La tuberculose tue environ 2 millions de personnes chaque année, principalement à cause d’un diagnostic tardif ou inefficace ou de soins trop tardifs. Les techniques de diagnostic les plus efficaces sont souvent coûteuses et complexes à mettre en oeuvre dans les pays en voie de développement, régions de plus forte incidence de la maladie. L’objectif de ce projet est l’élaboration de capteurs luminescents à base de matériaux nanoporeux élaborés par procédé Sol-Gel pour détecter un métabolite très spécifique de Mycobacterium tuberculosis, l’acide nicotinique (AN), présent dans l’haleine des malades à des concentrations de quelques dizaines à quelques centaines de ppq, et de le discriminer vis-à-vis d’autres métabolites.Un complexe de nitrate de terbium (III) a été choisi comme molécule-sonde car la luminescence du terbium (III) peut être exaltée en présence de certains ligands organiques, notamment l’acide nicotinique. Une première étape a consisté à déterminer en solution les conditions de pH les plus favorables à la formation de complexes luminescent Tb(III)/AN. Ainsi l’établissement d’un pH de 6,4 dans un milieu tampon à base d’hexamine permet d’optimiser la formation du complexe Tb(III)/AN et le transfert d’énergie du ligand vers le cation. Le dosage de l’acide nicotinique est possible dans ces conditions dans une gamme de concentration de 400 nmol.L-1 à 100 μmol.L-1, soit de 7,2 ppb à 1,8 ppm.La seconde étape a consisté à produire des matrices nanoporeuses à base d’alcoxydes de silicium en vue d’obtenir des matrices à pH intrapore similaire ou proche de 6,4. Les variations de pH intrapore des matrices lors du piégeage de vapeur d’eau et/ou de dioxyde de carbone, deux interférents présents à des concentrations élevées dans l’haleine, ont été étudiées au moyen d’un colorant sensible au pH, le bleu de bromothymol. Les matrices ont été élaborées à partir de deux précurseurs de silice, dont un possédant une chaîne aminopropyle lui conférant un caractère basique. L’exposition des matrices à de la vapeur d’eau jusqu’à saturation a montré que le pH intrapore des matrices contenant 3% du précurseur aminé varie entre 6,5 et 6, gamme de pH optimisée pour la formation du complexe Tb(III)/AN.Dans la dernière étape, des matrices à 3% de précurseur aminé, dopés de terbium et tamponnées à pH 6,4 avec de l’hexamine ont été élaborées. Des mesures de luminescence de matrices exposées de manière statique à des vapeurs d’acide nicotinique pur ou provenant d’une solution aqueuse saturée ont montré une augmentation de la luminescence des matrices, preuve d’un piégeage effectif de l’acide nicotinique et de la formation in situ de complexes luminescents Tb(III)/AN. Malgré la présence d’eau qui désactive partiellement l’état excité de Tb3+, le piégeage de l’acide nicotinique et la formation de complexes Tb(III)/NA dans ces matrices demeure efficace. .Les études d’interférence ont permis de montrer que la présence de marqueurs secondaires, comme le nicotinate de méthyle, affecte la luminescence des complexes Tb(III)/AN uniquement par absorption compétitive du rayonnement d’excitation. Des solutions permettant de s’affranchir des interférences des métabolites secondaires sont à l’étude. / Tuberculosis kills nearly 2 million people each year, mainly because of late or inefficient diagnostic or late cures. The most efficient methods are often too expensive and too complex to implement in developing countries, areas of greater incidence of the disease. The aim of this project is the design of luminescent sensors for the detection of a very specific tuberculosis metabolite, nicotinic acid, detected in concentration ranging from around ten to hundred ppq, present in sick people’s breath, and to discriminate it from other metabolites.A terbium nitrate complex is used as its luminescence can be sensitized by organic ligands, as nicotinic acid. A first step was the optimization of the pH of aqueous solution to enhance the complexation between Tb3+ ion and nicotinic acid. A solution buffered at pH 6,4 using hexamine allows optimization of the complex formation and energy transfer from nicotinic acid to terbium. Sampling of nicotinic acid can be done in the range 400 nM-100 μM, or from 7,2 to 1,8 ppm.The second step was to design nanoporous matrices from silicon alcoxydes to obtain matrix with an intraporous pH of 6,4. We studied the changes of the matrix intraporous pH while trapping water vapor or carbon dioxide, present in high concentration in breath, using bromothymol blue as pH indicator. The matrices were produced from 2 silicon precursors, one of them containing an aminopropyle carbon chain, conferring an alkaline nature. Changes of the matrix pH between 6,5 and 6 were observed following the exposure of a silica matrix containing 3 % of the aminated precursor to water vapor to saturation. This range of pH value is optimized to favor Tb3+-nicotinic acid complex formation.In the last step, silica matrix containing 3% of the aminated precursor, doped with terbium and buffered at pH 6,4 with hexamine were designed. Luminescence measurements made on matrix exposed to vapors from pure nicotinic acid or saturated aqueous solution, showed an increase of the matrix luminescence, proof of the trapping of nicotinic acid in the nanoporous matrix and of the complexation between nicotinic acid and Tb3+. Trapping of nicotinic acid and subsequent complexation with Tb3+ are lowered by the presence of water vapor, which can partially deactivate the luminescent excited state of Tb3+. Interference studies showed that secondary metabolites as methyl nicotinate can only affect the luminescence of Tb3+/AN complex by competitive absorption of the excitation radiation. Detection methods free of interferences from the secondary metabolites are studied.

Aims: LDL oxidation plays an important role in the initiation and progression of atherosclerosis. HDL impedes oxidation, glycation and glycoxidation in vitro and there is evidence to suggest paraoxonase-1 (PON1) plays an important role in this. 1. In patients with dyslipidaemia treated with statins, I assessed the relationship of serum PON1 activity with in vitro HDL antioxidant capacity, susceptibility of LDL to oxidation and the protection offered by HDL. 2. I studied the effect of the presence and severity of obstructive sleep apnoea (OSA) in morbidly obese patients on HDL anti-oxidant and anti-inflammatory functions. 3. I investigated the influence of extended release niacin/ laropiprant (ERN/LRP) versus placebo in patients who had persistent dyslipidaemia despite receiving high doses of potent statins. I assessed the effect of ERN/LRP on mediators of vascular inflammation and HDL's in vitro anti-oxidant function. Methods: 1. LDL isolated from dyslipidemic patients was incubated with and without HDL, in the presence of Cu2+. Similarly isolated HDL was incubated alone. Lipid peroxides (LPO) generated over 3 hours were measured. Patients were divided into 2 groups based on median serum PON1 activity. 2. 41 morbidly obese patients were divided into two groups based on the presence or absence of OSA ("OSA" and "no OSA" group) or on severity of OSA (high or low apnoea-hypoapnoea index (AHI) groups). I studied HDL's ability to protect itself from in vitro oxidation and measured serum PON1 activity, tumor necrosis factor alpha (TNFalpha) and intercellular adhesion molecule 1 (ICAM1). 3. This was a randomised double blind cross over trial, where I studied the effect of ERN/LRP compared to placebo in 27 patients who had high LDL-C inspite of maximum tolerated doses of statins. I measured lipid profile, apolipoproteins, cholesteryl ester transport protein (CETP) activity, paraoxonase 1 activity (PON1), oxidised LDL (oxLDL) and related mediators of vascular inflammation. I also examined the capacity of HDL to protect LDL from in vitro oxidation. Results and conclusion: 1. In statin treated dyslipidemic patients the capacity of HDL to protect itself and LDL from oxidation in vitro is significantly better in individuals with higher serum PON1 activity. 2. The capacity of HDL to protect itself from in vitro oxidation in morbidly obese patients is reduced with onset and severity of OSA. The differences in TNFalpha and ICAM1 levels may suggest endothelial dysfunction due to OSA. Oxidative damage of PON1 attributable to OSA could be a mechanism for HDL and endothelial dysfunction. 3. Treatment with ERN/LRP resulted in a significant improvement in HDL-C but did not affect HDL's in vitro anti-oxidant function in patients who had persistent dyslipidaemia despite high doses of potent statins. For the first time I have shown that ERN/LRP reduces mediators of vascular inflammation.

A vareniclina é uma substância química sintética utilizada para o tratamento de tabagismo; atua como agonista de receptores colinérgicos nicotínicos, em especial, como agonista parcial em receptores α4β2 e α3β4, e como agonista total do receptor α7. Levando em consideração que há uma tendência de ampliação do uso clínico da vareniclina para o tratamento da dependência à diversas substâncias de padrão abusivo e que há poucos estudos relacionados aos seus efeitos sobre o comportamento, cognição e sistema motor, tornam-se necessários mais estudos sobre essa substância. Assim, no presente trabalho foram estudados os efeitos da exposição prolongada (28 30 dias) de ratos à vareniclina, avaliando-se o consumo de água e de ração, o ganho de peso e o comportamento animal, por meio dos testes de campo aberto, labirinto em cruz elevado, interação social, comportamento estereotipado, labirinto de Barnes e esquiva passiva. Ainda foram feitas as avaliações dos níveis de neurotransmissores e seus metabólitos em diferentes estruturas cerebrais, bem como avaliações hematológicas, bioquímicas séricas, urinárias e estudos anatomopatológicos e histopatológicos. Foram utilizadas três doses de vareniclina: 0,03 (dose terapêutica para o ser humano), 0,1 e 0,3 mg/kg, por via oral (gavagem). Os resultados mostraram que a exposição prolongada de ratos à diferentes doses de vareniclina não provocou toxicidade, uma vez que não houve alteração no consumo médio de água e de ração e no ganho de peso avaliados semanalmente. Quanto às avaliações comportamentais, observou-se leve aumento da atividade geral no campo aberto, bem como diminuição do tempo de interação social, não sendo capaz de alterar parâmetros neuroquímicos, hematológicos, bioquímicos séricos, urinários, anatomopatológicos e histopatológicos de ratos expostos à vareniclina. / Varenicline is a synthetic chemical used for the smoking addiction treatment; it acts as an agonist of nicotinic cholinergic receptors, in particular, as a partial agonist of receptors α4β2 and α3β4 and as a full agonist of the α7 receptor. More studies about this substance are necessary, given that its clinical use is increasingly being applied to the treatment of addiction to a variety of abusive drugs. Moreover, there are few studies on vareniciline effects on behavior, cognition and the motor system. Thus, in this study the effects of prolonged (28-30 days) exposure of rats to varenicline were evaluated. It was analyzed the water and food consumption, the weight gain and the animal behavior, through open field, elevated plus maze, social interaction, stereotyped behavior, Barnes maze and passive avoidance tests. The neurotransmitter levels and their metabolites in different brain structures were measured and hematological, serum biochemistry, urinary evaluations and pathological and histological studies were carried out. We used three doses of varenicline: 0.03 (therapeutic dose for humans), 0.1 and 0.3 mg/kg orally (gavage). The results showed that prolonged exposure of rats to different doses of varenicline did not cause toxicity, since there were no changes in average weekly consumption of water or food nor body weight gain, which were measured weekly. As for behavioral assessments, there was a slight increase in overall activity in the open field as well as decreased time of social interaction. Varenicline was not able to change neurochemical, hematological, serum biochemical, urinary, pathology and histopathology parameters of rats.

While cancer chemotherapy continues to significantly contribute to the number of cancer survivors, exposure to these drugs can often result in chemotherapy-induced peripheral neuropathy (CIPN), a consequence of peripheral nerve fiber dysfunction or degeneration. CIPN is characterized by sensory symptoms in the hands and feet, such as numbness, burning, and allodynia, resulting in an overall decrease in quality of life. Paclitaxel (Taxol), a microtubule poison that is commonly used to treat breast, lung, and ovarian cancers, has been found to cause CIPN in 59-78% of cancer patients. There is currently no effective preventative or therapeutic treatment for this side effect, which can be a dose-limiting factor for chemotherapy or delay treatment. Our collaborators in the laboratory of Dr. M. Imad Damaj have shown that nicotine, a nicotinic acetylcholine receptor (nAChR) agonist, and R-47, an α7 nAChR silent agonist, can prevent and reverse paclitaxel-induced peripheral neuropathy in mice. With regard to cancer, this work demonstrates that nicotine and R-47 do not enhance A549 and H460 human non-small cell lung cancer cell viability, colony formation, or proliferation alone, and they do not attenuate paclitaxel-induced growth arrest, apoptosis, or DNA fragmentation. Most importantly, nicotine and R-47 do not increase the growth of A549 tumors or interfere with the antitumor activity of paclitaxel in tumor-bearing mice. These data suggest that targeting nAChRs may be a safe and efficacious approach for the prevention and treatment of CIPN in cancer patients.

Abstract not available

Doctor of Philosophy(PhD) / The aims of this project were to contribute to the elucidation of the role of the cholinergic system in attention and memory, two cognitive processes severely compromised in Alzheimer’s disease (AD), and to evaluate and develop tools for the functional molecular imaging of this system with a view to improving knowledge of AD and other neurological disorders. Towards the first aim, the specific anti-cholinergic toxin 192 IgG-saporin (SAP) was administered to female Sprague-Dawley rats via either an intracerebroventricular (icv) or an intracortical route and animals were tested with a vibrissal-stimulation reaction-time task and an object recognition task to evaluate their attentional and mnemonic function, respectively. The second aim was approached in two ways. Firstly, relative neuronal densities from animals with icv lesions were assessed with both ex vivo and in vitro autoradiography with the specific cholinergic radiopharmaceuticals [123I]iodobenzovesamicol (123IBVM) and 125I-A-85380, ligands for the vesicular acetylcholine transporter and the nicotinic acetylcholine receptor, respectively. Secondly, a number of in vivo and in vitro studies were performed on a novel and unique molecular imaging system (TOHR), with which it had been hoped initially to image eventually SAP-lesioned animals, with a view to measuring and ameliorating its performance characteristics and assessing its in-principle suitability for small-animal molecular imaging. The behavioural studies support a critical role for the cholinergic system in normal attentional function. Additionally, in accord with literature evidence, no significant impairment was observed in mnemonic function. It is postulated however that the results observed in the intracortically-lesioned animals support the published hypothesis that cholinergic projections to the perirhinal cortex are critical for object-recognition memory. In autoradiographic studies, SAP-lesioned animals demonstrated reduced uptake of 123IBVM in multiple regions. A reduction of nicotinic receptors was also seen in SAP-lesioned animals, a novel finding supportive of the excellent characteristics of radioiodinated I-A-85380. Examination of the performance characteristics of the TOHR support in principle its utility for targeted small-animal molecular imaging studies.

<p>This thesis examined neurotoxic effects of nicotine exposure during a defined critical period of neonatal brain development in mice.</p><p>In our environment there are numerous hazardous contaminants that an individual can be exposed to during its entire lifetime. In many mammalian species the neonatal period is characterised by a rapid development of the brain. The present studies have identified a defined critical period during the neonatal brain development in mice, where exposure to low doses of nicotine causes permanent disturbances in the cholinergic nicotinic receptors and altered behaviour response to nicotine at adult age. This adult reaction to nicotine, a hypoactive response, was the opposite of that observed in control animals and animals exposed to nicotine before or after this period. Animals showing a hypoactive response to nicotine lacked nicotinic low affinity binding sites in the cerebral cortex. Furthermore, neonatal exposure to nicotine affected learning and memory in adult animals, an effect that was time-dependent. This thesis also showed that neonatal exposure to nicotine increased adult susceptibility to a repeated exposure of nicotine, manifested as an even more pronounced effect in spontaneous behaviour after challenging doses of nicotine. In these animals the nicotinic receptors in the cerebral cortex, assayed by a-bungarotoxin, was decreased. </p><p>Neonatal exposure to nicotine was also shown to increase adult susceptibility to the organophosphate paraoxon, a known cholinergic agent, and to the brominated flame retardant 2,2´,4,4´,5-pentabromodiphenyl ether, a novel environmental agent, at adult age. This was seen at doses that did not affect behaviour in control animals, and was manifested as deranged spontaneous behaviour and reduced habituation, aberrations that also worsened with age. </p><p>The results indicate that differences in adult susceptibility to environmental pollutants are not necessarily an inherited condition. Rather they may well be acquired by low dose exposure to toxic agents during early life.</p>

<p>Impulses coming from peripheries are modified in the spinal cord and transmitted to the brain. Several neurotransmitters have been involved in the processing of impulses in the spinal dorsal horn. Acetylcholine (ACh) is one of many neurotransmitters involved in the regulation of nociception in the spinal cord. In this study we investigated the role of nicotinic, muscarinic, serotonergic and GABA receptors in the regulation of spinal ACh release since these receptors are reported to be involved in spinal nociceptive processes.</p><p>Different receptor ligands were infused intraspinally via microdialysis and the spinal ACh release was measured by on-line HPLC. Receptor-ligand binding studies were performed with spinal cord homogenates as well as receptors expressed in cells.</p><p>In the first study, we found that nicotine and some of the nicotinic antagonists used increased ACh release suggesting that spinal ACh release is regulated by different nAChRs. Nicotine and nicotinic agonists may act on different types of receptors with different affinity to produce the observed net effect of increased ACh release. We propose the possibility of an involvement of three different nicotinic receptor subtypes in the regulation of spinal ACh release. </p><p>The effect of epibatidine, which is regarded as a nicotinic agonist, on muscarinic receptors was investigated in the second study. We propose that epibatidine, in μM concentrations, is a partial muscarinic receptor agonist that may interact with spinal muscarinic receptors to increase ACh release. The dual action on both nAChRs and mAChRs may explain the potent analgesic effect observed after intra-spinal epibatidine administration.</p><p>In the third study, we investigated the role of serotonin receptor involvement in ACh release control. The results suggest that only 5-HT_1A and 5-HT_2A receptors are involved in spinal ACh release. Considering current knowledge, the most probable location of 5-HT_2A receptors is on cholinergic neurones. On activation of the 5-HT_2A receptors the cellular excitability of cholinergic neurones is increased which results in an increasing ACh release. The 5-HT_1A receptors might be located on cell bodies of GABA neurones which inhibit the firing rate of the GABA neurones when activated by serotonin. </p><p>In the fourth study, we investigated the GABA receptor involvement in the regulation in spinal ACh release. We found that GABA_A receptors are tonically inhibiting spinal ACh release. The results further suggest that GABA_B receptors also are involved in the regulation of spinal ACh release. However, unlike GABA_A antagonists, GABA_B antagonists do not increase ACh release. This suggests that GABA_B receptors are not tonically regulating the spinal ACh release. </p>

This thesis examined neurotoxic effects of nicotine exposure during a defined critical period of neonatal brain development in mice. In our environment there are numerous hazardous contaminants that an individual can be exposed to during its entire lifetime. In many mammalian species the neonatal period is characterised by a rapid development of the brain. The present studies have identified a defined critical period during the neonatal brain development in mice, where exposure to low doses of nicotine causes permanent disturbances in the cholinergic nicotinic receptors and altered behaviour response to nicotine at adult age. This adult reaction to nicotine, a hypoactive response, was the opposite of that observed in control animals and animals exposed to nicotine before or after this period. Animals showing a hypoactive response to nicotine lacked nicotinic low affinity binding sites in the cerebral cortex. Furthermore, neonatal exposure to nicotine affected learning and memory in adult animals, an effect that was time-dependent. This thesis also showed that neonatal exposure to nicotine increased adult susceptibility to a repeated exposure of nicotine, manifested as an even more pronounced effect in spontaneous behaviour after challenging doses of nicotine. In these animals the nicotinic receptors in the cerebral cortex, assayed by a-bungarotoxin, was decreased. Neonatal exposure to nicotine was also shown to increase adult susceptibility to the organophosphate paraoxon, a known cholinergic agent, and to the brominated flame retardant 2,2´,4,4´,5-pentabromodiphenyl ether, a novel environmental agent, at adult age. This was seen at doses that did not affect behaviour in control animals, and was manifested as deranged spontaneous behaviour and reduced habituation, aberrations that also worsened with age. The results indicate that differences in adult susceptibility to environmental pollutants are not necessarily an inherited condition. Rather they may well be acquired by low dose exposure to toxic agents during early life.

Impulses coming from peripheries are modified in the spinal cord and transmitted to the brain. Several neurotransmitters have been involved in the processing of impulses in the spinal dorsal horn. Acetylcholine (ACh) is one of many neurotransmitters involved in the regulation of nociception in the spinal cord. In this study we investigated the role of nicotinic, muscarinic, serotonergic and GABA receptors in the regulation of spinal ACh release since these receptors are reported to be involved in spinal nociceptive processes. Different receptor ligands were infused intraspinally via microdialysis and the spinal ACh release was measured by on-line HPLC. Receptor-ligand binding studies were performed with spinal cord homogenates as well as receptors expressed in cells. In the first study, we found that nicotine and some of the nicotinic antagonists used increased ACh release suggesting that spinal ACh release is regulated by different nAChRs. Nicotine and nicotinic agonists may act on different types of receptors with different affinity to produce the observed net effect of increased ACh release. We propose the possibility of an involvement of three different nicotinic receptor subtypes in the regulation of spinal ACh release. The effect of epibatidine, which is regarded as a nicotinic agonist, on muscarinic receptors was investigated in the second study. We propose that epibatidine, in μM concentrations, is a partial muscarinic receptor agonist that may interact with spinal muscarinic receptors to increase ACh release. The dual action on both nAChRs and mAChRs may explain the potent analgesic effect observed after intra-spinal epibatidine administration. In the third study, we investigated the role of serotonin receptor involvement in ACh release control. The results suggest that only 5-HT1A and 5-HT2A receptors are involved in spinal ACh release. Considering current knowledge, the most probable location of 5-HT2A receptors is on cholinergic neurones. On activation of the 5-HT2A receptors the cellular excitability of cholinergic neurones is increased which results in an increasing ACh release. The 5-HT1A receptors might be located on cell bodies of GABA neurones which inhibit the firing rate of the GABA neurones when activated by serotonin. In the fourth study, we investigated the GABA receptor involvement in the regulation in spinal ACh release. We found that GABAA receptors are tonically inhibiting spinal ACh release. The results further suggest that GABAB receptors also are involved in the regulation of spinal ACh release. However, unlike GABAA antagonists, GABAB antagonists do not increase ACh release. This suggests that GABAB receptors are not tonically regulating the spinal ACh release.