yellow light conditions, and showed a defect in responding to auxin in hypocotyl elongation in yellow light. Compared to wild type, both shl1-1 and shl2-2 showed increased hypocotyl length in response to cytokinin in white light. Gibberellin (GA) partially recovered shl1-1 mutant phenotype in yellow light, whereas showed no effect on hypocotyl elongation of shl2-2 in this light condition. These altered responses of shl1-1 and shl2-2 to multiple phytohormones in different light regimes suggests that cross-talks among light and hormones regulate SHL1 and SHL2. One of the SHL genes, SHL2 was cloned by map-based positional cloning and shown to be allelic to the previously identified locus designated murus3(mur3) and katamari1(kam1). MUR3/KAM1 encodes a XyG galactosyltransferase. Sequence analysis demonstrated that our original EMS generated reference allele shl2-2 is probably not a null mutant, therefore the phenotypes of T-DNA insertion null mutant in SHL2, SALK_074435 were studied in different light conditions. Unlike shl2-2, SALK_074435 had a slightly short hypocotyl phenotype in the dark (though not to the extent of the det/cop/fus mutants). A consideration of the phenotypes and molecular lesions of shl2-2 and mur3 alleles, along with the phenotypes of null alleles kam1 and SALK_74435, suggests that SHL2/MUR3/KAM1 may be involved in hypocotyl elongation in low light through the modification of xyloglucan in the plant cell wall, and may play a role in hypocotyl elongation in the dark through proper organization of the endomembrane.

Together with the advancements in animal cloning, the ability to efficiently target specific genes in somatic cells would greatly enhance several areas of research. While it has been possible for quite some time to target specific genes in the germ cells of mice, the advancements in somatic cell gene targeting has been slowed for two main reasons. First, the finite lifespan of somatic cells, due mainly to the inability of the somatic cells to regenerate or maintain their telomeres, poses a major problem given the lengthy selection process needed to identify a targeting event. The second problem is the overall inefficiency of homologous recombination. A double strand break or introduction of foreign DNA into a cell can be processed either through the homologous recombination or non-homologous end joining pathways. Of these two, non-homologous end joining is dominant in somatic cells. A two plasmid recombination system was used to study the effects of the manipulation of several non-homologous end joining and homologous recombination pathway molecules on the rates of homologous recombination in porcine fetal fibroblasts. In addition, the effect of telomerase expression, cell synchrony, and DNA nuclear delivery was examined. Results indicate a strong positive relationship between inactivation of p53, cell synchronization, and efficient DNA nuclear delivery in enhancing the rate of homologous recombination. These findings were then applied to an actual locus in the pig, the α1,3 galactosyltransferase gene. Results from these transfections are compared to published accounts of successful targeting at this locus and possibilities for the differences found are discussed.

No description available.

No description available.