La nicotine est considérée comme étant la « molécule » addictogène de la cigarette et du tabac. Mais différentes études cliniques, utilisant notamment des substituts nicotiniques, débouchent pratiquement toutes sur une même conclusion : efficacité ne dépassant que de peu celle d’un placebo, et très limitée dans le temps, contrastant avec le pouvoir hautement addictif du tabac, qu'il soit chiqué, prisé ou fumé. Dans ce travail de thèse, nous avons essayé de mettre en évidence le rôle que pourraient avoir certains des autres composés présents dans le tabac ou produits par pyrolyse. Nous avons d’abord utilisé des extraits aqueux de fumée et de tabac pour approcher un aspect global de ce que les fumeurs absorbent chaque fois qu’ils fument une cigarette, nous rapprochant ainsi des conditions physiologiques du fumeur. Puis nous avons choisi un certain nombre de substances. La cotinine, métabolite de la nicotine. L’harmane, une ß-carboline, synthétisée au cours de la combustion et dans l’organisme des fumeurs. La norharmane, une ß-carboline, présente en partie dans le tabac et synthétisée dans la fumée par pyrolyse. La technique utilisée tout au long de ce travail est l’enregistrement électrophysiologique. Cette technique s’applique très bien à l’étude in vivo de différents systèmes neuronaux y compris le système dopaminergique. Nous l’avons utilisée chez le rat, la souris WT et la souris Knockout ß2 (ß2KO). Nous nous sommes intéressés à deux aspects de l’activité cellulaire des neurones dopaminergiques de l’aire tegmentale ventrale : la fréquence de décharge (le firing) et les bouffées (bursts). En parallèle, nous avons conduit des expériences de liaison (binding) sur des cultures de cellules exprimant le récepteur nicotinique α4ß2. Nos résultats les plus significatifs ont montré que : Les bursts sont le plus souvent absents après les injections d’extraits de tabac et de fumée. Cela pourrait, entre autres, impliquer qu’il existe dans le tabac et la fumée des composés autres que la nicotine qui bloquent les effets de la nicotine sur les bursts. Les effets des extraits de tabac et de fumée sur le firing et les bursts ne sont plus présents chez les souris ß2 KO, ce qui implique que l’ensemble des composés du tabac agit essentiellement sur les récepteurs nicotiniques porteurs de la chaine ß2, même si des hypothèses alternatives existent. L’harmane a des effets activateurs très puissants sur le firing des neurones dopaminergiques, et ces effets sont bloqués à 80% par la mécamylamine, ce qui démontre qu’un des principaux composés du tabac et de la fumée autre que la nicotine agit par un mécanisme essentiellement nicotinique. Les expériences de binding confirment que les effets du tabac et de la fumée impliquent les récepteurs nicotiniques d’une façon majeure, mais d’une façon qui diffère légèrement de celle de la nicotine.Les résultats que nous avons obtenus montrent que les effets pharmacologiques du tabac ne se résument pas à ceux de la seule nicotine. Ils peuvent constituer un point de départ pour d’autres travaux, notamment pour étudier de plus près les effets des ß-carbolines. Il est nécessaire d’identifier les types de récepteurs sur lesquels elles se fixent, en utilisant des agonistes et antagonistes de récepteurs aux neurotransmetteurs contrôlant l’activité des neurones dopaminergiques. Des expériences sur des souris transgéniques chez lesquelles différents types de sous-unités de récepteurs nicotiniques ont été supprimés doivent également être envisagées, pour déterminer les mécanismes d’action des composants autres que la nicotine contenus dans le tabac et sa fumée sur les neurones dopaminergiques / Nicotine is generally considered as the sole tobacco addictive compound. However, nicotine replacement therapy studies almost all end with the same conclusion: the effectiveness of nicotine replacement is very limited on the short-term, and hardly exceeds that of placebo on the long-term. In addition, studies dealing with the effects of denicotinized cigarettes have provided evidence that these cigarettes have an addictive potential. In the present work, we tried to determine the behavioral role of some tobacco or smoke compounds other than nicotine at the neuronal level. We first compared the effects of nicotine with those of whole tobacco and smoke extracts, given that these preparations closer mimic the smoking situation than nicotine alone. We then examined the effects of a number of selected tobacco or smoke compounds. Cotinine, a major nicotine metabolite. Harmane and norharmane, two ß-carbolines synthesized in smoke as well as in the body of smokers. The technique used consists in the in vivo recording of the firing rate and bursts of dopamine neurons in the ventral tegmental area after intravenous injections of compounds in rats and mice. This electrophysiological technique is known to be a useful way to investigate the properties of selected compounds. In the case of mice, we used wild type and ß2 KO mice. We also made a series of in vitro experiments investigating the binding properties of the compounds on cells expressing high densities of α4ß2 nicotinic receptors. The main results of our studies are the following: Bursts are absent most of the times after the injection of the extracts. These results suggest that tobacco and smoke extracts contain compounds that inhibit the burst-promoting effects of nicotine. Increased firing is no longer present in ß2 KO mice treated with tobacco or smoke extracts, indicating that tobacco and smoke components, as a whole, primarily acts on nicotinic receptors that carry the ß2 chain, although alternative hypotheses may exist. Harmane very strongly activates the firing of dopaminergic neurons. Up to 80% of this effect is blocked by mecamylamine, demonstrating that that a major component of tobacco and smoke other than nicotine acts primarily through a nicotinic mechanism. The binding experiments confirm that the effects of tobacco and smoke involve nicotinic receptors in a major way, but in a way that slightly differs from that of nicotine. Our results may constitute a new starting point for further work, especially for a closer look at the effects of ß-carbolines. Attempts to identify the types of receptors involved in these effects are needed, using agonists and antagonists of neurotransmitter receptors that control the activity of dopamine neurons. Experiments on transgenic mice with deletion of different types of subunits of nicotinic receptors should also be made, to determine the different mechanisms of action of tobacco and smoke compounds other than nicotine on dopaminergic neurons

Exposure cigarette smoke (CS) during prenatal life is the leading cause of preventable premature death. In this study, we explored the hypothesis that in vitro exposure of fetal lung cells to cigarette smoke extract (CSE) may result in the alteration of apoptosis through activation of caspase-3. Alongside we compared the responses of fetal lung cells with A549 cells and rat periodontal ligament (PDL) fibroblasts exposed to CSE in a dose dependent manner. Caspase-3 activity and inhibition was measured using a fluorometric assay. Cell viability in smoke exposed cells was measured using MTT formazan assay. Caspase-3 expression and cellular localization was detected by western blot analysis and immunofluorescence. Our results indicate that caspase-3 activity was significantly (p < 0.05) elevated and cell viability was significantly inhibited in fetal rat lung cells exposed to 10% or 15 % (v/v) CSE. No significant differences were observed in the caspase-3 activity or cellular viability in A549 cells and rat PDL fibroblasts exposed to 5%, 10% or 15% (v/v) CSE. Activation of caspase-3 in fetal lung connective tissue and alveolar epithelial cells may be one of the reasons for the developmental pulmonary toxicity induced by CSE.

Exposure cigarette smoke (CS) during prenatal life is the leading cause of preventable premature death. In this study, we explored the hypothesis that in vitro exposure of fetal lung cells to cigarette smoke extract (CSE) may result in the alteration of apoptosis through activation of caspase-3. Alongside we compared the responses of fetal lung cells with A549 cells and rat periodontal ligament (PDL) fibroblasts exposed to CSE in a dose dependent manner. Caspase-3 activity and inhibition was measured using a fluorometric assay. Cell viability in smoke exposed cells was measured using MTT formazan assay. Caspase-3 expression and cellular localization was detected by western blot analysis and immunofluorescence. Our results indicate that caspase-3 activity was significantly (p < 0.05) elevated and cell viability was significantly inhibited in fetal rat lung cells exposed to 10% or 15 % (v/v) CSE. No significant differences were observed in the caspase-3 activity or cellular viability in A549 cells and rat PDL fibroblasts exposed to 5%, 10% or 15% (v/v) CSE. Activation of caspase-3 in fetal lung connective tissue and alveolar epithelial cells may be one of the reasons for the developmental pulmonary toxicity induced by CSE.

Cystic fibrosis (CF) is the most common genetic disease affecting Canadian newborns (1 in 3,850) and is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. This gene encodes for CFTR, a phosphorylation-dependent ion channel localized at the apical membrane. Phosphorylation of CFTR by the cyclic adenosine monophosphate (cAMP)-dependent enzyme protein kinase A activates its activity, facilitating the transport of chloride and bicarbonate ions across the epithelial membrane. CFTR contributes to ion and airway surface liquid regulation, crucial for maintaining host defenses. The inheritance of CFTR mutations leads to a variety of respiratory complications, including impaired mucociliary clearance, excessive mucus production, persistent airway infections, and heightened inflammation, ultimately causing lung damage. While there is currently no cure for CF, the development of CFTR modulators, targeting the defective CFTR protein directly, has significantly improved the quality of life for many CF patients. Despite these advancements, many patients remain unresponsive to current treatment options. It has been well-established that combination therapies outperform monotherapies, emphasizing the need for alternative or complementary therapeutic strategies for CF management. Furthermore, CFTR dysfunction extends beyond CF and has been implicated in other respiratory diseases, such as chronic obstructive pulmonary disease, which is primarily linked to tobacco smoke exposure. This Ph.D. thesis explores a complementary therapeutic approach, targeting proteins within the CFTR-containing macromolecular signaling complex to elevate intracellular cAMP levels, thereby enhancing CFTR function. We hypothesized that synergistic use of cAMP modulators, alongside CFTR modulators, will serve as an effective therapeutic strategy for CF and other respiratory diseases. Collectively, our studies highlight the potential of cAMP and CFTR modulation as a therapeutic strategy for improving the treatment of CF and other respiratory diseases, warranting further investigation, offering insights for future studies, and contributes to the ongoing pursuit of improved combination treatments. / Dissertation / Doctor of Philosophy (PhD) / Cystic fibrosis (CF) is the most common genetic condition affecting Canadian newborns, caused by inheritance of mutations in the CF transmembrane conductance regulator (CFTR) gene. These mutations result in respiratory issues, including breathlessness, excess mucus, and susceptibility to infections, causing lung damage and premature death. Despite progress in CF drug development, some patients remain unresponsive to existing drug combinations, highlighting the need for new combinations to improve the quality of life for all CF patients. CFTR function is also compromised in other respiratory diseases like chronic obstructive pulmonary disease, a lung disease that shares many characteristics with CF and is mainly caused by tobacco smoke exposure. This Ph.D. thesis explores the effectiveness of a new drug strategy targeting proteins interacting with CFTR. By investigating drugs to complement existing treatments, we aim to improve CFTR function. This research offers a promising strategy to improve treatment for CF and other respiratory diseases.

Monocytes exhibiting a pro-inflammatory phenotype play a key role in adhesion and development of atherosclerotic plaques. As an alternative to smoking, next-generation tobacco and nicotine products (NGP) are now widely used. However, little is known about their pro-inflammatory effects on monocytes. We investigated cell viability, anti-oxidant and pro-inflammatory gene and protein expression in THP-1 monocytes after exposure to aqueous smoke extracts (AqE) of a heated tobacco product (HTP), an electronic cigarette (e-cig), a conventional cigarette (3R4F) and pure nicotine (nic). Treatment with 3R4F reduced cell viability in a dose-dependent manner, whereas exposure to alternative smoking products showed no difference to control. At the highest non-lethal dose of 3R4F (20%), the following notable mRNA expression changes were observed for 3R4F, HTP, and e-cig respectively, relative to control; HMOX1 (6-fold, < 2-fold, < 2-fold), NQO1 (3.5-fold, < 2-fold, < 2-fold), CCL2 (4-fold, 3.5-fold, 2.5-fold), IL1B (4-fold, 3-fold, < 2-fold), IL8 (5-fold, 2-fold, 2-fold), TNF (2-fold, 2-fold, < 2-fold) and ICAM1 was below the 2-fold threshold for all products. With respect to protein expression, IL1B (3-fold, < 2-fold, < 2-fold) and IL8 (3.5-fold, 2-fold, 2-fold) were elevated over the 2-fold threshold, whereas CCL2, TNF, and ICAM1 were below 2-fold expression for all products. At higher doses, greater inductions were observed with all extracts; however, NGP responses were typically lower than 3R4F. In conclusion, anti-oxidative and pro-inflammatory processes were activated by all products. NGPs overall showed lower responses relative to controls than THP-1 cells exposed to 3R4F AqE.