As the plasma membrane of the cardiac muscle cell (sarcolemma) plays a major role in the cardiac physiology and homeostasis, the aim of this study is to identify using mass spectrometry (MS) and data base mining the integral and peripheral proteins that are making its composition and to uncover putative novel proteins and to ultimately reveal the differences in protein composition between the sarcolemma of control rat hearts and hearts submitted to ischemia and ischemia-reperfusion to find disease-specific markers. Toward this goal we have developed a new method to purify cardiac sarcolemma using an approach that avoids denaturing conditions and combines subcellular fractionation and immunoadsorption: An initial cardiac sarcolemma preparation obtained by sucrose gradient centrifugation was enriched 27-fold in 5-nucleotidase activity and 5-12-fold in sarcolemmal specific markers. Subsequently, this enriched preparation was incubated with antibodies directed against intercalated disc proteins, N-cadherin and Connexin43, and then immunoadsorbed to superparamagnetic beads (Dynabeads Pan Mouse IgG). Samples from the enriched sarcolemma and immunoabsorbed fractions were separated by 1-D SDS-PAGE and in-gel digested with trypsin. The tryptic fragments were identified, analyzed and assigned to proteins using tandem MS-MS spectrometry in combination with Mascot search software. Out of the 518 identified proteins, 40% were attributed to the sarcolemma and associated cytoskeleton, of which 65% were peripheral proteins and the remainders were either membrane spanning or lipid anchored proteins. Further functional classification of the sarcolemma proteins indicates that the majority (65%) are involved in signaling, trafficking and cell adhesion. The MS analysis also reveals the presence of 71 novel proteins, which are currently being evaluated as relevant candidates for further study. Comparing the sarcolemma and immunoselected intercalated disc preparations; we noticed that the distribution of protein abundance in all of the localization and functional categories follows almost the same trend. This led us to conclude that in order to have an easy, fast and a reliable technique to compare different heart states in terms of sarcolemma protein composition the sarcolemma preparation would serve just as well as an immunoselected preparation with less time, effort and materials. In order to find out differences in protein composition between normal sarcolemma and sarcolemma submitted to global ischemia/ischemia-reperfusion; sarcolemma fractions (SF) obtained from Langendorff rat heart models were analyzed by mass spectrometry. The results of this study revealed that global ischemia induced an increase in calcium binding proteins (i.e. annexins) concomitantly with a decrease in transport proteins (i.e. Na/K ATPase). After ischemia, the signaling proteins category showed a decrease followed by a return to the control level after reperfusion. The amount of G alpha inhibiting 2 protein, caveolin, flotilin and myosin varied significantly during the ischemia/reperfusion protocol.

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The main objective of this study was to determine the distribution of these perforatorial polypeptides in the head of the rat spermatozoon. / Immunoblotting demonstrated that seven perforatorial polypeptides tested in this study, had epitopes in common. Immunogold labeling of spermatozoa showed that the distribution of the 34, 43, 57 and 63 kDa polypeptides corresponded exactly to the regions of the perinuclear theca immunolabeled with the whole perforatorium serum. However, antibodies affinity purified against the 13, 13.4 and 16 kDa polypeptides were restricted in their localization to the thicker apical portion of the perforatorium and to the inner layer of the ventral spur. / These results suggest (1) the perforatorium is biochemically distinct from the postacrosomal sheath with the exception of a restricted region referred to as the ventral spur; (2) there are regional differences in protein composition of the perforatorium, of the outer periacrosomal layer and of the postacrosomal sheath; and (3) that perforatorial polypeptides may not necessarily be restricted to the subacrosomal region, by may also compose portions of the outer periacrosomal layer, and postacrosomal sheath; and (4) Actin is not part of the perforatorium in mature rat spermatozoa.

The lectins Ulex europaeus 1 (UEA 1), Ricinus communis 1 (RCA 1), Wheat Germ Agglutinin (WGA), and Lens culinaris agglutinin (LCA) which are specific for L-fucose, D-galactose, N-acetylglucosamine, and D-mannose respectively, have been used to localize cytochemically the presence of these sugar residues in certain human, mammalian, and amphibian cell types. Thin sections of Lowicryl K4M-embedded human distal colon, rat duodenum, and frog dorsal root ganglion were incubated with UEA 1-gold, RCA 1-gold, WGA/ovomucoid-gold, or LCA-gold. UEA-1, RCA-1, and LCA binding sites were present in the nuclei of human colonic cells, rat duodenal goblet and columnar cells and frog dorsal root ganglion neurons, satellite cells, and Schwann cells. Most of the nuclear binding sites were localized in the euchromatin compartment, while extranuclear labelling was largely restricted to sites known to contain glycoproteins. WGA binding sites were present in the nuclei of rat duodenum columnar cells and frog dorsal root ganglion neurons, satellite cells, and Schwann cells, but no labelling of human colonic cell nuclei was observed. Thus, we report the presence of these sugars in the nuceloplasm of human colonic cells (with the exception of WGA), rat duodenal villous columnar cells, and frog dorsal root ganglion cells examined.

Cres is a member of the cystatin superfamily of cysteine protease inhibitors; however, it differs from typical cystatins in that it is a serine protease inhibitor. In the testis, Cres exhibits expression solely in spermatids; while in the epididymis, Cres is secreted by principal cells of the initial segment to associate with sperm in the lumen. In the cauda region Cres is endocytosed by clear cells. The presence of Cres mRNA in the efferent ducts by RT-PCR analysis is suggestive of its synthesis in the region. In Cres-/- mice, statistically significant reductions were noted in tubule, epithelial, and luminal profile areas in the testis and epididymis, as compared to wild-type mice. Cres -/- mice revealed degenerating germ cells in the testis. Immature germ cells were present in the epididymal lumen and the epithelial principal cells contained large irregularly shaped lysosomes, unlike the small spherical ones of wild type mice, suggesting of lysosomal storage diseases. Taken together the data indicate the importance of Cres for sperm production and maintenance of epithelial integrity of the testis and epididymis. / Cres est un membre de la superfamille des cystatines. Les cystatines sont reconnues pour être des inhibiteurs de protéases de cysteine. Cres diffère des autres membres car il s'agit d'un inhibiteur de protéases de serines. Dans le testicule, Cres est exprimé seulement par les spermatides. Dans l'épididyme, Cres est secrété par les cellules principales du segment initial pour ensuite s'associer aux spermatozoïdes présents dans la lumière. Dans la région de la queue, Cres est incorporé par endocytose dans les cellules claires. La présence de l'ARN messager de Cres dans les canaux efférents, qui a été démontrée par RT-PCR, suggère qu'il est produit dans cette région. Dans les souris Cres -/-, une réduction significative de la taille des tubules, de la lumière et de l'épaisseur de l'épithélium, en comparaison avec les souris contrôles (sauvages), a été observée dans le testicule et l'épididyme. Une dégénération des cellules germinales dans le testicule a aussi été observée chez les souris Cres -/- et des cellules immatures étaient présentes dans la lumière de l'épididyme. De plus, les cellules principales possédaient de larges lysosomes de forme irrégulière, très différents des petits lysosomes sphériques qui sont généralement observés dans l'épididyme. Ces informations indiquent l'importance de Cres dans le maintien de l'intégrité des épithéliums du testicule et de l'épididyme, et donc dans la spermatogenèse et la maturation des spermatozoïdes.

A morphological study was done of Tomes' process, the organic matrix and the hydroxyapatite crystals of enamel. The membrane of Tomes' process where secretion of rod and interrod material occurs is extensively infolded. The interrod secretion site extends circumferentially around the ameloblast and granules fuse with deep membrane infoldings. The infoldings associated with secretion sites can be abolished by injecting vinblastine. Newly secreted enamel proteins do not accumulate extracellularly. So-called "stippled material" is an artifact of fixation and results from degradation of previously calcified enamel. Simultaneous visualization of the organic matrix and hydroxyapatite crystals is not possible because the aqueous stains needed to reveal the organic matrix dissolve the young enamel crystals. Osmium tetroxide, however, protects against beam damage and dissolution by water. When visualized separately the organic matrix seems to be contained within the space occupied by the crystal. Using stereo electron microscopy, crystals appear as thin flat ribbons. Images previously described as hexagonal cross-cut crystals are due to visualizing a three-dimensional structure in two dimensions.

Anatomically, the stomach-proper of the adult mouse is composed of two parts, the corpus and pyloric antrum. By serially sectioning entire stomachs, and histologically reconstructing the stomach-proper, it has been shown that both areas contain numerous finger-like, epithelial infoldings or gastric units. Special attention was given to the units of the antrum, which by three-dimensional reconstruction appeared as tubes, generally having a single pit, a gland and connecting these an isthmus. Using light and electron microscopy, with or without ('3)H-thymidine radioautography, the renewal of gland cells was investigated. The mucous cells, making up about 82% of the gland cells were produced by the division of cells in the isthmus and upper gland, and displayed immature features. With time, mucous gland cells migrated toward the base of the gland, and began to assume the features of differentiated cells. Not all mucous gland cells, however, reached the gland base. Some were lost by cell death and phagocytosed by neighbouring cells, at various levels of the gland. While some mucous gland cells had a fairly short lifespan of several or more days, a few lived in excess of 60 days.

Glial cells are actively produced during the growth of the mouse central nervous system, during young adult life, but it is not known whether this cell production continues throughout the life of an animal. This problem was examined by radioautography of the corpus callosum of aged, 9 to 10-month old and adult, 4-month old mice, each given an intraperitoneal pulse injection or continuous subcutaneous infusion of 3H-thymidine and then randomly assigned to one of the following experimental groups: aged and adult mice, a 2 hour group of 5 and 6 mice, respectively, which received a single intraperitoneal pulse injection of 5 uCi per g body weight and were sacrificed 2 hours later; aged mice, a 30 day group in which 5 mice were continuously infused with 3H-thymidine at the rate of 1 uCi per g body weight per day and were sacrificed by perfusion at day 30; aged mice, a 30 + 60 day group of 2 mice and a 30 + 180 day group of 3 mice which were continuously infused with 3H-thymidine for 30 days and then left untreated for 60 or 180 days, respectively, before sacrifice; adult mice, a 7 and 15 day group in which 6 and 5 mice, respectively, were continuously infused with 3H-thymidine at the rate of 1 uCi per g body weight per day and were sacrificed by perfusion at day 7 and 15, respectively. A total of 11,114 and 14,169 glial cells were counted in the corpus callosum of aged and adult mice using toluidine-blue stained preparations. The results for the aged mice are as follows. In the 2 hour group, the labelled cells were found to be immature glial precursor cells with a labelling index of 8.15%. These cells showed a labelling index of 18.69% in the 30 day group and 0% and 0%, respectively, in the 30 + 60 and 30 + 180 day groups. These results indicate "renewal" of the precursor cells. Mature oligodendrocytes or astrocytes were not labelled in the 2 hour group. In the 30 day group their labelling index was 1.12% and 11.90%, respectively. The oligodendrocyte index remained at

A strong relationship between bone macrostructural morphology and bone mechanical function has been well documented and is an essential component of many vertebrate biomechanical studies. However, a vastly richer data set could be had if the relationship between bone microstructure and bone function were as well understood. This thesis enumerates the bone microstructure-function relationship in a statistically consistent manor in extant endotherms. / Phylogeny, physiology and function have been shown to independently contribute to bone microstructure morphology. However, rarely have two or more of these factors been examined in combination. In this work the author used various statistical and experimental techniques to quantify the contribution of each of these factors to bone microstructure. / This work is organized into four parts: First, a review of methods used to quantify bone microstructure is given and a new method for quantifying vascular orientation proposed. This method allows the researcher to observe vascular orientation as an unbiased continuous measure and therefore complete more extensive statistical testing. Second, an analysis of the use of skeletochronology for aging three species of extant carnivores is given. This technique, although rarely used in extant endotherms, is commonly used for aging specimens from palaeontological findings. Upon discovering a significant discordance between organismal age and skeletochronology in the carnivorans studied here, I discuss the validity of its use in palaeontology. Third, using a sample of seven carnivoran species, the impact of phylogeny, function and physiology on bone microstructure was tested using a variance partitioning method. It was found that phylogeny has a large and significant impact on bone microstructural characteristics but only in conjunction with functional and physiological variables. When considering the effects of the three "pure" factors I found that physiological factors are the major drivers of bone microstructure. To further explore these findings, the final chapter presents an experimental study on the effects of biomechanical function and repeated loading on the humerus and tibiotarsus in Helmeted GuineaFowl. It was found that the type of strain and the repetition of strain from exercise both significantly impact bone microstructure but the relationship between tensile, compressive and shear strains to microstructure is complex with no obvious correlation. / Il existe une forte relation entre la morphologie de la structure macroscopique des os et leurs caractéristiques fonctionnelles au niveau mécanique. Cette relation est bien documentée et est un aspect essentiel de plusieurs études sur la biomécanique des vertébrés. Cependant, un ensemble de données beaucoup plus étoffé serait disponible si la relation entre la morphologie de la microstructure des os et leur fonction était mieux comprise. La présente thèse comporte une énumération des relations entre la microstructure des os et leurs caractéristiques fonctionnelles chez certaines espèces actuelles d'endothermes, en suivant une approche statistique cohérente. / Il a été démontré que la phylogénie, la fonction et la physiologie contribuent séparément à la morphologie de la microstructure des os. Cependant, les effets combinés de deux ou plusieurs de ces facteurs ont rarement été examinés. Dans la présente étude, l'auteur a utilisé plusieurs méthodes statistiques et expérimentales afin de quantifier l'impact respectif de chacun de ces facteurs sur la microstructure des os. / Cette thèse est organisée en quatre parties. D'abord, une revue des méthodes utilisées pour quantifier la microstructure des os est présentée et une nouvelle méthode pour quantifier l'orientation vasculaire est proposée. Cette nouvelle méthode permet d'observer l'orientation vasculaire d'une manière continue et non-biaisée, et permet donc une analyse statistique plus approfondie. Ensuite, l'utilisation de la squelettochronologie pour la détermination de l'âge de trois espèces de carnivores est analysée. Cette technique, bien que rarement utilisée pour déterminer l'âge chez les endothermes actuels, est communément employée pour les espèces paléontologiques. À la suite de la découverte d'une discordance significative entre l'âge des organismes et la squelettochronologie chez les carnivores étudiés ici, la validité de cette technique en paléontologie est discutée. En troisième partie, à partir d'un échantillon de sept espèces de carnivores et au moyen d'une analyse de partition de variance, l'impact de la phylogénie, de la fonction et de la physiologie sur la microstructure des os a été testé. Il a été découvert que la phylogénie avait un impact important sur la microstructure des os, mais seulement en conjonction avec les variables liées à la fonction et à la physiologie. Lorsque les effets des trois facteurs « purs » étaient considérés, la physiologie était le facteur qui contribuait le plus à la variabilité observée dans la microstructure des os. Afin d'examiner ces résultats plus en détail, le chapitre final présente une expérience investiguant les effets d'une charge répétée et de la fonction biomécanique sur l'humérus et le tibiotarse de la pintade de Numidie (Numida meleagris). Le type d'effort et la répétition de l'effort imposé par l'exercice avaient tous les deux un impact significatif sur la microstructure des os, mais les relations entre les forces de tension, de compression et de cisai