La ciguatéra, ichtyosarcotoxisme majoritairement rencontré dans la zone intertropicale, trouve son origine dans la consommation de poissons tropicaux contaminés par des neurotoxines marines, les ciguatoxines (CTXs). Ces polyéthers polycycliques synthétisés par les micro-algues du genre Gambierdiscus, se fixent de façon privilégiée aux canaux sodiques dépendant du potentiel. Cependant, ce seul mode d'action n'explique pas tous les symptômes observés dans la ciguatéra notamment les manifestations particulières de type inflammatoire. L'effet de deux toxines ciguatérigènes, la P-CTX-1B et la P-CTX-3C, ainsi que l'activité d'un analogue, la brévétoxine PbTx-3, sur la modulation des médiateurs de l'inflammation que sont les cytokines, a été caractérisé sur un modèle d'étude in vitro de cellules de macrophages murins RAW 264.7. Le suivi de l'expression génique au moyen de techniques de PCR quantitative a révélé que la P-CTX-1B est capable d'induire les cytokines pro-inflammatoires interleukine (IL)-1β, IL-6 et le Tumor Necrosis Factor (TNF)-α, et l'anti-inflammatoire IL-10. Par ailleurs, alors que les traitements proposés par la médecine occidentale sont essentiellement symptomatiques, la médecine traditionnelle propose l'utilisation de nombreuses plantes dont certaines sont capables de réduire les effets des CTXs in vitro et in vivo. La nécessité d'enrichir l'arsenal thérapeutique de la ciguatéra ainsi que l'implication probable des mécanismes inflammatoires dans cette pathologie nous ont conduits à caractériser le potentiel anti-inflammatoire de quatre plantes parmi celles traditionnellement employées dans le Pacifique. Les effets d'extraits aqueux de Cerbera manghas, Euphorbia hirta, Heliotropium foertherianum et Vitex trifolia sur la cinétique de synthèse des ARNm ainsi que sur la production des protéines cytokiniques, ont été étudiés sur le modèle d'induction de macrophages murins stimulés par un lipopolysaccharide bactérien (LPS). Les résultats montrent que E. hirta et V. trifolia possèdent les activités anti-inflammatoires les plus intéressantes et cette dernière a été sélectionnée pour de plus amples tests. L'extrait aqueux de V. trifolia montre la capacité à inhiber les cytokines pro-inflammatoires IL-1β, IL-6 et TNF-α et à induire l'anti-inflammatoire IL-10 dépendant du LPS. La finalité du projet doit permettre l'étude de principes actifs isolés des plantes sélectionnées et les composés d'intérêt pourront être testés sur modèle animal avant d'être validés sur l'homme. Ainsi de nouveaux traitements thérapeutiques de la ciguatéra pourront-ils être proposés. Mots clés : ciguatéra, ciguatoxine, cytokine, PCR quantitative, ELISA, physiopathologie, remèdes traditionnels, Vitex trifolia, Euphorbia hirta, Cerbera manghas, Heliotropium foertherianum.

<p>Insulin release from single islets of Langerhans is pulsatile. The secretory activities of the islets in the pancreas are coordinated resulting in plasma insulin oscillations. Nutrients amplitude-regulate the insulin pulses without influencing their frequency. Diabetic patients show an abnormal plasma insulin pattern, but the cause of the disturbance remains to be elucidated. Ithe present thesis the influence of the cytoplasmic calcium concentratio([Ca²⁺]_i) and cell metabolism on pulsatile insulin release was examined in single islets of Langerhans from <i>ob/ob</i>-mice. Glucose stimulation of insulin release involves closure of ATP-sensitive K⁺ channels (K_ATP channels), depolarization, and Ca²⁺ influx in β-cells. In the presence of 11 mM glucose, pulsatile insulin secretion occurs in synchrony with oscillations i[Ca²⁺]_i. When [Ca²⁺]_i is low and stable, e.g. under basal conditions, low amplitude insulin pulses are still observed. When [Ca²⁺]_i is elevated and non-oscillating, e.g. when the β-cells are depolarized by potassium, high amplitude insulin pulses are observed. The frequency of the insulin pulses under these conditions is similar to that observed when [Ca²⁺]_i oscillations are present. By permanently opening or closing the K_ATP channels with diazoxide or tolbutamide, respectively, it was investigated if glucose can modulate pulsatile insulin secretion when it does not influence the channel activity. Under these conditions, [Ca²⁺]_i remained stable whereas the amplitude of the insulin pulses increased with sugar stimulation without change in the frequency. Metabolic inhibition blunted but did not prevent the insulin pulses. The results indicate that oscillations in metabolism can generate pulsatile insulin release when [Ca²⁺]_i is stable. However, under physiological conditions, pulsatile secretion is driven by oscillations in metabolism and [Ca²⁺]_i, acting in synergy.</p>

Groundnut or peanut (Arachis hypogaea L.) is an important legume cultivated in several developing countries in the tropics and subtropics. It plays a significant role as a food crop in regions with alarming population growth rates. The disease “peanut clump”, which is caused by viruses in the genus Pecluvirus, has been reported from India and from several countries of West Africa. In India, the causal agent is the Indian peanut clump virus (IPCV), which is transmitted by a soil-borne root parasite, Polymyxa graminis. The virus is also transmitted by infected seed and so far no economical method of control has been found. Therefore efforts have been concentrated on understanding the epidemiology of peanut clump disease with the aim of devising cultural methods of control. The work addressed in this thesis describes how investigation in various aspects of clump disease epidemiology, including identification of alternative hosts of the virus and the vector, and of factors that contribute to survival and spread of inoculum, has led to formulation of simple cultural practices that could reduce disease incidence.

Groundnut or peanut (Arachis hypogaea L.) is an important legume cultivated in several developing countries in the tropics and subtropics. It plays a significant role as a food crop in regions with alarming population growth rates. The disease “peanut clump”, which is caused by viruses in the genus Pecluvirus, has been reported from India and from several countries of West Africa. In India, the causal agent is the Indian peanut clump virus (IPCV), which is transmitted by a soil-borne root parasite, Polymyxa graminis. The virus is also transmitted by infected seed and so far no economical method of control has been found. Therefore efforts have been concentrated on understanding the epidemiology of peanut clump disease with the aim of devising cultural methods of control. The work addressed in this thesis describes how investigation in various aspects of clump disease epidemiology, including identification of alternative hosts of the virus and the vector, and of factors that contribute to survival and spread of inoculum, has led to formulation of simple cultural practices that could reduce disease incidence.

This thesis describes studies directed towards development of novel antiadhesive agents, with particular emphasis on compounds that prevent attachment of bacteria to a host-cell. Three different proteins involved in the assembly or function of adhesive pili in uropathogenic Escherichia coli have been targeted either by rational structure based design or statistical molecular methods. A library of substituted galabiose (Galα1-4Gal) derivatives was screened for binding to the E. coli adhesin PapG in an assay based on surface plasmon resonance, and for inhibition of Streptococcus suis adhesins PN and PO in a hemagglutination assay. The results were used to generate QSAR models which had good predictive powers and provided further insight in the structural requirements needed for high affinity binding. 2-pyridones and amino acid derivatives were modelled into the binding site of chaperones involved in pilus assembly in E. coli and a heuristic method, VALIDATE, was used for affinity prediction. The affinity of the compounds for the chaperones PapD and FimC were assessed in assays based on surface plasmon resonance and relaxation-edited NMR spectroscopy. Their ability to disrupt chaperone/subunit complexes was investigated in vitro through a FPLC assay and their capacity to inhibit pilus formation in vivo was determined via hemagglutination and confirmed with atomic force microscopy. Statistical molecular design was used to design a diverse peptide library targeting pili subunits, and an ELISA was developed to investigate the ability of the peptides to inhibit chaperone/subunit complexation. The resulting QSAR model provided extensive information regarding binding of the peptides to the subunits. Because the peptides were suggested to bind in an extended β-strand formation, β-strand mimetics consisting of oligomeric enaminones were designed. Finally, new methods to synthesize enaminone building blocks were developed using microwave assisted chemistry. The projects described have generated compounds that besides their value as leads for developing novel antibacterial agents, also constitute new chemical tools to study the mechanisms underlying bacterial virulence.

<p>This study shows that herbs can be effectively screened for potiential bio-activity using in vitro methods. Further studies will be needed to better explore Artemisia afra&rsquo / s effect on immunoregulation, particularly long term effects of the herb on the immune system and its effect on other disease states.</p>

Insulin release from single islets of Langerhans is pulsatile. The secretory activities of the islets in the pancreas are coordinated resulting in plasma insulin oscillations. Nutrients amplitude-regulate the insulin pulses without influencing their frequency. Diabetic patients show an abnormal plasma insulin pattern, but the cause of the disturbance remains to be elucidated. Ithe present thesis the influence of the cytoplasmic calcium concentratio([Ca2+]i) and cell metabolism on pulsatile insulin release was examined in single islets of Langerhans from ob/ob-mice. Glucose stimulation of insulin release involves closure of ATP-sensitive K+ channels (KATP channels), depolarization, and Ca2+ influx in β-cells. In the presence of 11 mM glucose, pulsatile insulin secretion occurs in synchrony with oscillations i[Ca2+]i. When [Ca2+]i is low and stable, e.g. under basal conditions, low amplitude insulin pulses are still observed. When [Ca2+]i is elevated and non-oscillating, e.g. when the β-cells are depolarized by potassium, high amplitude insulin pulses are observed. The frequency of the insulin pulses under these conditions is similar to that observed when [Ca2+]i oscillations are present. By permanently opening or closing the KATP channels with diazoxide or tolbutamide, respectively, it was investigated if glucose can modulate pulsatile insulin secretion when it does not influence the channel activity. Under these conditions, [Ca2+]i remained stable whereas the amplitude of the insulin pulses increased with sugar stimulation without change in the frequency. Metabolic inhibition blunted but did not prevent the insulin pulses. The results indicate that oscillations in metabolism can generate pulsatile insulin release when [Ca2+]i is stable. However, under physiological conditions, pulsatile secretion is driven by oscillations in metabolism and [Ca2+]i, acting in synergy.

Contagious bovine pleuropneumonia (CBPP) is a severe infectious disease caused by Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC) and is a vast problem in Africa. Current CBPP prevention is based on attenuated live strain vaccines, but these are limited by factors such as short-term immunity, cold-chain dependence and retained virulence. CBPP can be diagnosed using post-mortem examination, identification of the agent using culture and PCR based methods as well as serological diagnostic methods, but the latter are generally not sensitive enough and there is also demand for an inexpensive, pen side field test.The research presented in this thesis was focused on using recombinantly expressed surface proteins from M. mycoides SC to characterize humoral immune responses to CBPP. Thereby candidate proteins to be used in development of serological diagnostic methods and possibly subunit vaccines could be identified. As a first step, five putative variable surface proteins of M. mycoides SC were expressed and purified from E. coli in Paper I. These proteins were analyzed using immunoblotting techniques and results showed that one protein, MSC_0364, was variably expressed on the surface of M. mycoides SC in vitro. Paper II presents expanded efforts including cloning and expression of 64 recombinant surface proteins and an assay for high throughput analysis of protein-specific IgG, IgA and IgM titers in hundreds of sera using a bead-based screening assay. The assay was evaluated by protein-specific inhibition experiments, comparisons to Western blotting and monitoring of immune responses over time in a study with sera taken from eight animals over 293 days from a previous vaccine trial.Papers III and IV present applications using the recombinant proteins and bead-based screening assay wherein proteins for diagnostic and vaccine development were identified. In Paper III, the assay was used to screen 61 proteins using well-characterized serum samples from cattle with CBPP and healthy controls, resulting in selection of eight proteins suitable for diagnostic use. These proteins were combined and evaluated in a proof-of-concept ELISA with a discriminative power that enabled 96% correct classification of sera from CBPP-affected and CBPP-free bovines. Paper IV reports the results and protein-specific analyses of a vaccine trial using the recombinant putative variable surface proteins presented in Paper I as a subunit vaccine. The vaccine conferred no protection, but a weak vaccine response could not be excluded as the cause of failure. In an effort to identity other protein candidates to be used in a subunit vaccine, protein-specific analysis of humoral immune responses elicited by the currently approved live strain vaccine, T1/44, were investigated. Here, five proteins with high IgG titers associated to immunity were identified: LppQ, MSC_02714, MSC_0136, MSC_0079 and MSC_0431. These proteins may be important in the development of a novel subunit vaccine against CBPP. / QC 20100719

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.

Clostridium chauvoei is a gram-positive, spore-forming anaerobic bacterium. It is the pathogenic agent of blackleg, a disease causing serious toxemia and high mortality in cattle, sheep and many other domestic and wild animals. It is considered the most important Clostridium producing economic losses in livestock. Typically, animals infected with blackleg die rapidly without any signs of illness. Animals quickly die within 12 to 48 hours after contracting the disease. Therefore, the control of this disease is done by commercial vaccines consisting of whole formolized cultures. Immunity against C. chauvoei is associated with whole cell, including its somatic and flagellar antigens while in other clostridial diseases, protective immunity is obtained by the use of vaccines containing toxoids. Moreover, it is essential to obtain new information about the somatic antigens of C. chauvoei. Proteomics is the study of the proteome, the protein complement of the genome. The proteome has been defined as the entire complement of proteins expressed by a cell, organism, or tissue type, and accordingly, proteomics is the study of this complement expressed at a given time or under certain environmental conditions. 2-DE with Immobilized pH Gradients (IPGs) combined with protein identification by Mass Spectrometry (MS) is currently the workhorse for proteomics. Much of information about immunogenic component can be derived from proteomics coupled to Western blotting, namely immunoproteomics. Our study constitutes the first immunoproteomic analysis of C. chauvoei to identify candidate immunogenic antigens for development of new vaccines. Analyses were performed by Western blot and dot blot techniques against the whole cell extract proteins of C. chauvoei separated by 2-DE. Firstly, the growth conditions of two different strains, C. chauvoei ATCC 11957 and C. chauvoei 20 were optimized. After mice immunization studies with experimental vaccines prepared, sera were obtained for evaluation of the immunoglobulin G antibody level by ELISA. After high level of antibody response determination, 1-DE, 2-DE and immunoblot studies were performed for the characterization of immunogenic proteins. In the study, a total of 460 protein spots could be detected on the 2-DE gels by the help of Delta2D image analysis software and 30 of them were reacted with polyclonal antibodies against inactivated whole cells of C. chauvoei. Among these 30 spots, and 8 of them could be characterized by MALDI-TOF MS analyses. Of these 8 spots revealed four different gene products (distinct ORFs). Ornithine decarboxylase, methionine adenosyltransferase, glucose-6-phosphate isomerase, and flagellin protein FliB (C) are the characterized proteins. Glucose-6-phosphate isomerase has been identified as an immunogenic protein for a pathogenic microbe and in C. chauvoei for the first time. Methionine adenosyltransferase and ornithine decarboxylation were identified as immunogenic for C. chauvoei for the first time. The last defined protein is the flagellin protein FliB(C) which is known to be major immunogenic protein of C. chauvoei.