The exine ultrastructure of Williamsoniella coronata Thomas from the Bajocian of Yorkshire (United Kingdom) was investigated with light, scanning electron, and transmission electron microscopy. The pollen averages 16.5 μm alongitsshort axisand24.5μmalongitslongaxis andismonosulcate, and thenonapertural sculpturingisdistinctly verrucate. The pollen wallishomogeneous, and the sulcus membraneiscomposedofthin exine withscattered small granules. The pollen grains differ in exine sculpturing and pollen wall ultrastructure from pollen grains of the bennettitalean taxa Cycadeoidea dacotensis (MacBride) Ward and Leguminanthus siliquosis (Leuthardt) Kraeusel. They are similar todispersed pollen grainsof Granamonocolpites luisae Herbst from the Triassic Chinle Formation of the United States, supporting the bennettitalean affinity of these dispersed pollen grains. The Bennettitales are palynologically characterized by monosulcate "boat-shaped" pollen with a homogeneous or granular pollen wall ultrastructure.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18893号 / 医博第4004号 / 新制||医||1009(附属図書館) / 31844 / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 羽賀 博典, 教授 瀬原 淳子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Liverworts are speciose, morphologically diverse, and members of an ancient lineage that is now recognized as the sister group to all other land plants. Spermatozoid ultrastructural characters have provided insight on interrelationships among plant groups as well as the puzzling placement of some taxa in molecular-based phylogenies. With completion of a comprehensive molecular phylogeny of liverworts based on nuclear, plastid, and mitochondrial sequences, changes in morphology of spermatozoids may be readily tracked across lineages. The research presented herein was conducted to fill in critical data on spermatogenesis in major clades of liverworts and to evaluate the evolutionary changes in these complex cells throughout the phylum. Ultrastructural studies of the locomotory apparatus in mid-stage spermatids and mature spermatozoids were conducted on Aneura pinguis, Scapania nemorea, Calypogeia mulleriana, Bazzania trilobata, and Porella platyphylla. The locomotory apparatus of the taxa examined exhibits the typical liverwort architecture, with a multilayered structure and two staggered flagella that are attached to the spline by dimorphic basal bodies. The locomotory apparatus of Aneura is unique among liverworts in that the two basal bodies are inserted at nearly the same location near the anterior of the MLS. The spermatozoid of all taxa are streamlined and coiled, and contain a long cylindrical nucleus, two mitochondria and a starch-filled plastid. Spermatozoids of Scapania, Porella and Bazzania coil 1.75 revolutions, while Aneura spermatozoids coil nearly 4 revolutions. The plastid terminates the spermatozoid in all but Aneura, where the plastid and nucleus overlap to the terminus. A data matrix was compiled from published data and the present studies, and a list of 21 characters scored for 11 of the most completely studied taxa. Representative taxa were selected from all major clades within the liverwort phylogeny, as well as one moss, one hornwort, and two tracheophytes as outgroups. Mesquite was used to perform an ancestral state reconstruction using maximum likelihood with Mk1and AsymmMk parameter models. Haplomitriopsida taxa (Haplomitrium and Treubia) shared several characters including more than one plastid, more than two mitochondria, wide spline width, and left lateral curve of the lamellar strip; however, no character states were calculated to have a significant proportional likelihood value at the ancestral node. Marchantiopsida taxa (Blasia, Marchantia, Sphaerocarpos) shared several characters including a three microtubules-wide spline aperture, a notch in the lamellar strip, and a right and left taper in shape to the lamellar strip, all of which were supported with significant proportional likelihood values at the ancestral node. Jungermanniopsida taxa (Pellia, Pallavicinia, Aneura, Porella, Bazzania, Scapania) possessed a right to left taper of the lamellar strip and a spline that attached tangentially to the nucleus and were supported by significant proportional likelihood values at the ancestral node. BayesTraits was used to give estimated values of characters for the liverwort ancestor using both the directional and random walk models. Values included a spline width of 48 microtubules, a lamellar strip length of 0.933 µm, and lamellar strip width of 1.427 µm. In addition to possessing features that have not been documented, Aneura spermatozoids share features with distantly related Haplomitriopsida including the absences of a Fibrillenscheide and the lack of a narrow spline shank.

La recherche des marqueurs biologiques de la tendreté a fait l’objet de nombreux travaux chez les animaux producteurs de viande et en particulier les bovins. A l’issue de ces études, une expression différentielle de la protéine Hsp27 entre des groupes de tendreté extrême a été mise en évidence. Cette protéine est présente à un « carrefour » biologique de l’interactome lié à la tendreté. Comprendre les mécanismes d’action de la protéine Hsp27 dans la tendreté de la viande bovine est l'un des défis de recherche dans le domaine de la production de viande. Dans cette optique, mon travail de thèse (2010-2013) avait pour objectif d’analyser le rôle de Hsp27 dans le développement du muscle et son implication dans le déterminisme des caractéristiques des tissus liés à la qualité de la viande. La première étape de ce travail a consisté à produire un modèle de souris présentant une inactivation du gène de la protéine Hsp27 (KO HspB1) et d’analyser leur phénotype comparativement à des témoins. Les souris KO HspB1 sont viables, fertiles et ne présentent aucune anomalie majeure, mais ont un format plus petit que celui de leurs témoins. L’analyse de leurs caractéristiques musculaires par une technique immunohistoligique mise au point spécifiquement (Publication 1) n’a pas révélé de différences. Au niveau ultrastructural, l'observation du muscle des souris par microscopie électronique à transmission a révélé des différences ultrastructurales entre les deux génotypes à T0 post-mortem avec des écarts entre les myofibrilles très espacées chez les souris KO HspB1 et un appareil contractile musculaire moins organisé. Ces différences sont encore plus marquées à T72 heures post-mortem. Ainsi le phénotype musculaire fin des souris KO HspB1 est plus altéré (Publication 2). Une analyse bio-informatique a été réalisée dans l'objectif de compléter la liste des interacteurs de la protéine Hsp27 et des gènes cibles de l’invalidation d’HspB1 susceptibles de participer à des différences de structure du muscle et de la tendreté. Les partenaires ou cibles prédits de Hsp27 sont des protéines impliquées dans différentes fonctions, comme des Heat shock proteines, des régulateurs de l'apoptose, des facteurs de traduction, des protéines du cytosquelette et des antioxydants. Les abondances de 15 protéines ont été quantifiées par Western-bloting dans deux muscles (m. Soleus, m. Tibialis). Elles sont modifiées chez les souris dépourvues de Hsp27 principalement dans le muscle le plus oxydatif. Cette étude démontre l'existence de liens fonctionnels entre Hsp27 et ses cibles prédites qui pourraient participer au phénotype fin des souris (Publication 3). Pour compléter cette étude, une analyse protéomique du muscle Tibialis anterior a été menée en utilisant la technique d’électrophorèse bidimensionnelle couplée à la spectrométrie de masse. La comparaison des protéomes spécifiques de ces deux génotypes a permis de mettre en évidence des profils d’expression différents pour plusieurs protéines. Elle confirme l’effet muscle spécifique du KO et révèle un lien avec le métabolisme du calcium et des Hsps différentes de celles mises en évidence dans le muscle oxydatif (Publication 4). L'ensemble des données issues de cette étude réalisée dans une espèce modèle apporte des connaissances nouvelles susceptibles d’éclairer sur les mécanismes moléculaires impliqués dans l’établissement de la tendreté de la viande bovine. Elle suggère que le statut en Hsp, les processus apoptotiques et la protection contre le stress oxydatif contribuent à l'évolution de l'ultrastructure post-mortem des muscles et à la tendreté de la viande. Ces nouvelles connaissances seront validées ultérieurement sur muscle bovin. / Thanks to genomics, we have previously identified markers of beef tenderness, and computed a bioinformatic analysis that enabled us to build an interactome in which we found Hsp27 at a crucial node. Understanding the role of Hsp27 in the development of muscle and in the determinism of beef tenderness is one of the research challenges in meat production. In this context, my pHDthesis (2010-2013) aimed to analyze the role of Hsp27 in muscle development and its involvement in the determination of the characteristics related to the quality of the meat tissue. In this study, we generated mice devoid of Hsp27 protein by homologous recombination of the HspB1 gene as an animal model. The HspB1-/ - mice were viable and fertile, showing no apparent abnormality but a smaller than their control format. The muscle structure of animals was examined by optical microscopy and transmission electron microscopy. The first approach, made by a developed immunohistochemical classification (Publication 1), did not reveal any differences in the characteristics of muscle fibers (contractile and metabolic type, shape, perimeter, cross-sectional area) but a trend for a higher proportion of small fibers. Different myosin heavy chains electrophoretic profiles were also observed in HspB1-/- mice. At the ultrastructural level, examination of the myofibrillar material showed destructured myofibrils and higher gaps between myofibrils in HspB1-/-, and a greater disintegration of myofibrils at 72h postmortem (Publication 2). We have used a network-based approach for understanding the contribution of Hsp27 to tenderness through the prediction of its interactors related to tenderness. We have revealed the direct interactors of Hsp27. The predicted partners of Hsp27 included proteins involved in different functions e.g. members of Hsp families, regulators of apoptosis, translation factors, cytoskeletal proteins and antioxidants. The abundances of 15 proteins were quantified by Western blotting in two muscles of HspB1-null mice and their controls. We observed changes in the amount of most of the Hsp27 predicted targets in mice devoid of Hsp27 mainly in the most oxidative muscle (Soleus. Our study demonstrates the functional links between Hsp27 and its predicted targets. It suggests that Hsp status, apoptotic processes and protection against oxidative stress are crucial for post-mortem muscle metabolism, subsequent proteolysis, and therefore for beef tenderness (Publication 3). To complete this study, we performed a proteomic analysis of m. Tibialis anterior (glycolytic muscle), using 2D gel electrophoresis, to detect changes in protein abundance subsequent to the invalidation of HspB1 gene. This study confirms the muscle specific effect of HspB1 invalidation and reveals a new list of Heat shock proteins different from those highlighted in oxidative muscle and relationships with calcium (Publication 4). All together, these results provided from a model species showed the very important role of Hsp27 for muscle ultrastructure and revealed its implication in different muscle biological pathways. This provided new elements for understanding the crucial role for Hsp27 in the modulation of the tenderizing process of muscle during meat ageing that will be further examined in beef.

Call number: LD2668 .T4 1979 O26 / Master of Science

La biologie de l'ovocyte canin est très différente de celle des autres espèces mammifères : en particulier, la fin de la maturation ovocytaire dans le follicule se déroule en présence d'une forte concentration en progestérone (lutéinisation préovulatoire) et l'ovulation libère un ovocyte en prophase I, qui n'atteindra le stade métaphase II qu'au bout de 48 à 60 heures de transit oviductal. Parallèlement, les taux de maturation obtenus in vitro chez la chienne sont très faibles. Le but de ce travail était de fournir une description ultrastructurale de la maturation ovocytaire canine in vivo (avant le pic de LH jusqu'à 105 heures post ovulation) et d'y comparer l'évolution des ovocytes mis en maturation in vitro. In vivo, nos résultats en microscopie électronique à transmission montrent que l'ovocyte canin suit globalement le modèle de maturation ovocytaire des mammifères, à l'exception de l'existence d'une période de réactivation transcriptionnelle entre 24 et 48 heures post ovulation. Nous avons mis en évidence, grâce à une étude quantificative de l'incorporation de BrUTP, une activité transcriptionnelle très intense pendant cette période, activité à la fois nucléoplasmique et nucléolaire. Ni le pic de LH ni l'ovulation ne se révèlent suivis de modifications ultrastructurales importantes. Ils ne sont associés respectivement qu'à l'expansion et à la recompaction du cumulus. Les signaux de régulation de la méiose chez la chienne restent donc inconnus. In vitro, l'examen ultrastructural montre que les ovocytes issus d'ovaires d'anoestrus sont très immatures. La comparaison avec les formes observées in vivo indique que les ovocytes qui n'achèvent pas leur méiose in vitro (VG et métaphase I) sont simplement interrompus dans leur maturation et ne présentent pas de formes aberrantes. Quant aux ovocytes ayant atteint le stade métaphase II in vitro, ils montrent un très faible degré de maturation cytoplasmique.

No abstract available. / Thesis (M.Sc.)-University of Durban-Westville, 1986.

This research was an attempt to characterize previously constructed mutants with a specifically altered psbO gene which encodes a 33 kDa protein active in photosynthesis. This polypeptide was believed to function in stabilization of manganese ions during photolysis of water at the photosystem II. The initial phase of this work was concerned with determining the manganese content of the genetically manipulated PS II particles of the photosynthetically active cyanobacteria.We found however, that the results of the isolation procedure for PS II particles of photosynthetically active cyanobacteria as described by Burnap et al. was not reproducible in our research organism. This prevented the chemical characterization of function of these particles as had been planned.In the second phase of the research sequencing of the mutated gene was to be performed for several clones in order to determine the kinds of specific alterations that had been made. The mutated genes had been cloned into both pUC1 20 and pPGV5 vectors which were transformed into Escherichia OR (EQQJi) and the cyanobacterium Synechococcus PCC 7942, respectively.Several attempts were mad o isolate plasmid DNA from both the transformed E QQJI and cyanobacterium. Isolation of pUC120 DNA was not achieved due to the toxicity of the 33 kDa protein product of the psbO gene in sgJj. The pPGV5 plasmid isolation was successful and PCR-sequencing was performed. However, the sequencing did not result in a readable sequence. Instead, banding patterns showed more than one nucleotide per lane. Since pPGV5 contains a strong constitutive promoter, a large amount of mutant protein was being produced. Our findings suggested that transformed cyanobacteria may have been under pressure to revert the altered gene to wild-type. Thus, upon growth of a single colony to a larger volume, a heterogeneous population of cells with different sizes of plasmids may have resulted. Restriction analysis of isolated plasmid DNA confirmed the presence of multiple-sized plasmid molecules. Therefore, this research has shown that the previously constructed mutants are not stable enough to characterize for alterations in manganese binding. / Department of Biology

The 33 kDa manganese stabilizing protein (MSP) has been proposed to provide ligands to stabilize Mn ions in the water lysis reaction of photosystem II of photosynthesis. In previous research site-directed mutagenesis had been performed on regions of the psbO gene encoding two aspartic acid residues of MSP which were thought to have the potential to form carboxyl bridges with Mn ions. The purpose of this research was to analyze these mutants. Plasmids pUC120-33 (#1,3,5,7,9,11,15) containing mutant psbO genes could not be isolated from E.coli because the expressed MSP was toxic to the cells. However, a psbO mutant gene carried in pPGV5-33 (#7) was isolated from E.coli and transformed into cyanobacterium Svnechococcus PCC 7942. Cyanobacterial cells carrying the MSP mutant showed a susceptibility to intensive light (100 footcandles) with a decrease of 30% in the growth rate within the first 100 hours after inoculation. This result suggested a possible function of the MSP in protecting the oxygen evolving complex from intensive light exposure. However, the mutant appeared to revert after this time probably due to homologous gene recombination with the wild type gene. In order to further analyze the function of mutants without recombination occurring, the construction of an MSP deletion was attempted using insertion of a kanamycin cartridge into the middle of the psbO gene. The inactivated psbO gene was transformed into E.coli and transformants were selected by kanamycin resistance. However, plasmid DNA carrying the interrupted genes could not be isolated, probably due to toxicity of the expression product in E.coli cells. Thus, future studies should be directed to reconstruction of a deletion mutant by direct transformation into cyanobacterial cells. Once a deletion mutant has been constructed analyses of the site-directed mutations could be performed in cyanobacteria. / Department of Biology

Em bovinos, o desenvolvimento embrionário é caracterizado pelo surgimento de duas camadas de células distintas, o trofectoderma e a massa celular interna. Esta última sofrerá diferenciação para formar o disco embrionário constituído pelo epiblasto e hipoblasto. Os trabalhos de caracterização morfológica do epiblasto e hipoblasto em bovinos são necessários uma vez que podem ajudar a elucidar as causas de perdas gestacionais, principalmente nos casos de embriões derivados de produção in vitro. Assim, o objetivo deste estudo foi caracterizar ultraestruturalmente o embrião bovino em diferentes fases do desenvolvimento com ênfase nas células do hipoblasto. Para isso, os embriões bovinos com 7, 14 e 16 dias de gestações derivados de técnicas de produção in vitro foram fixados para processamento e realização de microscopia eletrônica de transmissão. De acordo com os resultados obtidos observou-se que os embriões derivados de transferência nuclear de células somáticas e de partenogênese apresentaram grandes modificações na estrutura macro e microscópica. Nestes embriões, o tamanho estava reduzido, a massa celular interna não se apresentou de forma definida. Além disso, as organelas destes embriões, responsáveis por processos de absorção, comunicação, crescimento e metabolismo celular estavam em menor quantidade e tinham modificações quanto à forma, quando comparados aos resultados vistos nos embriões derivados de fertilização in vitro. Assim, verificamos que os blastocistos D7 derivados de transferência nuclear e partenogênese apresentaram graves modificações morfológicas, assim como verificou-se também nas células do hipoblasto dos embriões D14 e D16. / In cattle, the embryo development is characterized by the apperance of two distinct cell layers, the trophectoderm and the inner cell mass. This latter one will siffer a differentiation to form the embryonic disc constituted by the epiblasto and hypoblast. The works on the morphological characterization of the epiblasto and hypoblast in cattle are needed because they may help to elucidate the causes of pregnancy loss, especially in cases of embryos derived from in vitro production. Considering this, the objective of this study was to characterize ultrastrcturally the bovine embryo at different stages of development with emphasis on the hypoblast cells. To do so, bovine embryos at 7, 14 and 16 days of pregnancies derived from in vitro production techniques were fixed for processing and performing transmission electron microscopy. According to the results observed that embryos derived from the nuclear transfer of somatic cells and from parthenogenesis showed significant changes in the macroscopic and microscopic structure. In these embryos, the size was reduced, the inner cell mass has not show a defined shape. Furthermore, the organelles from such embryos, responsible for the absorption processes, communication, growth and cellular metabolism were fewer in number and have changes in shape, when compared to the results seen in embryos derived from in vitro fertilization. Thus, we observed that the blastocyst D7 derived from nuclear transfer and from the parthenogenesis, showed severe morphological changes, as well as for the hypoblastic cells in the D14 and D16 embryos.