Localization and functional characterization of iridoid biosynthetic genes in Catharanthus roseus /

Catharanthus roseus is the sole biological source of the medicinal compounds vinblastine and vincristine. These chemotherapeutic compounds are produced in the aerial organs of the plant, however they accumulate in small amounts constituting only about 0.0002% of the fresh weight of the leaf. Their limited biological supply and high economical value makes its biosynthesis important to study. Vinblastine and vincristine are dimeric monoterpene indole alkaloids, which consists of two monomers vindoline and catharanthine. The monoterpene indole alkaloids (MIA's) contain a monoterpene moiety which is derived from the iridoid secologanin and an indole moiety tryptamine derived from the amino acid tryptophan. The biosynthesis of the monoterpene indole alkaloids has been localized to at least three cell types namely, the epidermis, the laticifer and the internal phloem assisted parenchyma. Carborundum abrasion (CA) technique was developed to selectively harvest epidermis enriched plant material. This technique can be used to harvest metabolites, protein or RNA. Sequencing of an expressed sequence tagged (EST) library from epidermis enriched mRNA demonstrated that this cell type is active in synthesizing a variety of secondary metabolites namely, flavonoids, lipids, triterpenes and monoterpene indole alkaloids. Virtually all of the known genes involved in monterpene indole alkaloid biosynthesis were sequenced from this library.This EST library is a source for many candidate genes involved in MIA biosynthesis. A contig derived from 12 EST's had high similarity (E'^') to a salicylic acid methyltransferase. Cloning and functional characterization of this gene revealed that it was the carboxyl methyltransferase imethyltransferase (LAMT). In planta characterization of LAMT revealed that it has a 10- fold enrichment in the leaf epidermis as compared to the whole leaf specific activity. Characterization of the recombinant enzyme revealed that vLAMT has a narrow substate specificity as it only accepts loganic acid (100%) and secologanic acid (10%) as substrates. rLAMT has a high Km value for its substrate loganic acid (14.76 mM) and shows strong product inhibition for loganin (Kj 215 |iM). The strong product inhibition and low affinity for its substrate may suggest why the iridoid moiety is the limiting factor in monoterpene indole alkaloid biosynthesis. Metabolite profiling of C. roseus organs shows that secologanin accumulates within these organs and constitutues 0.07- 0.45% of the fresh weight; however loganin does not accumulate within these organs suggesting that the product inhibition of loganin with LAMT is not physiologically relevant. The limiting factor to iridoid and MIA biosynthesis seems to be related to the spatial separation of secologanin and the MIA pathway, although secologanin is synthesized in the epidermis, only 2-5% of the total secologanin is found in the epidermis while the remaining secologanin is found within the leaf body inaccessable to alkaloid biosynthesis. These studies emphasize the biochemical specialization of the epidermis for the production of secondary metabolites. The epidermal cells synthesize metabolites that are sequestered within the plant and metabolites that are secreted to the leaf surface. The secreted metabolites comprise the epidermome, a layer separating the plant from its environment.

The encounter with the real and post-Soviet trauma : fantasy construction in Russian popular cinema /

This thesis examines the impact of the Soviet Union's collapse on the Russian Symbolic as represented through popular cinema of the post-Soviet period. The disintegration of the USSR in 1991 became one of the most traumatic experiences for many Russian people. The trauma of the collapse of the Soviet Union penetrated the everyday reality of the Russian Symbolic, leaving the traces-symptoms in different cultural fonns like literature, arts, television and cinema. Because popular culture usually reacts very quickly to any social, political and economical shifts in society, it is an excellent barometer for deeper changes in society. Focusing on postSoviet popular cinema, this thesis analyzes the symptoms of cultural and individual trauma occasioned by the momentous changes of the 1990's. This study is grounded in post-analytic theory of Jacques Lacan and its interpretation by Slavoj Zizek, which emphases the traumatic encounter with the Real as a "hard core" of our reality. According to this paradigm, a new chain of signifiers is structured around the traumatic breach in the Symbolic, initiating a process of fantasy construction to deal with consequences of trauma and, thus, to support our Symbolic order. This thesis examines three major fantasy constructions - drinking, traveling to a "happy land" and family reunion and money - in popular films by Alexander Rogozhkin, Yurij Mamin, Georgij Shengelia, Dmitrij Astrakhan, Valerij Todorovskij, Alexej Balabanov, Sergej Bodrov Jr. and Petr Buslov. According to Zizek, enjoyment underlies any fantasy constructions, and that is why after the intrusion of the Real every individual and culture should go through the process of fantasizing about some substitutes which can help to minimize the traumatic effect and which can lead to a partial enjoyment. By analyzing the fantasies about drinking, "happy land", reconstruction of the family bonds and money in Russian popular cinema since 1991, this thesis demonstrates how the traumatic engagement with the Real affected the everyday lives of Russian people, and how individuals tried to fill the gap, the lack, in the post-Soviet Symbolic and "return" the lost feeling of unity and plenitude.

Upper Lakes Shipping Ltd. fonds 1955-1990, n.d. (non-inclusive)

In March 1931, Captain Bruce Angus was sent to Sarnia by Gordon C. Leitch, general manager of Toronto Elevators. He was sent to inspect the Sarnian to ensure it was still seaworthy. Leitch was a savvy business man, who had been active in the business community for a number of years. Leitch began his career with a partner in the lumber business. When that went under he moved into graineries and worked for the Winnipeg Wheat Pool for 12 years. After Winnipeg he moved to Toronto, which was closer to his home town of Ridgetown, Ontario. In Toronto Leitch became manager of the Toronto branch of the Canadian Wheat Pool. While managing the wheat pools in Toronto Leitch became aware of huge costs associated with shipping the grains from the praries into the Toronto area. He felt that there was no need for such costs and decided to do something to make them better and cheaper for the business. Originally the grain was loaded onto Lakers that would bring the grain from the praries to Lake Huron and Georgian Bay. It was stored there until needed by the Toronto graineries and then hauled across land by either truck or train. The land journey was the most expensive and the one which Leitch wanted to eliminate. This was a fine plan except for 2 obstacles that were quickly overcome. First of all the Welland canals were not large enough to accommodate the large carriers that were bringing in the grain. This was changing as the expansion and widening of the canals was already underway. The second issue was the lack of storage in Toronto for the grain. The grain elevators had been destroyed by fire in the late 1880s and never replaced. Leitch propsed his company built its own storage elevators along the water front to allow not only for easier access to the grain, and more timely production of products. The elevators would aslo create a reduction in shipping costs and an overall more competitoive price for the customers of the grainery. The company refused, so Leitch went elsewhere to friends and contacts within the grain industry. The elevators were built and Leitch quit his job with the Canadian Wheat Pool and became the general manager of the elevators. Although the elevators were built and ready for storage the next issue was filling them. None of the carriers wanted to do business with Leitch because the competition in Georgian Bay threatened to cancel their contracts if they did. Leitch saw no way around this, but to provide his own transportation. This is when he sent Captain Bruce Angus to scout out potential ships. The ship was purchased for $37,000 and after another $30,000 was spent to fix it up, it was ready for business. The need for transportation and the finding of a seaworthy ship, lead to the beginnings of the Northland Steamship Company. The Sarnian proved to not be enough for the business underway. Leitch decided another ship was necessary. He joined forces with James Norris the owner of the Norris Grain Company. He proposed they join forces to create a more economical means of transportating their products.