A fumaça do cigarro é composta por mais de 4.700 substâncias, muitas das quais tóxicas. O sistema nervoso central (SNC) possui poucas defesas antioxidantes, além de ser rico em lipídeos facilmente oxidáveis e de conter alto teor de metais de transição envolvidos na formação de radicais livres. Existem evidências de que a fumaça do cigarro causa alterações nas enzimas antioxidantes em roedores adultos, mas pouco se sabe sobre os efeitos do tabaco no SNC ainda em desenvolvimento. Assim, este trabalho tem como objetivo avaliar os possíveis efeitos da fumaça do cigarro no SNC de camundongos jovens. Camundongos BALB/c foram expostos a uma mistura de fumaça central e lateral do cigarro (Souza Cruz - Derby Vermelho) dentro de uma câmara de polipropileno acoplada a um sistema Venturi. Além da exposição aguda, realizada no 18º dia de vida, os animais foram expostos a partir do 5º dia de vida por 13 ou 35 dias, durante 2 horas por dia, 7 dias por semana. Os animais foram então eutanasiados por deslocamento cervical imediatamente ou três horas após a última exposição e foram realizadas as determinações das atividades enzimáticas da glutationa peroxidase, glutationa redutase, glutationa S-transferase, além da quantificação de malonaldeído (MDA) e 3-nitrotirosina no cerebelo, córtex frontal, estriado, hipocampo e hipotálamo. Nossos resultados indicam que o SNC em desenvolvimento é susceptível à fumaça do cigarro. Foram detectadas alterações nas enzimas antioxidantes em diferentes estruturas encefálicas imediatamente após a última exposição sugerindo diferença de sensibilidade das diferentes áreas à fumaça do cigarro. Contudo, essas perturbações não são persistentes, uma vez que a maior parte delas desapareceu três horas após a exposição. O cerebelo parece ser a estrutura mais resistente enquanto o córtex frontal e o estriado, as mais sensíveis. No córtex frontal as alterações enzimáticas são mais persistentes e houve aumento de MDA no grupo agudo e eutanásia 3 horas após a exposição, enquanto no cerebelo, estriado e hipocampo houve aumento de MDA no grupo agudo e eutanásia imediatamente após a exposição. Esses resultados sugerem uma resposta mais tardia do córtex frontal e uma possível adaptação dos tecidos ao xenobiótico. É importante destacar que a eutanásia realizada imediatamente após a exposição crônica à fumaça do cigarro não reflete somente o efeito da última exposição, já que tanto o padrão encontrado na alteração das enzimas quanto na determinação de MDA foi diferente do observado após exposição aguda. / Cigarette\'s smoke is composed of more than 4.700 substances, many of them are toxic. The central nervous system (CNS) has few antioxidant defenses, is rich in easily oxidable lipids and contains high levels of transition metals which are involved in the formation of free radicals. There are evidences that tobacco smoke causes changes in the antioxidant enzymes in adult rodents, but little is known about its effects during CNS development. Thus, the aim of this work was to evaluate the possible effects of cigarettes smoke in the CNS of young mice. BALB/c mice were exposed to a mixture of mainstream and sidestream smoke of cigarette (Souza Cruz Red Derby) in a polypropylene chamber attached to a Venturi system. Besides acute exposure, performed at the 18th day of life, the animals were exposed from the 5th day of life for 13 or 35 days, 2 hours a day, 7 days in a week. The animals were then euthanized by cervical dislocation, immediately or three hours after the last exposure. The determinations of enzymatic activities of glutathione peroxidase, glutathione reductase, glutathione S-transferase, and the quantification of malonaldehyde (MDA) and 3-nitrotyrosine in cerebellum, frontal cortex, striatum, hippocampus and hypothalamus were performed. Our results indicate that the CNS in development is susceptible to cigarettes smoke. Alterations in antioxidant enzymes in different brain structures were detected immediately after the last exposure suggesting differences in sensitivity of different areas to cigarette\'s smoke. However, these disturbances are not persistent, as most of them disappeared three hours after exposure. Cerebellum seems to be the more resistant structure, while frontal cortex and striatum the most sensitive. Enzyme changes in frontal cortex were more persistent and there was an increase of MDA only in the acute group and euthanasia 3 hours after exposure, whereas in cerebellum, striatum and hippocampus, MDA increased only in acute group and immediately euthanasia after exposure. These results suggest a delayed response of the frontal cortex and a possible adaptation of tissues to xenobiotics. It is important to point out that euthanasia performed immediately after chronic exposure to cigarette smoke not only reflect the effect of the last exposure, since the pattern found in the modification of enzymes and in the determination of MDA was different from that observed after acute exposure.

A nicotina é um alcalóide extraído da planta do tabaco e age como agonista em neurônios e outras células que expressam receptores nicotínicos. Essa substância é capaz de atravessar a barreira hematoencefálica e mimetizar os efeitos da acetilcolina endógena ao interagir com os diferentes subtipos de receptores. O hipocampo e o cerebelo são regiões encefálicas ricas em receptores nicotínicos como o alfa7 e o alfa42 que estão também envolvidos na progressão de doenças neurodegenerativas, como a doença de Alzheimer, e neuropsiquiátricas, como a esquizofrenia, ambas apresentando um contexto inflamatório. O lipopolissacarídeo (LPS) é um indutor inflamatório que age via receptor toll-4 (TLR-4) ativando, dentre outros, o fator de transcrição NF-capaB. Vários estudos têm demonstrado tanto in vitro quanto in vivo que a exposição crônica à nicotina inibe a resposta imune. Uma vez que o fator de transcrição NF-capaB regula a expressão gênica de diversos fatores que medeiam a plasticidade e o desenvolvimento neuronal e inflamação, nosso objetivo foi avaliar os efeitos da nicotina crônica sobre a via de sinalização induzida pelo LPS. Sendo assim, avaliou-se a translocação nuclear do NF-capaB, a expressão de indutores inflamatórios (TNF, IL-1 e iNOS) e também do receptor TLR-4 tanto no hipocampo quanto no cerebelo. Além disso, foram determinados os níveis plasmáticos das citocinas TNF, IL-1, MIP-1alfa, CINC2alfa/. Nossos resultados mostram que, perifericamente, o LPS induziu a produção das citocinas plasmáticas, mas a nicotina não foi capaz de reverter seus efeitos. Já no cerebelo e no hipocampo, a nicotina crônica (0,1 e 1,0 mg/kg) interferiu significativamente na translocação do NF-capaB induzida pelo LPS, levando inclusive a um bloqueio importante da expressão do mRNA de TNF, IL-1, iNOS no hipocampo, e de IL-1 no cerebelo. Ademais, no hipocampo, a nicotina também interferiu na expressão do receptor TLR-4. A mecamilamina, antagonista heteromérico não-seletivo não foi capaz de reverter os efeitos da nicotina sobre o NF-capaB no hipocampo, mas o fez no cerebelo. Já o antagonista seletivo de receptor alfa 7, metililaconitina, bloqueou a ação da nicotina no hipocampo. / Nicotine is an alkaloid extracted from Tobacco plants, and it acts as an agonist in neurons and other cells that express nicotinic receptors. This substance crosses the blood brain barrier and mimics endogenous acetylcholine in interacting with various receptor subtypes. The hippocampus and cerebellum are brain regions rich in nicotinic receptors including 7, 42 and other subtypes, involved in neurodegenerative and neuropsychiatric disorders, including Alzheimer disease and schizophrenia, respectively, in a neuroinflammatory context. The lipopolysaccharide (LPS) is a known inflammatory inducer that exerts its effects through the Toll-like receptor 4 (TLR-4) and activates the transcription factor NF-capaB. In the present work, we evaluate the effects of chronic nicotine on the nuclear translocation of NF-capaB, on the expression of NF-capaB regulated inflammatory inducers (TNF, IL-1 and iNOS) and on the LPS-induced TLR4 mRNA expression in response to chronic nicotine treatment in hippocampus and cerebellum. In addition, we accessed the production of peripheral cytokines (TNF, IL-1, MIP-1, CINC 2/). Our results demonstrate that LPS induced the production of peripheral cytokines but nicotine treatment did not interfere in their plasma levels. However, in cerebellum and hippocampus, chronic nicotine 1.0 and 0.1 mg/kg interfered in the LPS-induced NF-capaB leading to a deficient mRNA expression of its related genes TNF, IL-1 and iNOS in hippocampus and of IL-1, in cerebellum. Chronic nicotine (0.1 mg/kg, s.c.) significantly interfered in the TLR4 mRNA expression in LPS-challenged animal group in hippocampus. The non-selective heteromeric antagonist mecamylamine (1.0 mg/kg s.c.) reverted the nicotine effects over NF-capaB nuclear translocation in cerebellum, but not in hippocampus. On the other hand, the 7 antagonist methyllycaconitine (MLA) (5.0 mg/kg i.p.) reverted the nicotine effects over NF-capaB in hippocampus but not in cerebellum. Nicotine prevents LPS effects, both in cerebellum and hippocampus, once it reduced significantly the nuclear translocation of NF-capaB and the expression of related pro-inflammatory cytokines. Besides, the nicotinic effects seem to be mainly mediated by heteromeric receptors in cerebellum and through 7 receptors in hippocampus.

<p>The duodenal lumen is exposed to aggressive factors with a high potential to cause damage to the mucosa. Bicarbonate secretion by the duodenal mucosa is accepted as the primary important defense mechanism against the hydrochloric acid intermittently expelled from the stomach.</p><p>The present thesis concerns the influence of the central nervous system and the effects of the hormone melatonin on bicarbonate secretion in anesthetized rats in vivo. Effects of melatonin on intracellular calcium signaling by duodenal enterocyte in vitro were examined in tissues of both human and rat origin. The main findings were as follows:</p><p>Melatonin is a potent stimulant of duodenal mucosal bicarbonate secretion and also seems to be involved in the acid-induced stimulation of the secretion. Stimulation elicited in the central nervous system by the α1-adrenoceptor agonist phenylephrine induced release of melatonin from the intestinal mucosa and a four-fold increase in alkaline secretion. The melatonin antagonist luzindole abolished the duodenal secretory response to administered melatonin and to central nervous phenylephrine but did not influence the release of intestinal melatonin. Central nervous stimulation was also abolished by synchronous ligation of the vagal trunks and the sympathetic chains at the sub-laryngeal level. </p><p>Melatonin induced release of calcium from intracellular stores and also influx of extracellular calcium in isolated duodenal enterocytes. Enterocytes in clusters functioned as a syncytium.</p><p>Overnight fasting rapidly and profoundly down-regulated the responses to the duodenal secretagogues orexin-A and bethanechol but not those to melatonin or vasoactive intestinal polypeptide.</p><p>In conclusion, the results strongly suggest that intestinal melatonin plays an important role in central nervous elicited stimulation of duodenal mucosal bicarbonate secretion. Sensitivity of this alkaline secretion to some peripheral stimulators markedly depends on the feeding status.</p>

The duodenal lumen is exposed to aggressive factors with a high potential to cause damage to the mucosa. Bicarbonate secretion by the duodenal mucosa is accepted as the primary important defense mechanism against the hydrochloric acid intermittently expelled from the stomach. The present thesis concerns the influence of the central nervous system and the effects of the hormone melatonin on bicarbonate secretion in anesthetized rats in vivo. Effects of melatonin on intracellular calcium signaling by duodenal enterocyte in vitro were examined in tissues of both human and rat origin. The main findings were as follows: Melatonin is a potent stimulant of duodenal mucosal bicarbonate secretion and also seems to be involved in the acid-induced stimulation of the secretion. Stimulation elicited in the central nervous system by the α1-adrenoceptor agonist phenylephrine induced release of melatonin from the intestinal mucosa and a four-fold increase in alkaline secretion. The melatonin antagonist luzindole abolished the duodenal secretory response to administered melatonin and to central nervous phenylephrine but did not influence the release of intestinal melatonin. Central nervous stimulation was also abolished by synchronous ligation of the vagal trunks and the sympathetic chains at the sub-laryngeal level. Melatonin induced release of calcium from intracellular stores and also influx of extracellular calcium in isolated duodenal enterocytes. Enterocytes in clusters functioned as a syncytium. Overnight fasting rapidly and profoundly down-regulated the responses to the duodenal secretagogues orexin-A and bethanechol but not those to melatonin or vasoactive intestinal polypeptide. In conclusion, the results strongly suggest that intestinal melatonin plays an important role in central nervous elicited stimulation of duodenal mucosal bicarbonate secretion. Sensitivity of this alkaline secretion to some peripheral stimulators markedly depends on the feeding status.

Drug dependence is a serious and increasing problem in our society, especially among adolescents. The use of the large variety of substances available can result in a range of physiological and psychological adverse effects on individuals and negative consequences on the society overall. Several different types of drugs induce neurotoxicological damages, which in turn can generate impairment in for example the reward system and affect cognitive parameters.  The drug gamma-hydroxybutyrate (GHB) is usually considered a harmless compound among abusers, but has now shown to be highly addictive. Furthermore, GHB can cause memory impairments in both humans and animals. On the contrary, growth hormone (GH) and its main mediator insulin-like growth factor 1 (IGF-1) have recently been suggested to improve memory and learning in several studies. The hormones exhibit certain neuroprotective capabilities and have also previously been demonstrated to reverse opioid induced apoptosis in hippocampal cells. These effects and the fact that GHB is shown to increase GH secretion, which attracted considerable attention among body builders, led us to initiate studies on GHB and its impact on relevant systems in the central nervous system (CNS). Thus, the main purpose of the present investigation was to elucidate some of the underlying mechanisms that could account for the effects exerted by GH and GHB in the CNS. We found that a) GH affects the density and functionality of GABAB-receptors and opioid receptors in the male rat brain, b) GHB induces cognitive deficits and down-regulates GABAB-receptors, c) GHB treatment creates an imbalance between the endogenous opioids Met-enkaphalin-Arg6Phe7 (MEAP) and dynorphin B and increases the levels of MEAP in regions of the brain that are associated with drug dependence, and d) GHB affects the expression of IGF-1 receptors but not the plasma levels of IGF-1. In conclusion, the present work demonstrates that GH interacts with both opioid and GABAB-receptors in the male rat CNS and that GHB has an impact on brain regions associated with cognition and the development of dependence. These observations may be of relevance in many aspects related to addiction and might be translated into humans.

Spinal manipulative therapy (SMT) is a therapeutic procedure employed by various healthcare practitioners for alleviating acute and chronic musculoskeletal complaints. This form of treatment is also delivered to enhance the performance and augment the rehabilitation of athletes. However, despite research findings alleging the strength-modulating effects of SMT alongside numerous professional athletes’ positive anecdotal claims concerning its results, the physiological processes to explain its effects remain largely unexplained. Therefore, the purpose of this work was to investigate the effects of SMT in a college-aged sample population with two experiments. The first study examined the effect of SMT targeting the lumbosacral region on concentric force production of the knee extensors and flexors. A randomized, controlled, single-blind crossover design was utilized with 21 subjects. Isometric and isokinetic peak torques (Nm) were recorded during maximal voluntary isometric contractions (MVIC) or maximal voluntary contractions (MVC) post-treatment of either SMT or a sham manipulation. The second study incorporated the same experimental design with 20 subjects to examine the effects of SMT on central nervous system (CNS) excitability. This was accomplished by assessing postactivation potentiation (PAP), measured with the Hoffmann Reflex (H-reflex). PAP is an enhanced neuromuscular response to prior contractile activity, and the H-reflex is the electromyographic (EMG) recording of submaximal electrical stimulation of the Ia monosynaptic reflex pathway. Subsequent to SMT and/or a plantar flexion MVIC, EMG amplitudes and isometric twitch torque generation of the gastrocnemius and soleus muscles were recorded during tibial nerve stimulations. The results of the first study indicate that SMT did not produce a significant strength-modulating effect during isometric and isokinetic contractions of neither knee extension nor flexion. Similarly, the second study revealed that SMT immediately preceding the MVIC to induce PAP did not significantly increase H-reflex EMG amplitudes of either muscle or the simultaneous isometric twitch torque generation compared to the MVIC only. These data from both investigations suggest that SMT does not enhance strength or PAP. The positive anecdotal claims of athletes who utilize SMT may be due to other factors, such as the clinical efficacy of the treatment in addressing musculoskeletal injuries or a placebo effect.

Myelin and oligodendrocytes (OLGs) are the apparent targets of the immune-mediated injury that underlies the development of multiple sclerosis (M8). Recovery from M8 clinical relapses likely reflects remyelination attributed to recruitment and differentiation of oligodendrocyte progenitor cells (OPCs), rather than to new process formation by previously myelinating OLGs. Newly emerging M8-directed immuno-modulatory therapies (statins and FTY720) can readily cross the blood-brain barrier and have been shown to impact signaling pathways implicated in cytoskeletal regulation, differentiation, migration, and survival; these are cellular events presumably important for myelin integrity and remyelination. / Statins inhibit the production of cholesterol (concentrated in the myelin membrane) and isoprenoids (post-translational attachments regulating the functions of proteins such as the Rho GTPases). We showed that treatment of human and rodent-derived OPCs with lipophilic statins induced an initial process extension associated with enhanced differentiation and impaired spontaneous migration, whereas prolonged treatment induced process retraction and cell death. Rodent and human mature OLGs demonstrated similar cytoskeletal and survival responses. Chronic simvastatin therapy of mice inhibited remyelination following demyelination induced by the OLG toxin, cuprizone, attributed to a block in OPC differentiation and consequent decrease in mature OLGs. Even fully myelinated animals treated with simvastatin over the long-term demonstrated a decrease in myelin in the brain by maintaining oligodendroglial cells in the pre-OLG state and preventing continual replacement of mature OLGs. / FTY720 is an agonist of G-protein-coupled receptors S1P1, 3, 4, and 5, that are associated with distinct receptor isotype-selective activation of Rho GTPases. In human OPCs, FTY720 could induce initial S1P3/5-dependent process retraction associated with an inhibition of differentiation, and subsequent S1P1-dependent process extension. Mature OLGs showed a dose-dependent cyclic modulation of process extension and retraction was observed over time. Both human OPCs and OLGs were rescued by FTY720 under death-promoting environments. Both cell types also demonstrated a cyclic and reciprocal modulation of S1P1 and S1P5 mRNA levels, reflected in the recurring receptor isotype-dependent functional responses over time. Studies using organotypic cerebellar slice cultures demonstrated that FTY720 did not impact myelin integrity under basal conditions, yet accelerated remyelination following lysolecithin-induced demyelination. Both treatment regimens were associated with an extension of OPC and mature OLG processes. / Our observations demonstrate that drug concentrations used to modulate immune function can have differential dose and time-dependent effects on OPCs, OLGs, as well as on myelin and remyelination processes. Our findings indicate the need to monitor the effects of putative immuno-modulatory therapies on myelin-related processes in MS patients.

The majority of the population in South Africa use traditional health care to treat various mental conditions. This thesis has two main objectives; to bring together a comprehensive and detailed record of psychotropic plants used in southern Africa by indigenous peoples for medicinal or cultural purposes. Secondly, this research attempts to investigate the validity and rationale of the use of these plants by screening them in various biological assays for psychotropic activity. Plants were selected, based on their traditional use and availability, and were screened in four assays, which detect biological activity of a useful nature. A number of in vitro enzymatic and neuronal signal transduction assays were employed in this thesis, the inhibition of the serotonin reuptake transporter protein (SERT); inhibition of catabolic enzymes (e.g. acetylcholinesterase, monoamine oxidase); GABAA- benzodiazepine receptor binding. The influence of legislation, past and present, on the state of traditional medicine is highlighted. Aspects of the philosophies and practises of the various practitioners of South African traditional medicine will be discussed. An annotated list compiled from available ethnobotanical literature of plants traditionally used for central nervous system-related purposes is provided. It contains more than 330 species, from 94 families, which are currently used or have been used for cultural, medicinal and recreational purposes related to the central nervous system (CNS). Where available, information pertaining to plant part used, preparation method, dosage, route of administration, known and potentially active constituents are included. Seventy five extracts from 34 indigenous plant species used in South African traditional medicine or taxonomically related to these were investigated for their affinity to the serotonin reuptake transport protein, making use of an in vitro [3H]-citalopram serotonin reuptake transport protein binding assay. Aqueous and 70% ethanolic extracts of various plant parts were screened and 45 extracts derived from 15 plant species showed affinity. The affinity of 12 extracts from four plants was characterized as high (more than 50% inhibition at 5, 1, and 0.5 mg/ml). Plant species with high affinity to the serotonin reuptake transport protein included Agapanthus campanulatus, Boophone disticha, Datura ferox and Xysmalobium undulatum. Agapanthus campanulatus yielded high activity in aqueous extracts from leaves and flowers. B. disticha showed high activity both in aqueous and ethanolic extracts of leaves and bulbs. D. ferox showed high activity in aqueous extracts from the seeds and X. undulatum showed high activity in the ethanolic extract of the whole plant. Two compounds, buphanadrine and buphanamine, were isolated by bioassay-guided fractionation on vacuum-liquid-chromatography (VLC) and preparative thin-layer-chromatography (TLC) from B. disticha. The structures of the compounds were determined by 1H and 13C NMR. Fractions were tested for affinity to the serotonin transporter in a binding assay using [3H]-citalopram as a ligand. The IC50 values of buphanidrine and buphanamine were 274 ìM (Ki = 132 ìM) and 1799 ìM (Ki = 868 ìM), respectively. The two alkaloids were also tested for affinity to the 5HT1A receptor, but only showed slight affinity. Aqueous and ethanol extracts of 43 plants that are traditionally used to treat against epilepsy and convulsions were initially tested in the GABAA-benzodiazepine receptor binding assay, where the binding of 3H-Ro 15-1788 (flumazenil) to the benzodiazepine site is measured. The GABAA-benzodiazepine receptor complex is involved in epilepsy and convulsions. Out of the 118 extracts tested, one aqueous and 18 ethanol extracts showed activity. The most active extracts were the ethanolic leaf extracts of Searsia tridentata, Searsia rehmanniana and Hoslundia opposita and the ethanolic corm extract of Hypoxis colchicifolia, which all showed good dose-dependent activity. A further forty-six ethanol extracts from another 35 species, both indigenous and exotic that are traditionally used predominantly as sedatives or to treat various CNS-related ailments were tested in the GABAA-benzodiazepine receptor-binding assay. Out of the 46 extracts tested, seven showed good activity and 10 showed moderate activity. The most active extracts were the ethanolic leaf extracts of Arctopus echinatus, Artemisa afra, four Helichrysum species and Mentha aquatica which all showed good dose-dependent activity. Two biflavonoids with activity in the 3H-Ro 15-1788 (flumazenil) binding assay were isolated by high pressure liquid chromatography (HPLC) fractionation of the ethanol extract of the leaves from Searsia pyroides. The structures of the two biflavonoids were elucidated by nuclear magnetic resonance spectroscopy (NMR) to be agathisflavone and amentoflavone. Agathisflavone and amentoflavone competitively inhibited the binding of 3H-Ro 15-1788 with a Ki of 28 and 37 nM, respectively. Extracts of Searsia dentata and Searsia pentheri were not as active as the extract from Searsia pyroides; both were found to contain apigenin and agathisflavone. The monomer apigenin, agathisflavone and amentoflavone were fitted into a pharmacophore model for ligands binding to the GABAA receptor benzodiazepine site. This reflected the affinities of the compounds in the [3H]-flumazenil binding assay. Mentha aquatica, a mint that is found in Europe and Africa, is used in Zulu traditional medicine for spiritual purposes. The ethanolic leaf extract showed a strong affinity to the GABA-benzodiazepine receptor. Viridiflorol from the essential oil and (S)-naringenin from an ethanolic extract was isolated by bioassay-guided fractionation using binding to the GABA-benzodiazepine site. Viridiflorol had an IC50 of 0.19 M and (S)-naringenin of 0.0026 M. Twenty plants used in Zulu traditional medicine for several CNS-related ailments were screened for MAO inhibition and specific MAO-B inhibition activity. MAO-B inhibitors are currently employed in the treatment of neurodegenerative related illnesses such as Parkinson's and Alzheimer's diseases. A photometric peroxidase linked assay was used to determine the inhibition of the oxidative deamination of tyramine by MAO isolated from rat liver. Ruta graveolens exhibited the best MAO inhibitory activity (ethyl acetate leaf extract = IC50 5 ± 1 ìg/ml, petroleum ether extract = 3 ± 1 ìg/ml) and specific MAO-B inhibition (ethyl acetate leaf extract = IC50 7 ± 6 ìg/ml petroleum ether extract = 3 ± 1 ìg/ml). Schotia brachypetala, Mentha aquatica and Gasteria croucheri also exhibited good MAO-B inhibition activity. Six extracts of varying polarity of Mentha aquatica were tested in a photometric peroxidase linked MAO bioassay. The 70% ethanol extract had highest inhibitory activity. (S)-Naringenin was isolated from the extract by bioassay guided fractionation on VLC and preparative TLC. The structure of the compound was determined by 1H, 13C and 13C-DEPT NMR and optical rotation. The IC50 values for MAO inhibition by naringenin were 342 ± 33 ìM for the rat liver mitochondrial fraction, 955 ± 129 ìM for MAO-A and 288 ± 18 ìM for MAO-B respectively. South African traditional medicine clearly utilizes many botanical species with CNS-related activity. Only a small number of the more than 330 southern African plant species reported to treat or alter the CNS have been scientifically evaluated. To date very few of the active compounds have been isolated and identified. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Les troubles anxieux sont parmi les troubles psychiatriques les plus souvent diagnostiqués chez les adolescents. Ces troubles sont souvent accompagnés de nombreuses comorbidités, dont des difficultés de sommeil. L’objectif principal de cette thèse est de caractériser l’activité corticale à l’éveil et pendant le sommeil à l’aide de l’EEG quantifié chez une population d’adolescents présentant un trouble anxieux, et de la comparer à un groupe témoin d’adolescents. Dans un second temps, on cherche à savoir si l’activité EEG des patients anxieux corrèle avec différentes mesures cliniques. Deux études permettent de répondre à ces objectifs, une première portant sur l’activité EEG au cours de l’éveil, et une seconde portant sur l’activité EEG au cours du sommeil (SL et SP). La première étude démontre que l’activité EEG des deux groupes ne présente pas de différence à l’EEG le soir. Par contre, le matin, les patients anxieux présentent une activité significativement supérieure à celle des contrôles aux électrodes centrales (0,75-10 Hz et 13-20 Hz) ainsi qu’aux électrodes occipitales (2,5-7,75 Hz). Dans la seconde étude, nous avons analysé l’activité EEG absolue et relative en SL et en SP. Nous avons trouvé une activité absolue significativement supérieure à l’EEG de la région centrale chez les participants du groupe anxieux : en SLP (stades 3 et 4) sur l’ensemble des bandes de fréquence, en stade 2 sur les bandes de fréquence thêta, alpha et beta seulement. Finalement, en SP, les différences sont trouvées en alpha et beta, et non en thêta et delta. Les résultats obtenus à ces deux études suggèrent la présence de mécanismes de synchronisation et de filtrage inadéquats au niveau de la boucle thalamo-corticale, entraînant une hypervigilance du SNC. Quant aux corrélations entre l’activité EEG et les mesures cliniques, les résultats obtenus dans les deux études révèlent que les fréquences lentes (thêta et delta) de l’activité d’éveil le matin corrèlent à la fois avec l’anxiété de trait et d’état et les fréquences rapides (Alpha et Beta) de l’EEG du sommeil corrèlent sélectivement avec l’anxiété d’état. Il semble donc exister un lien entre les mesures cliniques et l’activité EEG. Une hausse d’activité EEG pourrait être un indicateur de la sévérité accrue des symptômes anxieux. / Anxiety disorders are among the most diagnosed psychiatric disorders in the adolescent population. These disorders are often accompanied by different comorbidities, such as sleep problems. The main objective of this thesis is to characterize the cortical activity during wake and sleep, using quantified EEG, in a population of adolescents presenting an anxiety disorder, and to compare these results to those of a control group of adolescents. Secondly, we wish to verify if the EEG activity of the anxious participants correlates with different clinical measures. Two different studies are conducted in order to attain our objectives, the first one being on the EEG activity during wake, and the second being on the EEG activity during sleep (slow wave sleep and rapid eye movement sleep). The first study reveals that the EEG activity from both groups does not differ in the evening. However, in the morning, anxious participants display an increased activity on central electrodes (0.75-10 Hz and 13-20 Hz), and on occipital electrodes (2.5-7.75 Hz). In the second study, we demonstrate that anxious participants show an increased absolute EEG activity on central electrodes: in slow wave sleep (stages 3 and 4), it is found on all frequency bands, in stage 2, it is found on the theta, alpha and beta frequency bands. Finally, in rapid eye movement sleep, the differences are only in alpha and beta, and not in theta and delta. These data suggest the impairment of thalamo-cortical gating mechanisms in adolescents with anxiety disorders, leading to CNS hyperarousal. As for the correlations between the EEG activity and the clinical measures, the results from our studies reveal that the slow frequencies (theta and delta) of morning wake EEG correlate with both trait and state anxiety, while fast frequencies (alpha and beta) from the sleep EEG correlate specifically with state anxiety. Thus, there appears to be an association between EEG activity and clinical measures. An increased EEG activity could be an indicator of the severity of the anxious symptoms.

Identification, monitoring and quantification of biomolecules in the CNS is a field of growing interest for identifying biomarkers of neurological diseases. In this thesis, silicon needle-shaped multi-molecules sensing microprobes were developed. Our microelectrode array design comprises a needle length of 6mm with 100x50 µm2 cross-section bearing three platinum electrodes with a size of 40x200 µm and 200µm spacing between them. We have used these microprobes for simultaneous glucose and lactate monitoring, using the third electrode for control of non-specific current variations. Local microdroplet protein deposition on the electrode surface was achieved using a pneumatic picopump injection system. Enzyme immobilization on the electrode surface is a key step in microelectrode biosensor fabrication. We have developed a simple, low cost, non-toxic enzyme immobilization method employing poly(ethyleneglycol) diglycidyl ether (PEGDE). Successful biosensor fabrication was demonstrated with glucose oxidase, D-amino acid oxidase, and glutamate oxidase. We found that these biosensors exhibited high sensitivity and short response time sufficient for observing biological events in vivo on a second-by-second timescale. PEGDE-based biosensors demonstrated an excellent long-term stability and reliably monitored changes in brain glucose levels induced by sequential administration of insulin and glucose solution. We then carried out a comparative study of five enzyme immobilization procedures commonly used in Neuroscience: covalent immobilization by cross-linking using glutaraldehyde, PEGDE, or a hydrogel matrix and enzyme entrapment in a sol-gel or polypyrrole-derived matrices. Enzymatic microelectrodes prepared using these different procedures were compared in terms of sensitivity, response time, linear range, apparent Michaelis-Menten constant, stability and selectivity. We conclude that PEGDE and sol-gel techniques are potentially promising procedures for in vivo laboratory studies. The comparative study also revealed that glutaraldehyde significantly decreased enzyme selectivity while PEGDE preserved it. The effects that immobilization can have on enzyme substrate specificity, produce dramatic consequences on glutamate detection in complex biological samples and in the CNS. Our biosensor's results were systematically controlled by HPLC or capillary electrophoresis. The highly selective PEGDE-based biosensors allowed accurate measurements glutamate concentrations in the anesthetized and awaked rats at physiological conditions and under pharmacological and electrical stimulations. The microfabricated multielectrodes based on silicon needles coupled to the simple, non-toxic and mild immobilization method based on PEGDE, open new possibilities for specific neurotransmitter detection in the central nervous system and the study of cell-cell communication in vivo.