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Hepatic adrenoceptors were examined in different thyroid states. Activation of glycogen phosphorylase in hepatocytes from euthyroid and hyperthyroid rats was mediated by alpha adrenoceptors. In hypothyroid rats there was a significant beta receptor contribution to this response and enhanced beta receptor-mediated accumulation of cAMP. Responses to glucagon, a hormone which also activates phosphorylase via cAMP, were not similarly affected. The increase in beta receptor-mediated responses in the hypothyroid rat was accompanied by decreased alpha receptor activity as indicated by a marked suppression of phenylephrine-stimulated phosphorylase activation and calcium efflux. Similar effects produced by vasopressin and the calcium ionophore, A23187, were changed only slightly or not at all. Alpha(,1) receptors in liver plasma membranes were identified by {('3)H}prazosin binding. Hypothyroidism did not influence the binding affinity of agonists or {('3)H}prazosin, but reduced the density of alpha(,1) receptors. This change was opposite to the increase in density of beta receptors in hypothyroid rat liver reported by others. All the changes observed in hypothyroidism were partially reversed by in vivo thyroid hormone replacement. It is concluded that hypothyroidism produces a selective enhancement of beta and suppression of alpha receptor responses in rat liver. These changes are most likely the result of corresponding alterations in the density of membrane receptor sites.

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To examine the possibility that the diverse functions for which simian virus 40 (SV40) large tumor (T) antigen is required are carried out by the different structural subclasses of the protein, an electrophoresis system that uses nondenaturing polyacrylamide gradient gels was devised. It resolves the monomeric and various homologous and heterologous oligomeric forms of large T antigen into distinct bands which retain enzyme activity and immunoreactivity. The large T antigen analogue, D2T antigen, showed primarily a monomeric and three higher order bands on such gels. These were assayed for ATPase activity and protein kinase activity and found to exhibit different levels of these activities. / One hypothesis for transformation by SV40 is that high molecular weight complexes between large T antigen and the cellular encoded p53 tumor protein play an essential role. The complexes are identified by their sedimentation rates through sucrose gradients and species sedimenting at 16S and 23S and above have been characterized. These species were examined further in SV40-transformed mouse (SVT2) and human (SV80) cells by nondenaturing gradient gel electrophoresis. The T antigen species resolved on the gels were visualized by Western blotting employing anti-D2T serum. The 16S and 23S peaks of large T antigen were resolved into three high molecular weight bands, two of which also reacted with antibodies specific for p53, indicating that SV40-transformed cells contain two forms of the large T antigen/p53 complex. The various bands were also reacted with antibodies against biologically distinguishable populations of the viral protein. The results obtained with these antibodies, together with those obtained on the enzyme activities of the different forms of D2T antigen, support the hypothesis that oligomerization yields subclasses of large T antigen with distinct sets of functions and biological activities.

Our knowledge of limb development has increased dramatically in the last decades. However, our understanding of how gene expression is translated into morphology remains very limited. Using a variety of experimental approaches, I attempted to uncover the mechanistic factors underlying variation of digit number in tetrapods during my dissertation research. Firstly, classical embryology experiments were used to better understand how anterior and posterior axes contribute to autopodial formation. These experiments have shown that from stage HH23 in the chick (Gallus gallus), both anterior and posterior axes contribute independently to skeletal elements in the manus, which opposes some models of the evolution of limb development. To complement our understanding of limb patterning, I have also observed the earliest stage of skeletal formation, mesenchymal condensation, in a taxonomically broad sample of amniotes. These observations have allowed me to record the developmental groundstate of crocodilians, the closest extant relatives of birds. It was previously shown that the chick initially develops five digital anlagen, two of which fail to ossify. This has now been shown as well in Alligator mississippiensis, excluding the possibility that the anteriormost condensation in the chick manus is a vestigial remnant from a polydactylous ancestor. The comparative analysis of initial limb patterning has also shed new light on previous accounts of skeletogenesis, mostly based on chondrification patterns. Early stages of limb formation follow a much more proximodistal course than subsequent chondrogenesis, which has been described as following a posterior to anterior sequence in the autopodium. Furthermore, skeletal elements seem to originate from a continuous mass of condensed mesenchyme, particularly uniform in the carpal area. Individual chondrification foci initiate within the pre-existing condensations, and do not follow patterns of branching as previously described. This / Malgré que nos connaissances ne cessent d'augmenter sur le développement des membres, notre compréhension de comment les interactions génétiques et cellulaires donnent lieu a la morphologie reste limitée. A l'aide de diverses techniques expérimentales, j'ai exploré les divers mécanismes qui peuvent causer la réduction du nombre de doigts chez les tétrapodes au cours de cette thèse. Tout d'abord, des expériences d'embryologie classique on testé un modèle selon lequel les structures antérieures de l'autopode dépendent du développement initial des structures postérieures. Ces expériences ont démontré qu'à partir du stade HH23 chez l'embryon de poulet (Gallus gallus), l'axe antérieur et postérieur se développent indépendamment, puisque l'ablation de chacun de ces axes ne perturbe pas le développement de l'autre. J'ai aussi observé les patrons des stades précédant la chondrification, lors de la condensation du mesenchyme, chez plusieurs espèces ayant un nombre différent de doigts. Ces observations ont permis d'établir que cinq doigts étaient présents dans la configuration initiale de l'autopode chez les crocodiliens, les plus proches parents des oiseaux, comme il a été démontré chez l'embryon de poulet. Ceci permet d'éliminer la possibilité que la condensation antérieure présente dans le développement initial de l'aile ne soit un vestige d'ancêtres plus lointains, qui avaient plus de cinq doigts. Ces observations ont aussi permis de mieux comprendre comment s'établissent les patrons initiaux de développement des membres. Il a été trouvé que la condensation du mésenchyme progresse dans une séquence proximo-distale, ce qui est différent des séquences de chondrification précédemment décrites. Le développement du carpe survient donc avant le développement des doigts, ceux-ci prolongeant les zones de condensation à partir d'une masse cellulaire uniforme. Les blastèmes de chondrification se développent à l'int

RNA interference (RNAi) is an evolutionarily conserved me chanism for silencing gene expression mainly through small interfering RNAs (siRNAs) and microRNAs (miRNAs). SiRNAs have revolutionized experimental biology and gained recognition as a powerful tool for functional genomics studies. The sequence-specific selectivity and robust capacity of siRNA to inactivate genes in vivo has also led to the development of novel RNAi-based therapeutics. Recently, miRNA has attracted considerable attention due to its important roles in the regulation of various cellular processes, especially its link with cancer. Understanding of RNAi has advanced remarkably worldwide. In light of such progress, this project aimed to develop a high throughput approach for forward screening of the most effective siRNAs in mammalian cells, and to comprehensively analyze sequence variations in both miRNA targets and miRNA genes in human tumor tissues and characterize their roles in tumorigenesis. To develop a genetic forward approach for screening of siRNAs as the first objective, siRNA expression libraries were directly delivered into mammalian cells via bacterial invasion. The target genes were fused with the suicide gene, cytosine deaminase, and stably expressed in mammalian cells. The most effective siRNAs were conveniently and robustly identified in surviving cells under drug selection without the prior assay of the RNAi potency of individual siRNAs in all wells that is required by other siRNA screening methods. Significantly, the potent siRNAs isolated from the surviving cells proved to have noncellular toxicity. Hence, this approach can robustly enrich the most potent and nontoxic siRNAs in surviving cells, and thus has considerable potential to facilitate RNAi applications by minimizing the dose-dependent and sequence-nonspecific side effects of siRNAs. To investigate whether single-nucleotide polymorphisms (SNPs) located at miRNA-binding sites affect the expression of miRNA targets / RNA interférence (RNAi) est un mécanisme conservé au cours de l'évolution,les gènes de silence sont exprimés principalement par les petits RNAs interférents (siRNAs) et les microRNAs (miRNAs). Les petits RNAs interférents ont expérimentalement révolutionné la biologie, et ont obtenu la reconnaisance comme un outil puissant sur les études utiles génomiques. La sélectivité de la séquence spécifique et la capicité robuste de siRNA à inactiver les gènes in vivo a aussi dirigé le développement de la nouvelle thérapeutique de RNAi-based. Récemment, miRNA a attiré une attention considérable grâce à ses rôles importants dans la régulation de divers processus cellulaires, surtout ses liens avec le cancer. La compréhension de RNAi a progressé remarquablement dans le monde entire. À la lumière de tel progrès, ce projet s'est efforcé de développer une approche de haut rendement pour filtrer les siRNAs les plus effecaces dans les cellules mammifères, analyser d'une manière globale les variations des séquences dans les miRNAs de cibles et de gènes dans les tissus des tumeurs humains, et caractériser leurs rôles dans la cancérogénèse. Pour développer une approche génètique pour filtrer les siRNAs comme le premier objectif, les bibliothèques d'expression de siRNA ont été livrées directement aux cellules mammifères via les vecteurs bactériens. Les gènes de cibles ont été fondus avec le gène de suicide, et ont été exprimés avec stabilité dans les cellules mammifères. Les siRNAs les plus efficaces ont été identifiés commodément et puissamment dans la survie des cellules sous la sélection de médicament, sans l'essai préalable sur le pouvoir de RNAi de siRNAs individuel dans toutes les cellules qui sont demandées par les autres méthodes de filtrage de siRNA. Évidemment, les siRNAs puissants isolés de la survie des cellules prouvent qu'ils ont la toxicité non-cellulaire. De là, cette approche peut enrichir p

Previous studies on the impacts of cattle on the aquatic environment have mainly focused on cold water systems with high intensity grazing, and may be of limited relevance for assessing impacts of cattle grazing on low gradient, low intensity sites such as those in eastern Ontario. The present study looks at changes to the aquatic habitat following the removal of cattle from a watercourse. Biomonitoring was completed at an aquatic restoration site over a four year period encompassing pre- and post-implementation conditions. The initial results indicated modest improvements in the habitat and in the benthic macroinvertebrate and fish communities following exclusion of cattle from the watercourse. Trends over time suggested an increase in the proportion of sensitive benthic macroinvertebrates, a decrease in tolerant benthic species and an increase in fish density. Longer-term monitoring is required to observe changes to the aquatic communities following the growth of woody riparian vegetation. / Par la passé, plusieurs scientifiques ont étudié l'effet du bétail sur le milieu aquatique utilisant des sites où se trouve une haute densité de vaches dans des endroits où les cours d'eaux ont une forte pente. Puisque nous utilisons des sites dans l'Est Ontarien, les résultats de ces recherches auront peu de pertinence en ce qui concerne cette présente étude car la majorité des sites de la région sont ceux où l'on retrouve peu de vaches et des cours d'eaux ayant une faible pente. Cette thèse examine les changements du milieu aquatique suivant l'enlèvement des vaches à proximité du cours d'eau, et ce, depuis les quatre dernières années, incluant les conditions pré et post implémentation. Les résultats indiquent une amélioration modeste d'habitat et des communautés de macroinvertébrées benthiques et de poissons. Les tendances au fil du temps ont suggérées qu'il y eu une amélioration des proportions de macroinvertébrées benthiques sensible, une diminution de macroinvertébrées benthiques insensible et un accroissement dans le nombre de poissons. Plusieurs années seraient nécessaires pour étudier les effets de la croissance des arbres et arbustes sur les communautés aquatiques.