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Food intake is essential to all animals. However, when energy consumption through food overtakes energy expenditure, obesity can result. Obesity has been identified as a worldwide health problem associated with diseases such as type 2 diabetes and hypertension. Thus, it is essential to find effective anti-obesity therapies. The aim of this thesis was to determine whether the histaminergic system could be pharmacologically manipulated to alter food intake and whether in particular the H3R is a suitable therapeutic target. Histamine is a central neurotransmitter that plays a major role in controlling energy balance by acting through specific hypothalamic sites. Injections of histamine receptor-1 (H1R) antagonists into the ventromedial hypothalamic nucleus (VMN) cause hyperphagia, whereas antagonism of presynaptic histamine receptor-3 (H3R) causes hypophagia, leading to the hypothesis that selective antagonists or inverse agonists might be potential treatments for obesity through their actions on central H3R. My aim was to assess the precise mode of action of histamine and H3R drugs to affect acute, appetitive behaviour. Using feeding and behavioural studies I demonstrated the acute anorexigenic actions of histamine (ICV) and the H3R inverse agonist, thioperamide (ICV or IP), in rats without disrupting the behavioural satiety sequence. In accordance with predictions, the H3R agonist, imetit (ICV or IP), increased feeding. The actions of both thioperamide and imetit were blocked by the drug proxyfan, which in our model is acting as a neutral H3R antagonist. Interestingly, both thioperamide and imetit caused anorexia in mice. C-Fos functional immunostaining revealed that systemic administration of thioperamide and imetit increased the activity of neurones in the key feeding nuclei of the hypothalamus, including the VMN. To further investigate the mode of action of histaminergic drugs, I carried out extracellular electrophysiological recordings from neurones of the rat VMN in vitro. Of the 197 VMN cells recorded, 62% were histamine-responsive, with 97% of these showing an increase in neuronal firing rates in response to histamine. The excitatory response to histamine was blocked in 90% of instances by pyrilamine, a selective H1R antagonist. Neurones that responded to histamine previously were treated also with thioperamide. 88% of these neurones also responded with an increase in firing. The effect of thioperamide was blocked in all cases by co-administrating pyrilamine, proving that H3R in the VMN are presynaptic autoreceptors, rather than heteroreceptors modulating the release of other transmitters. Imetit had an inhibitory effect on VMN neuronal firing in 86% of recorded cells. Proxyfan was able to block the changes in neuronal firing that both thioperamide and imetit caused. This suggests it is acting as a neutral H3R antagonist in both our in vivo and in vitro models. Thus, using a neutral H3R antagonist we have proven the effects imetit and thioperamide had on feeding and neuronal VMN firing were a direct result of activating a H3R and, therefore, these compounds are receptor-specific for the H3R. In conclusion, our results support a role for histaminergic receptors, including postsynaptic H1R and presynaptic H3R autoreceptors in the VMN, to modulate feeding.

Thirty-four dogs were anesthetized with sodium pentothal i.v. and maintained with i.v. alpha-chloralose. Neuro-muscular blockade was accomplished with gallamine triethiodide (Flaxedil). Respiratory PCO₂ was monitored continuously while artificial ventilation at a rate of 1 5 cpm and appropriate tidal volume was adjusted to maintain expiratory P CO₂ between 38 and 40 mm Hg. Blood gas analysis (P CO₂, PC₂ and pH) allowed maintenance of blood pH between 7.35 and 7.45 by periodic administration of i.v. sodium bicarbonate. Blood volume was maintained with Dextran 75 when necessary. Body temperature was monitored continuously with an esophageal thermister and maintained automatically with heating elements in the operating table. Arterial vascular isolation of the hindlimbs was accomplished by ligating all major branches of the aorta below the renal arteries except the external iliac arteries. The dog's own blood, taken from a cannula in the abdominal aorta just distal to the renal arteries, was perfused at constant flow into cannulae in the external iliac arteries through separate pumps. Each external iliac artery pressure was monitored separately (Fig. 1). A bilateral laminectomy allowed access to the L₅, ₆ and ₇ spinal segments for electrical stimulation of their ventral roots after section of the corresponding dorsal root. In 26 dogs monophasic square wave stimulation (3 to 10V, 3 msec, 8 to 20 Hz) of the ventral root of L₅, L₆ or L₇ induced:1) a decrease in the perfusion pressure (PP) in the ipsilateral hindlimb (-41.8 - 2.7 mm Hg; mean - SE); 2) a decrease in the PP in the contralateral hindlimb (-32.2 - 2.7); 3) a fall in the aortic pressure (-15.6 - 0.7). (Fig.. 3). Similar effects were observed on stimulation of the peripheral stump of the ventral root. The above described vascular effects of ventral root stimulation were resistant to intra-arterial injections of cholinergic and beta-adrenergic blocking agents administered directly into the hindlimb perfusion lines. The effectiveness of the blockades was tested with direct intra-arterial injections of the appropriate agonists. Antihistaminics (diphenhydramine and mepyramine) similarly administered and tested did abolish the response in doses which did not suppress vascular-reactivity to acetylcholine or isoproterenol. These experiments do not provide a clear explanation of the mechanisms responsible for the contralateral vasodilatation or the fall in aortic pressure. The presence of significant anastomotic channels connecting either the two hindlimbs and/or the hindlimbs with the rest of-the body was excluded. Contralateral vasodilatation might perhaps be explained by the presence of nerve fibres crossing the midline in. the fused impar ganglion of the dog. The drop in aortic pressure was not due to the activation of afferent fibres coursing in the ventral roots, nor to the peripheral release of a vasodilator substance since the onset of the phen-omenom was too fast to be explained on these grounds. The possibility exists that the drop in aortic pressure is due to the activation by the stimulated efferent fibres of some afferent nervous pathways carrying inhibitory impulses to the vasomotor centers. The present experiments, however, do not provide data supporting or excluding this hypothesis. The experimental results strongly suggest that the described vasodilatation may be mediated by histamine released directly or indirectly by the activation of fibres coursing into the lower-ventral roots. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

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The early phase of allergic reactions is largely dominated by IgE-mediated degranulation of mast cells and basophils. Mast cells and basophils release mediators which include histamine, prostaglandins, leukotrienes, cytokines, etc. Histamine is a biogenic compound that can directly cause physiological changes that ultimately contribute to allergy and asthma symptoms. The current literature focused on the study of histamine production from “professional histamine producers”, such as mast cells and basophils in allergic diseases. Previous research had shown that activation of allergic specific T cells led to airway narrowing independent of basophil activation 6-hour post exposure. It was therefore possible that the activation of T cells may release histamine which contributed to the airway narrowing observed. This project aimed to determine expression of histidine decarboxylase (HDC) in T lymphocytes, which is the gene encoding the enzyme solely responsible for the production of histamine. We found that HDC is expressed in rare T cell populations by the bioinformatic analysis of publicly available datasets, and we found that the activation of human primary CD4+ T cells by anti-CD3/CD28 did not lead to the upregulation of HDC by qPCR. The activation of CD4+ T cells in non-allergic donors led to a LOG2FC of HDC to B2M housekeeping gene of: -1.0 +/- 0.48 at 6-hour, -1.3 +/- 0.23 at 24-hour, -2.2 +/- 0.32 at 72-hour. The activation of CD4+ T cells in allergic donors led to a LOG2FC of HDC of: -0.48 +/- 0.13 at 6-hour, -2.1 +/- 0.35 at 24-hour, -4.1 +/- 1.1 at 72-hour. In conclusion, HDC expressing T cells were rare and of low expression level. The activation of CD4+ T cells did not upregulate HDC and therefore it was unlikely that T cell derived histamine contribute to allergic manifestations. / Thesis / Master of Science in Medical Sciences (MSMS)

Les amines biogènes (AB) sont des substances physiologiquement actives résultant de la décarboxylation enzymatique des acides aminés libres. Elles participent, dans le corps, aux processus de transmissions d'informations et de régulation de fonctions physiologiques mais peuvent aussi, à doses élevées, avoir des effets néfastes pour la santé. De nombreuses AB sont répertoriées dans la littérature, telles que l'histamine, la cadavérine et la putrescine. Mais, seul le contrôle d'histamine dans la chair de certains poissons de la famille des scombridés est actuellement réglementée (CE n° 2073/2005). Du fait de cette réglementation, les techniques d'analyses chimiques de l'histamine sont largement documentées. Elles peuvent être semi-quantitatives (chromatographie sur couche mince) ou encore quantitatives (fluorescence moléculaire) mais, la chromatographie liquide haute-performance reste, à ce jour, l'unique méthode homologuée au niveau des instances de santé européenne. En marge de ces méthodes traditionnelles, l'enjeu d'un dispositif rapide, peu onéreux et permettant un contrôle plus systématique des aliments anime à la fois la communauté scientifique et le monde économique. Dans ce contexte, la pertinence à recourir à des Quantum Dots (QD) fonctionnalisés par un dérivé d'ortho-phthalaldéhyde (OPA) (utilisé en tant qu'agent de dérivation en fluorescence moléculaire pour les AB) a été posée. Lors de ce projet, deux éléments clés d'un tel dispositif ont été réalisés et caractérisés. Les premiers correspondent aux dérivés fonctionnalisés de l'OPA (4-Me-OPA et 4-HO-OPA). Ils ont été obtenus avec succès selon trois voies de synthèse. Les premières correspondent à des optimisations de synthèses référencées du 4-HO-OPA à partir d'alcool furfurylique ou du 3,4-diméthylphénol. Enfin, celle issue de l'acide 3-méthoxybenzoïque est une adaptation d'une synthèse référencée du 4-MeO-OPA. Dans un second temps, les propriétés du 4-MeO-OPA en tant qu'agent de dérivation vis-à-vis de l'histamine ont été évaluées et comparées à celles de l'OPA. Pour cela, les conditions optimales de complexation ont été étudiées par spectroscopies Ultra-Violet (UV) et de fluorescence moléculaire. Les seconds composants du dispositif sont des nanostructures fluorescentes (QD) dont les propriétés d'émission de fluorescence sont modulables. Notre choix s'est porté sur les QD hydrosolubles de cadmium et sélénium stabilisés par de l'acide mercaptosuccinique. Ces derniers ont été synthétisés par trois modes d'activation (thermique, micro-ondes, ultrasons) et caractérisés par diffractométrie de rayons X, spectrométrie à plasma à couplage inductif, spectroscopies UV et de fluorescence moléculaire. En conclusion, deux entités, le 4-HO-OPA et des QD possédant des ligants de type mercaptosuccinique, ont été réalisées en vue de la mise en place d'un dispositif innovant d'analyse d'histamine. / The biogenic amines (BA) are physiologically active substances resulting from the enzymatic decarboxylation of free amino acids. In the body, they are involved in the transmission of informations and in regulating physiological functions but, at high levels, they can also have damaging health effects. Many BA are recorded in literature, such as histamine, cadaverine and putrescine. Currently, the control of histamine in fish flesh is only regulated in the case of some Scombridae species (CE N° 2073/2005). As a consequence, histamine chemical analysis techniques are widely documented. They can be semi-quantitative (thin layer chromatography) or quantitative (molecular fluorescence). At present, high-performance liquid chromatography remains the only approved method by public heath authorities. Next to the commonly used methods, the challenge of a fast inexpensive system that allows a more systematic control of food animates both the scientific community and the economic world. In this context, a study of the relevance of using Quantum Dots (QD) functionalized by an ortho-phtalaldehyde (OPA) derivative (OPA), used as molecular fluorescence derivation agent for BA, was undertaken. During this project, two key elements of the system were realized and characterized. The first corresponds to OPA functionalized derivates (4-MeO-OPA and 4-HO-OPA). They have been successfully achieved through three synthesis ways. The first two are optimizations of 4-HO-OPA referenced syntheses from furfuryl alcohol or 3,4-dimethylphenol. Finally, the last one uses 3-methoxybenzoic acid and is a 4-Me-OPA reference synthesis adaptation. In a second part, the 4-MeO-OPA properties as a bypass agent for histamine analysis were evaluated and compared with OPA ones. In this case, optimal complexation conditions were analyzed by ultra-violet (UV) and molecular fluorescence spectroscopies. The second components are QD, which are fluorescent nanostructures with molecular properties. We selected water soluble cadmium and selenium QD which were stabilized by mercaptosuccinic acid. They were synthesized by three activation modes (thermal, microwave, ultrasound) and characterized by X-ray diffractometry, inductively coupled plasma spectrometry, UV spectroscopy and molecular fluorescence. In conclusion, two entities, 4-HO-OPA and QD, both having a mercaptosuccinic-type shell, were created as the innovative set up for histamine analysis.

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