Pseudomonas fluorescens est un bacille à Gram négatif ubiquitaire qui possède un fort potentiel adaptatif. En milieu hospitalier, cette bactérie est fréquemment retrouvée dans les solutions injectables, qu'elle contamine, mais émerge également comme agent pathogène responsable d'infections nosocomiales. Cependant, actuellement peu d'études permettent de déterminer le réel potentiel de virulence de cette bactérie, qui présente pourtant de nombreux traits relatifs à un pathogène opportuniste. <br />Afin d'appréhender le pouvoir pathogène de cette espèce, nous avons caractérisé et étudié une souche clinique, P. fluorescens MFN1032, isolée d'un patient atteint d'une infection pulmonaire. Cette souche est capable de pousser à 37°C et produits des facteurs de virulence. <br />Dans un premier temps, notre intérêt s'est particulièrement porté sur l'activité hémolytique de la souche, l'objectif étant de déterminer le ou les facteurs impliqués dans cette activité. Nous avons caractérisé une phospholipase C, PlcC, qui appartient à une classe de phospholipase C distincte de celles actuellement répertoriées. PlcC est impliquée dans l'activité hémolytique de MFN1032. Les résultats montrent que PlcC est étroitement liée à la production de biosurfactants, qui participent à l'activité hémolytique, via le régulateur transcriptionnel GntR.<br />D'autre part, le potentiel d'adaptation et de virulence de la souche est favorisé par des mécanismes de variations de phénotypes. Ainsi, différents variants phénotypiques ayant émergé de la souche MFN1032 ont été étudiés et comparés à la souche « sauvage », et les résultats montrent que l'activité hémolytique est directement concernée par cette variation. <br />D'un point de vue moléculaire, le phénotype d'une partie des variants est restauré par une complémentation en trans des gènes gacA ou gacS. La surexpression d'un des gènes gac diminue l'émergence des autres variants non complémentables par les gènes gac et présentant un phénotype « hyperadhérent ».<br />Le regroupement des différents résultats suggère que PlcC et GntR pourraient participer au processus d'émergence des variants hyperadhérents.

<p>Communication between neuronal and non-neuronal is called volume transmission when the released neurotransmitter (NT) acts via diffusion and affects several target cells. Both the neurosecretory and postsynaptic cell responses are linked to [Ca²⁺]_i elevations. </p><p>In the present thesis the role of pre-and postsynaptic Ca²⁺ elevations has been investigated in the reconstituted "synapse" model comprised of NGF-differentiated PC12 and HEL cells as well as in SH-SY5Y neuroblastoma cells. In PC12 cells, both 70mM K⁺ and nicotine triggered NT release, which could be detected as a secondary [Ca²⁺]_i increase in surrounding HEL cells. Both secretagogues shared the same voltage-dependent Ca²⁺ influx pathway as judged from the pharmacological profile blockers of voltage-gated Ca²⁺ channels. The coupling of electrical responses to the activation of Ca²⁺ signaling via muscarinic receptors in SH-SY5Y cells was also studied. These data revealed that depolarization caused a considerable potentiation of the muscarinic Ca²⁺ response. The potentiated Ca²⁺ increase was mainly dependent on the enhanced Ca²⁺ influx and to a lesser extent on [Ca²⁺]_i release from intracellular stores. A phospholipase C (PLC) activator, m-3M3FBS was used to further study the role of G-protein coupled receptor (GPCR)-coupled Ca²⁺ signaling. However, it was found that m-3M3FBS instead triggered [Ca²⁺]_i elevations independently of PLC activation. </p><p>In conclusion, the results indicate that the magnitude of NT release from PC12 cells is sufficient to cause a robust activation of neighboring target cells. Postsynaptic muscarinic signaling is amplified due to integration of electrical excitation and GPCR signaling. The PLC activator, m-3M3FBS is not suitable for studies of PLC-mediated signals in intact cells.</p>

Communication between neuronal and non-neuronal is called volume transmission when the released neurotransmitter (NT) acts via diffusion and affects several target cells. Both the neurosecretory and postsynaptic cell responses are linked to [Ca2+]i elevations. In the present thesis the role of pre-and postsynaptic Ca2+ elevations has been investigated in the reconstituted "synapse" model comprised of NGF-differentiated PC12 and HEL cells as well as in SH-SY5Y neuroblastoma cells. In PC12 cells, both 70mM K+ and nicotine triggered NT release, which could be detected as a secondary [Ca2+]i increase in surrounding HEL cells. Both secretagogues shared the same voltage-dependent Ca2+ influx pathway as judged from the pharmacological profile blockers of voltage-gated Ca2+ channels. The coupling of electrical responses to the activation of Ca2+ signaling via muscarinic receptors in SH-SY5Y cells was also studied. These data revealed that depolarization caused a considerable potentiation of the muscarinic Ca2+ response. The potentiated Ca2+ increase was mainly dependent on the enhanced Ca2+ influx and to a lesser extent on [Ca2+]i release from intracellular stores. A phospholipase C (PLC) activator, m-3M3FBS was used to further study the role of G-protein coupled receptor (GPCR)-coupled Ca2+ signaling. However, it was found that m-3M3FBS instead triggered [Ca2+]i elevations independently of PLC activation. In conclusion, the results indicate that the magnitude of NT release from PC12 cells is sufficient to cause a robust activation of neighboring target cells. Postsynaptic muscarinic signaling is amplified due to integration of electrical excitation and GPCR signaling. The PLC activator, m-3M3FBS is not suitable for studies of PLC-mediated signals in intact cells.

Thesis (Ph.D. in Microbiology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 201-223).

Oocyte activation deficiency (OAD) is an infertile condition observed in patients who have experienced recurrent total fertilisation failure (TFF) following intracytoplasmic sperm injection treatment. This condition was considered to be an idiopathic factor for a long time but strong clinical evidence now suggests that dysfunctional forms of phospholipase C zeta (PLC&zeta;) may be predominant causative factors for OAD. Genetic contribution has played a role in patients suspected of having OAD, as four PLC&zeta; exonic mutations have been discovered and characterised as being the cause of infertility. In this study, a novel nonsense mutation, PLC&zeta;K322Stop, was identified in the PLC&zeta; XY-linker region of Patient LR. This variant results in the truncation of approximately half of PLC&zeta;, therefore was non-functional when activity was tested. Patient LR, which also exhibited a previously reported mutation, PLC&zeta;H233L, may suggest that the patient is sub-fertile, as opposed to being infertile, as initially expected. Although research has purely focused upon the coding regions of PLC&zeta;, it was obvious that our knowledge of PLC&zeta; regulatory elements remain very limited. Next generation sequencing (NGS) was therefore employed to detect variants in the non-coding regions of PLC&zeta;, promoter and introns, which may have resulted in the observed phenotypic diversity of PLC&zeta; expression in fertile and infertile patients. As a result of mapping failure, an alternative approach was considered to identify variants within human PLC&zeta;, and this involved using the single nucleotide polymorphism (SNP) database. Over 2500 SNPs were localised in the intronic regions of PLC&zeta; and thus, it could be speculated that these variants may help elucidate the wide variation of PLC&zeta; expression reported. Additionally, two particular patients with TFF (79 and 107) were investigated in this study to identify an association with PLC&zeta; and their infertile state. For Patient 79, multiple PLC&zeta; immunofluorescence analysis was performed and a significant improvement in PLC&zeta; expression was observed one year after his first investigation. This may have been the result of an external factor, which influenced protein expression. As for Patient 107, a novel substitution mutation, PLC&zeta;V193E, was identified and was predicted to affect PLC&zeta; stability and folding. There is global interest to create a safer and alternative OAD therapy, namely a human recombinant PLC&zeta; protein (hrPLC&zeta;). The first method, using a bacterial cell line resulted in successful purification and identification but the product proved to be inactive following mouse oocyte microinjection. The second method involved production of a mammalian-expressed hrPLC&zeta;, which was successfully purified and identified but due to time restrictions, could not be tested for functionality. Concurrently, the findings in this thesis have reinforced the association between PLC&zeta; and OAD, and provided improved options for the diagnosis and treatment of OAD.

Les ligands orthostériques transmettent un signal complexe aux cellules en se fixant à l'intérieur de la pochette de liaison de leur récepteur cible. Le changement conformationel qui en résulte modifie l'efficacité du récepteur à recruter et activer les seconds messagers en amont des voies de signalisation, soit les protéines G hétérotrimériques dans le cas des récepteurs couplés aux protéines G (GPCRs). Ces variations entraînent une vaste gamme de modifications dans le milieu intracellulaire. Par exemple, l'activation de la protéine G q provoque entre autres l'activation de la phospholipase C? (PLC? ), la production d'inositol 1,4,5-trisphosphate (1P3), puis l'activation des protéines kinases C (PKC). L'activation de la protéine G s , pour sa part, stimule l'activité de l'adénylate cyclase (AC), ce qui entraîne la production d'AMPc et l'activation de la protéine kinase A (PKA). Un ligand n'influence pas nécessairement deux voies de signalisation indépendantes de façon similaire, ce qui lui confère la propriété de sélectivité fonctionnelle. Dans ce travail, nous avons caractérisé le profil pharmacologique de ligands orthostériques du récepteur FP de la PGF2? à l'aide d'un clone HEK-293-SL exprimant le récepteur FP de façon stable. La mesure de la production d'IP3 a permis d'évaluer la voie de la PLC alors que la mesure de la production d'AMP c a permis d'évaluer la voie de l'AC. Pour chacune d'entre elles, le fluprostenol s'est comporté comme un agoniste complet moins puissant que l'agoniste naturel. Le composé synthétique Al-8810 s'est comporté comme un agoniste partiel de la voie de la PLC, alors qu'il s'est avéré être un antagoniste de la voie de l'AC. Ces résultats démontrent que l'activité d'un ligand vis-à-vis un récepteur dépend du groupe d'effecteurs observé, ce qui illustre le concept de sélectivité fonctionnelle des ligands. L'étude des composés allostériques THG113 et THG113.824 démontre que ces derniers n'influencent pas la signalisation déclenchée en aval du récepteur FP par son agoniste naturel. Ces résultats suggèrent qu'ils agiraient comme antagoniste des effets de la PGF 2? par un mécanisme indépendant du récepteur FP. [Symboles non conformes]

The ƒÑ-toxin of Clostridium perfringens is a toxin involved in numerous diseases of humans and agriculturally important animals. One of these diseases is necrotic enteritis (NE), a sporadic enteric disease which affects avian species world-wide. This study involved the inactivation of alpha-toxin (ƒÑ-toxin) for use as a potential vaccine candidate to combat NE in chickens, and other diseases caused by C. perfringens type A. During the course of this research a number of ƒÑ-toxin recombinant proteins were developed through molecular inactivation of the ƒÑ-toxin gene, plc. Proteins plc316 and plc204 were developed by the deletion of the first three and seven ƒÑ-helices of the N-terminal domain respectively. These deletions resulted in proteins which were unstable in solution, constantly aggregated into insoluble masses and elicited lower overall antibody responses when administered to mice. A third protein, plcInv3 was developed from the deletion of part of the catalytic domain of the ƒÑ-toxin. PlcInv3 was highly soluble and upon immunisation of mice elicited a significant antibody response which was also capable of protecting mice against a live challenge of C. perfringens. The fourth and final protein developed was plc104. The smallest of the recombinant ƒÑ-toxin proteins, it consisted entirely of the C-terminal domain of ƒÑ-toxin. Its small size did not affect its ability to induce a strong antibody response when administered to mice, the antibodies of which were also protective during a challenge with C. perfringens. STM1, an attenuated strain of S. Typhimurium was used in the development of a vectored vaccine for the expression and oral delivery of plcInv3 and plc104 within the mouse host. The proteins were expressed within STM1 from expression plasmids containing the in vivo inducible promoters PhtrA and PpagC. A measurable humoral immune response against ƒÑ-toxin was absent following three oral vaccinations with the vectored vaccines, although, cytokine profiling of splenocytes from vaccinated mice revealed an increase in the number of interleukin-4 (IL-4)secreting cells and the lack of interferon-gamma (IFN-ƒ×) secreting cells. This indicated the stimulation of a T-helper type 2 (TH2) immune response which also lead to partial protection against a live C. perfringens challenge. This study demonstrates the feasibility of using STM1 as a carrier for the in vivo expression of the C. perfringens ƒÑ-toxin recombinant proteins plcInv3 and plc104. It is the first study to express C. perfringens antigens within an attenuated strain of S. Typhimurium, STM1.The partial protection of mice immunised with these vaccines indicates there is potential for this vectored vaccine system to be used in the protection of diseases caused by the ƒÑ-toxin of C. perfringens.

1. The effect of urotensin II (U-II) on spontaneous transmitter release was examined in the frog to see if the biological activity of this vasoactive peptide extended to neural tissues. 2. In normal Ringer solution, frog and human U-II (fU-II and hU-II, respectively) caused concentration-dependent, reversible increases in miniature endplate potential (MEPP) frequency, with hU-II about 22 times more potent than fU-II. hU-II caused a dose-dependent increase in MEPP amplitude, whereas fU-II caused an increase, followed by a decrease with higher concentrations. 3. Increasing extracellular Ca 2+ three-fold had no effect on the MEPP frequency increase to 25 μM hU-II. Pretreatment with thapsigargin to deplete endoplasmic reticulum Ca 2+ caused a 61% reduction in the MEPP frequency increase to 25 μM hU-II. 4. Pretreatment with the phospholipase C inhibitor U-73122 caused a 93% reduction in the MEPP frequency increase to 25 μM hU-II and a 15% reduction in the increase in MEPP amplitude. Pretreating with antibodies against the inositol 1,4,5-trisphosphate (IP 3) type 1 receptor using liposomal techniques reduced the MEPP frequency increase by 83% but had no effect on MEPP amplitude. 5. Pretreating with protein kinase C inhibitors (bisindolylmaleimide I and III) had no effect on the response to 25 μM hU-II, but pretreating with protein kinase A inhibitors (H-89 and KT5720) reduced the MEPP frequency increase by 88% and completely abolished the increase in MEPP amplitude. 6. Our results show that hU-II is a potent stimulator of spontaneous transmitter release in the frog and that the effect is mediated by IP 3 and cyclic AMP/protein kinase A.

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