The Trk family of tyrosine kinase receptors and the common p75NTR receptor are neurotrophin receptors. Nerve growth factor (NGF) binds TrkA, brain-derived neurotrophic factor (BDNF) binds TrkB, and neurotrophin-3 (NT-3) binds TrkC. The extracellular domain of the Trk receptor has five subdomains: a leucine-rich motif (D2), two cysteine-rich motifs (D1, D3) and immunoglobulin-like subdomains Ig-C1 (D4) and Ig-C2(D5). The Trk D4 subdomain regulates ligand-independent activation. The TrkA-D5 and TrkB-D5 subdomains regulate cognate ligand binding and Trk activation. However, the p75NTR receptor binds all neurotrophins and regulates ligand affinity and Trk signals. We showed that p75NTR affects Trk ligand - binding and activation of Trks by changing Trk subdomain utilization. When p75NTR is coexpressed, NGF can activate TrkA via the cysteine-1 subdomain (D1), and BDNF can activate TrkB via leucine-rich motif (D2) and cysteine-2 (D3) subdomains. We hypothesized conformational or allosteric regulatory mechanisms. To further study the interactions between ligands and Trks, we examined TrkA binding to NT-3 as a heterologous ligand because these interactions are biologically relevant. We found the TrkA “hot spot” functional docking sites used by NT-3. We demonstrate that TrkA-D5 has partially overlapping but distinct binding and activation “hot spots” for both, NGF and NT-3. Moreover, ligand - binding studies have identified additional NT-3 binding/allosteric site on TrkA-D4. NT-3 binding to both sites induces full agonism. Conversely, the TrkA-D5 NT-3 binding site is partially agonistic, but antagonizes NGF activity. Lasly, we address NT-3 binding and activation sites on the TrkC receptor by raising a monoclonal antibody that recognizes the juxtamembrane-linker domain of the TrkC receptor. This antibody is an artificial TrkC receptor agonist. The epitope of mAb 2B7 defines a previously unknown hot spot of TrkC. Binding to this “hot spot” induces survival but n / La famille de récepteurs de Trk tyrosine kinase et le récepteur p75NTR sont des récepteurs de neurotrophines. Le facteur de croissance nerveuse (NGF) intéragit avec le récepteur TrkA, le facteur neurotrophique dérivé du cerveau (BDNF) intéragit avec le récepteur TrkB et la neurotrophine-3 (NT-3) intéragit avec TrkC. Le domaine extracellulaire du récepteur Trk contient cinq sous-domaines: un motif riche en leucine (D2), deux motifs riches en cysteine (D1, D3) et des sous-domaines de type immunoglobuline Ig-C1(D4) et Ig-C2(D5). Le sous-domaine Trk D4 régule l'activation indépendante de ligand. Les sous-domaines TrkA-D5 et TrkB-D5 régulent la liaison de ligands endogènes ainsi que l'activation du récepteur Trk. Le récepteur p75NTR intéragit avec toutes les neurotrophines et régule l'affinité des ligands et les signaux issues de l'activation du récepteur Trk. Par ailleurs, nous avons démontré que le p75NTR affecte la liaison du ligand au récepteur Trk en changeant l'activation de l'utilisation des sous-domaines. Lorsque le recepteur de p75NTR est coexprimé, le NGF peut activer le récepteur TrkA via le sous-domaine cysteine-1 (D1) et BDNF peut activer TrkB via le motif riche en leucine (D2) ainsi que via le sous-domaine cysteine-2 (D3). Nous avons examiné la liaison d'un ligand hétérologue, NT-3 sur le récepteur TrkA afin d'étudier plus profondément les interactions entre les ligands et le récepteur TrkA. Ces interactions sont biologiquement pertinentes. Pour faire ceci, nous avons tout d'abord identifié les « points chauds » présents sur le récepteur TrkA qui servent des sites d'amarrage fonctionnels du ligand NT-3. Nous avons démontré que le sous domaine TrkA-D5 possède deux points chauds distincts, notamment un point chaud qui sert comme le site d'amarrage et d'activation du NGF et un point chaud qui sert comme le site d'amarrage et d'activation de la NT-3. Toutefois, ces deux sites d'amarrage se chevauchent partiellement. D

A growing line of evidence indicates that the proper control of DNA methyltransferases (DNMT) and DNA demethylases is critical for maintaining correct gene expression. In addition, misregulation of this machinery likely plays a role in the aberrant DNA methylation patterns and gene expression that is a hallmark of many pathologies such as cancer. A greater comprehension of the mechanisms involved in regulating the expression and activity of these proteins should provide new therapies aimed at restoring gene expression gone wrong.

While aging is generally associated with accumulation of DNA damage and decreased cell proliferation, the reason for age-related oligospermia remains unclear. The present study provides evidence that age-related oligospermia may be caused by the altered ability of testicular germ cells to undergo the first mitosis and meiosis. This is due to decreased DNA replication and/or repair in response to DNA damage. Young and aging rats were exposed to either saline or an alkylating agent, cyclophosphamide. The ability of the male germ cells to replicate and/or repair their DNA was estimated by examining expression of PCNA (an essential component of DNA replication and repair machinery) and incorporation of BrdU (analog of thymidine). PCNA expression correlated with that of BrdU incorporation. The ability of germ cells to replicate and/or repair their DNA was affected by both the aging process and cyclophosphamide exposure in a stage-specific manner. At mitosis 1, expression of PCNA and BrdU incorporation decreased with both age and drug exposure. Mitosis 2 was not affected by either age or cyclophosphamide. Meiosis was affected only by the aging process. These differential effects are due, probably, to the different types of DNA damage caused by aging and drug exposure: age produces a long term chronic damage while cyclophosphamide exposure produces an acute short term damage. The results of this study suggest that different mechanisms may be involved in mediating cell cycle arrest and DNA repair at different stages of spermatogenesis.

Calcitonin gene-related peptide (CGRP) is a 37 amino acid peptide arising from the alternative splicing of the calcitonin gene. The wide distribution of CGRP in the central and peripheral nervous systems suggests a role for CGRP in sensory processing. The primary focus of, this thesis was to study the involvement of CGRP and its binding sites in rat models of chronic inflammatory and neuropathic pain. The expression and binding sites of galanin and substance P were also examined to assess the specificity of the observed changes. / No changes were observed in CGRP, galanin and substance P immunostaining in the dorsal horn of the lumbar regions 4 and 5 (L4--L5) of the spinal cord at 24 hours, 4, 7 and 14 days following the injection of Freund's complete adjuvant or sciatic nerve constriction. However, binding sites at the L4--L5 levels were found to be modulated during the development of chronic pain. CGRP binding sites were found to be increased and decreased in models of inflammatory and neuropathic pain, respectively. Substance P binding sites were increased in both models while galanin binding sites were decreased only in this neuropathy pain model. / In conclusion, the changes in CGRP binding levels observed following the injection of Freund's complete adjuvant or constriction of the sciatic nerve were not mirrored by similar changes in substance P and galanin binding sites. This finding indicates differential involvement of these neuropeptides and their receptors in chronic pain mechanisms.

Purpose. There are reports indicating that diuretics may increase the risk of gastrointestinal bleeding. The study was conducted to verify this hypothesis and to assess whether the risk varies with the different types of diuretics. / Methods. Using the Saskatchewan health database, a nested case-control design was used, conducted within a population-based cohort of 47,865 new users of antihypertensive medications in Saskatchewan, from 1980 to 1983, and followed up to mid 1987. 753 subjects hospitalized for gastrointestinal bleeding were identified during this period, each of whom was matched with 10 randomly selected controls from a risk set formed at the index date, namely when a case was identified. / Results. The rate of hospitalization for gastrointestinal bleeding in this cohort was 2.83 cases per 1,000 subjects per year. The adjusted rate ratio of gastrointestinal bleeding for current use of any diuretic within the 30-day time window prior to the index date was 1.54 (95% confidence interval, CI 1.27 to 1.86) compared with no current use of antihypertensive medications. Among the different classes, potassium-sparing diuretic are associated with the highest adjusted rate ratios (2.64; 95% CI, 1.35 to 5.16), and current use of combination of thiazide diuretics and potassium sparing with the lowest (1.39; 95% CI, 1.11 to 1.73) in the 30-day time window. The adjusted rate ratio of hospitalization for gastrointestinal bleeding for current use of a daily dose of thiazide diuretics less than 50mg was 1.34; 95% CI, 0.37 to 4.90, and for a daily dose equal to 50 to 60 mg the rate ratio was 1.81; 95% CI, 0.93 to 3.54, while for a daily dose greater than 60mg the rate ratio was 2.99; 95% CI, 1.14 to 7.84 within the 30-day time window. Results were similar when a 60-day exposure time window was used. Higher doses of furosemide (loop diuretic) were positively associated with hospitalization for gastrointestinal bleeding. / Conclusion. Diuretic use appears to increase the risk of gastrointestinal bleeding.

We investigated the use of oral corticosteroids (OCS) among patients with chronic obstructive pulmonary disease (COPD) and the subsequent risk of death from all causes, COPD, cardiac problems, and pneumonia. We identified an incident cohort of 2050 patients with COPD using the Saskatchewan Health databases. We matched 668 case patients who died with 6742 controls with respect to date of entry into the cohort. A nested case-control analysis was then conducted using conditional multivariate regression. The matched adjusted rate ratio (RR) of all cause mortality for any use of OCS was 1.48 (95% CI 1.18--1.84). An increased risk of death was also shown with cumulative dose (RR 1.17; 95% CI 1.08--1.25) and average yearly dose (RR 1.31; 95% CI 1.19--1.43). A dose-related increase in the adjusted rate ratios for cumulative OCS doses above 250 mg was observed. The risk is particularly evident among patients filling prescriptions for OCS in the 12 months before death. An increased risk of death from COPD was found, but no significant association was found for cardiac and pneumonia mortality. Although the results suggest the use of OCS increases the risk death among COPD patients, the possibility of confounding by indication and channelling may have influenced the results.

The epigenetic modification of DNA by methylation at cytosine in CpG dinucleotides plays an important role in X-chromosome inactivation, genomic imprinting, host defense and cancer. In this thesis, I investigated the role of the predominant mammalian DNA methyltransferase, Dnmt1, in the establishment and maintenance of DNA methylation patterns in the germline. In a comparative study of the expression of Dnmt1 during male and female germ cell development, I identified putative windows in which Dnmt1 may set down sex-specific methylation patterns for imprinted genes, I also identified and characterized sex-specific exons in the 5' region of Dnmt1 which control the production and localization of enzyme during specific stages of gametogenesis by a mechanism that involves alternative splicing. An oocyte-specific 5' exon is associated with the production of a functional Dnmt1 protein that is truncated at the N-terminus, accumulates to high levels during oocyte growth and later persists through preimplantation development, while a spermatocyte-specific 5' exon leads to the production of a larger, nontranslated message. I further examined the dynamics in the expression of the oocyte-specific Dnmt1 isoform in populations of isolated oocytes obtained at different stages of development and their association with the methylation status of the maternally imprinted Snrpn gene. The data suggest that the tightly regulated expression of the oocyte-specific Dnmt1 during postnatal oogenesis may play a role in the establishment of maternal methylation imprints at this time, Furthermore, since the oocyte-specific isoform persists during preimplantation development, at a time when the somatic isoform of Dnmt1 is not detected, I propose that the oocyte isoform may also be important for the propagation of maternally- and paternally-derived imprints in the early embryo.

Prostaglandin (PG) E2 is involved in a number of physiological and path op hysiologicaI events in many tissues. PGE2 mediates its actions through interaction with specific plasma-membrane G-protein coupled receptors (GPCR's). There are four subtypes of PGE2 receptors: EP1, EP2, EP3 and EP4, classified by their different pharmacological profiles and signal transduction pathways. EP4 agonists have potential therapeutic benefit in diseases such as osteoporosis (EP4 mediates the bone anabolic actions of PGE 2), but possibly in other areas such as asthma. A characteristic feature of GPCR's is their ability to undergo agonist-induced desensitization and this response may limit the therapeutic utility of agonists due to tachyphylaxis. / This thesis outlines [1] the agonist-induced regulation of EP4 by PGE2; [2] the development of a novel selective agonist of EP 4 that also mediates EP4 desensitization; and [3] the discovery of a new role for EP4 in mouse models of allergic lung inflammation by employing this selective agonist. PGE2 challenge of EP 4 in human embryonic kidney cells results in desensitization of intracellular signalling responses (cAMP), phosphorylation independent of protein kinase A and sequestration. These events are mediated by sequences in the carboxyl-terminal tail of EP4. EP4 agonists were discovered by preparing analogues of PGE2 by replacing the hydroxycyclopentanone ring by a lactam. Optimized compound (19a) shows high potency at EP4 and is highly selective over the other seven prostaglandin receptors. In vivo stability was increased further by the addition of a gem di-fluro group on carbon 15 of the molecule. The resulting compound, EP 4_Ags, was employed to selectively elucidate the role of EP4 in mouse models of allergic lung inflammation. The EP4 agonist prevents ovalbumin (OVA)-induced inflammation, airways hyperreactivity (AHR) and T helper (Th) 2 cytokine increases. While interleukin (IL)-13-induced inflammation is not inhibited by the EP4 agonist, AHR is attenuated. These results demonstrate that an EP4 agonist has potent anti-inflammatory activity in a mouse model of allergen-induced lung inflammation. Additionally, EP4 may have effects on AHR independent of its anti-inflammatory activity. Interestingly, the rapid desensitization observed in-vitro did not appear to limit efficacy in-vivo. The role of EP4 in mouse models of allergic lung inflammation is a novel finding and the use of an EP4 agonist may have therapeutic benefit in the treatment of asthma.

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