Myasthenia Gravis is a disorder in humans characterized by abnormally rapid exhaustion, with loss of strength in the muscles under voluntary control and a return of strength, at least in part, after a period of rest. The term MG comes from the Greek mys (muscle), asthenia (weakness), and from the Latin gravis (heavy) and implies a marked or severe muscle weakness. Jolly in 1895 (1) first used the Dame ‘Myasthenia Gravis' to describe the syndrome. Be found upon repeated stimulation with faradic current a reaction of asthenia demonstrable by recording tracings.

When animals, including man, survive a bacterial infection, they generally become resistant to a second attack of the same pathogenic material. The serum of such individuals is capable of agglutinating or dissolving the bacteria or rendering them susceptible to phagocytosis. These reactions show a high degree of specificity: the serum reacts only with those micro-organisms whose previous invasion has been overcome. The active factor of the serum is referred to as antibody and the bacterium or its components which have elicited the production of the former is called an antigen.

The investigation to be described in this thesis forms part of a research project carried out in the Department of Biochemistry, McGill University on the problems of the preservation of human blood. This larger study was initiated early in 1940, under the guidance of Dr. O.F. Denstedt, and has been in progress since that time. Although the use of preserved blood dates back to World War 1 when O.H. Robertson used specimens stored up to twenty-two days in the treatment of battle casualties, little attention was paid to the question until the Russians revived it in 1930.

Significant chemical work on the hormones of the adrenal cortex dates from 1930 when Swingle and Pfiffner (l) and Hartman and Brownell (2) prepared the first extracts of cortical tissue which would maintain life in the adrenalectomised animal. In 1934 groups of chemists associated with Kendall and Mason, Wintersteiner and Pfiffner, and Reichstein, began investigations aimed at the isolation and characterization of the cortical hormones. Within a few years twenty-eight crystalline steroids were isolated and all groups of steroid hormones, namely, cortical, progestational, androgenic and estrogenic, were represented (3).

The dicholine ester of succinic acid (succinyldicholine; SDC) is a potent muscle-relaxant widely used in modern anaesthetic practice. The special properties of this drug are best considered in relation to those of other muscle relaxants. The clinical use of muscle relaxants in general and SDC in particular will therefore be reviewed. The paralytic properties of curare were well known to the South American Indiana who used it as a crude plant-extract painted on arrowheads.

The compound now known as cholesterol was first isolated by Poulletier de la Salle in 1770. The elucidation of the chemical structure of cholesterol was mainly due to the researches of Windaus and his associates, aided by the work of Wieland on the bile acids, and the studies of Suida and Mauthner on the derivatives of cholesterol. In 1919 Windaus arrived at a tentative formula which was changed, as a result of further work by Rosenheim, King, Dane, Diels and Gadke, and the final formulation as we know it today was decided upon in 1932.

Although allergy and immunity may appear, on superficial examination, to be two unrelated phenomena, studies have revealed that the mechanisms of the reactions involved in the manifestations of the immune and allergic states have common features. A brief survey of the fundamental aspects of immunology would therefore appear to be essential prior to a discussion of the mechanisms involved in the allergic reaction. The existence of immunity can be observed in an animal which, having recovered from a bacterial infection, is found to be resistant to a second infection with the pathogenic organism. The serum obtained from such an animal is capable of causing agglutination or lysis of the organism when incubated with it in vitro.

The work to be presented in this thesis forms part of a larger program of research carried out in this laboratory on mechanisms of homeostasis. The fact that the catecholamines play a part in these mechanisms is undisputed, yet much remains to be understood regarding their role. The more specific object of the present studies was to obtain information with respect to the part played by nutritional factors in the biosynthesis of adrenaline and noradrenaline and also to investigate the possibility that more than one pathway for their synthesis exists. Dietary deficiencies of riboflavin, pyridoxine and thiamine in young rats were investigated.

Engineered biocatalysts have become increasingly sought after as replacements for individual chemical synthetic steps. However, their implementation in multistep sequences as biosynthetic pathways has proven to be a significant challenge, in part due to a lack of broadly applicable methods for their assembly. One strategy for construction of a de novo biosynthetic pathway can be inspired by the theory of retrograde evolution, where pathway enzymes are recruited and optimized in the reverse order of biosynthesis. Herein, this process, termed bioretrosynthesis, is demonstrated through the construction and evolution of a pathway for in vitro production of the antiretroviral nucleoside analog didanosine (2,3 dideoxyinosine). Using methods in structure guided mutagenesis and directed evolution, we have evolved phosphopentomutase to accept 2,3-dideoxyribose 5 phosphate with a 700-fold change in substrate selectivity and 3-fold improved turnover in cell lysate as a retrograde extension from an engineered purine nucleoside phosphorylase. A second pathway extension via evolution of ribokinase resulted in a didanosine biosynthetic pathway with 9,500-fold change in nucleoside production selectivity. Surprisingly, the product of bioretrosynthesis in this step was not a retrograde extension from phosphopentomutase, but instead resulted in the discovery of a pathway shortening bypass via a new phosphorylation regiochemistry in the engineered ribokinase.

The G protein coupled receptor (GPCR) family is comprised of ubiquitous, membrane-bound proteins that are highly conserved in structure, yet varied in their cognate ligand. This allows for regulation of diverse physiological processes ranging from cardiac contractility and neurotransmission to olfaction and vision. Signaling events downstream of activated GPCRs are initiated through a conserved allosteric mechanism of receptor-mediated nucleotide exchange of GDP for GTP in the Gα subunit of their intracellular binding partner, the heterotrimeric G protein (Gαβγ). While much is known about GPCR and Gαβγ function, less is understood about the mechanism of GPCR-Gαβγ complex formation and subsequent receptor-mediated Gα activation. Biophysical characterizations have indirectly informed on the dynamic nature of the interaction. Direct investigations, however, have been thwarted due to the intrinsic instability of such complexes both in vivo and in vitro. In lieu of structural data on the GPCR-Gαβγ complex, models derived from structural studies of the Gα subunit mimicking receptor-activated states constitute our understanding of receptor-mediated Gα activation. Many of these studies focus on the endpoints of activation and information on transient features of GPCR-Gαβγ interactions remains sparse. The first part of this work aims to lower the barrier for structural studies on GPCR-G protein complexes by developing and characterizing model membrane systems that recapitulate the native environment of the signaling receptor where complex stability is optimized. In the second part of this work, these findings are applied to the structural characterization of the model system GPCR-G protein pair, rhodopsin-transducin. In the third part, I focus on the biochemical and structural characterization of an allosteric activation intermediate of Gαi1 to better understand how crosstalk between receptor and nucleotide binding sites is facilitated for promoting GDP release during Gα activation. Taken together, the work presented here offers insight into allosteric processes of GPCR-G protein complex formation, complex stabilization, and subsequent activation of G proteins for rhodopsin, transducin, and Gαi1. These results provide a framework for future investigations into the allosteric regulation of additional GPCR-G protein superfamily members.