Добірка наукової літератури з теми "Complex coupler activator"

Recently, a requirement for β-arrestin–mediated endocytosis in the activation of extracellular signal–regulated kinases 1 and 2 (ERK1/2) by several G protein–coupled receptors (GPCRs) has been proposed. However, the importance of this requirement for function of ERK1/2 is unknown. We report that agonists of Gαq-coupled proteinase–activated receptor 2 (PAR2) stimulate formation of a multiprotein signaling complex, as detected by gel filtration, immunoprecipitation and immunofluorescence. The complex, which contains internalized receptor, β-arrestin, raf-1, and activated ERK, is required for ERK1/2 activation. However, ERK1/2 activity is retained in the cytosol and neither translocates to the nucleus nor causes proliferation. In contrast, a mutant PAR2 (PAR2δST363/6A), which is unable to interact with β-arrestin and, thus, does not desensitize or internalize, activates ERK1/2 by a distinct pathway, and fails to promote both complex formation and cytosolic retention of the activated ERK1/2. Whereas wild-type PAR2 activates ERK1/2 by a PKC-dependent and probably a ras-independent pathway, PAR2(δST363/6A) appears to activate ERK1/2 by a ras-dependent pathway, resulting in increased cell proliferation. Thus, formation of a signaling complex comprising PAR2, β-arrestin, raf-1, and activated ERK1/2 might ensure appropriate subcellular localization of PAR2-mediated ERK activity, and thereby determine the mitogenic potential of receptor agonists.

Activation of GPCRs (G-protein-coupled receptors) leads to conformational changes that ultimately initiate signal transduction. Activated GPCRs transiently combine with and activate heterotrimeric G-proteins resulting in GTP replacement of GDP on the G-protein α subunit. Both the detailed structural changes essential for productive GDP/GTP exchange on the G-protein α subunit and the structure of the GPCR–G-protein complex itself have yet to be elucidated. Nevertheless, transient GPCR–G-protein complexes can be trapped by nucleotide depletion, yielding an empty-nucleotide G-protein–GPCR complex that can be isolated. Whereas early biochemical studies indicated formation of a complex between G-protein and activated receptor only, more recent results suggest that G-protein can bind to pre-activated states of receptor or even couple transiently to non-activated receptor to facilitate rapid responses to stimuli. Efficient and reproducible formation of physiologically relevant, conformationally homogenous GPCR–G-protein complexes is a prerequisite for structural studies designed to address these possibilities.

The HBx protein of hepatitis B virus (HBV) is a small transcriptional transactivator that is essential for infection by the mammalian hepadnaviruses and is thought to be a cofactor in HBV-mediated liver cancer. HBx stimulates signal transduction pathways by acting in the cytoplasm, which accounts for many but not all of its transcriptional activities. Studies have shown that HBx protein activates Ras and downstream Ras signaling pathways including Raf, mitogen-activated protein (MAP) kinase kinase kinase (MEK), and MAP kinases. In this study, we investigated the mechanism of activation of Ras by HBx because it has been found to be central to the ability of HBx protein to stimulate transcription and to release growth arrest in quiescent cells. In contrast to the transient but strong stimulation of Ras typical of autocrine factors, activation of Ras by HBx protein was found to be constitutive but moderate. HBx induced the association of Ras upstream activating proteins Shc, Grb2, and Sos and stimulated GTP loading onto Ras, but without directly participating in complex formation. Instead, HBx is shown to stimulate Ras-activating proteins by functioning as an intracellular cytoplasmic activator of the Src family of tyrosine kinases, which can signal to Ras. HBx protein stimulated c-Src and Fyn kinases for a prolonged time. Activation of Src is shown to be indispensable for a number of HBx activities, including activation of Ras and the Ras-Raf-MAP kinase pathway and stimulation of transcription mediated by transcription factor AP-1. Importantly, HBx protein expressed in cultured cells during HBV replication is shown to activate the Ras signaling pathway. Mechanisms by which HBx protein might activate Src kinases are discussed.

Neurite outgrowth is a complex differentiation process stimulated by many neuronal growth factors and transmitters and by electrical activity. Among these stimuli are ligands for G-protein-coupled receptors (GPCR) that function as neurotransmitters. The pathways involved in GPCR-triggered neurite outgrowth are not fully understood. Many of these receptors couple to Gαo, one of the most abundant proteins in the neuronal growth cones. We have studied the Go signaling network involved in neurite outgrowth in Neuro2A cells. Gαo can induce neurite outgrowth. The CB1 cannabinoid receptor, a Go/i-coupled receptor expressed endogenously in Neuro2A cells, triggers neurite outgrowth by activating Rap1, which promotes the Gαo-stimulated proteasomal degradation of Rap1GAPII. CB1-receptor-mediated Rap1 activation leads to the activation of a signaling network that includes the small guanosine triphosphate (GTP)ases Ral and Rac, the protein kinases Src, and c-Jun N-terminal kinase (JNK), which converge onto the activation of signal transducer and activator of transcription 3 (Stat3), a key transcription factor that mediates the gene expression process of neurite outgrowth in Neuro2A cells. This review describes current findings from our laboratory and also discusses alternative pathways that Go/i might mediate to trigger neurite outgrowth. We also analyze the role neurotransmitters, which stimulate Go/i to activate a complex signaling network controlling neurite outgrowth, play in regeneration after neuronal injury.

Abstract The adhesion molecule von Willebrand factor (vWF) activates platelets upon binding 2 surface receptors, glycoprotein (GP) Ib-V-IX and integrin IIbβ3. We have used 2 approaches to selectively activate GP Ib using either the snake venom lectin alboaggregin-A or mutant recombinant forms of vWF (▵A1-vWF and RGGS-vWF) with selective binding properties to its 2 receptors. We show that activation of GP Ib induces platelet aggregation, secretion of 5-hydroxy tryptamine (5-HT), and an increase in cytosolic calcium. Syk becomes tyrosine phosphorylated and activated downstream of GP Ib, and associates with several tyrosine-phosphorylated proteins including the Fc receptor γ-chain through interaction with Syk SH2 domains. GP Ib physically associates with the γ-chain in GST-Syk-SH2 precipitates from platelets stimulated through GP Ib, and 2 Src family kinases, Lyn and Fyn, also associate with this signaling complex. In addition, GP Ib stimulation couples to tyrosine phosphorylation of phospholipase Cγ2. The Src family-specific inhibitor PP1 dose-dependently inhibits phosphorylation of Syk, its association with tyrosine-phosphorylated γ-chain, phosphorylation of PLCγ2, platelet aggregation, and 5-HT release. The results indicate that, upon activation, GP Ib is physically associated with FcR γ-chain and members of the Src family kinases, leading to phosphorylation of the γ-chain, recruitment, and activation of Syk. Phosphorylation of PLCγ2 also lies downstream of Src kinase activation and may critically couple early signaling events to functional platelet responses.

The adhesion molecule von Willebrand factor (vWF) activates platelets upon binding 2 surface receptors, glycoprotein (GP) Ib-V-IX and integrin IIbβ3. We have used 2 approaches to selectively activate GP Ib using either the snake venom lectin alboaggregin-A or mutant recombinant forms of vWF (▵A1-vWF and RGGS-vWF) with selective binding properties to its 2 receptors. We show that activation of GP Ib induces platelet aggregation, secretion of 5-hydroxy tryptamine (5-HT), and an increase in cytosolic calcium. Syk becomes tyrosine phosphorylated and activated downstream of GP Ib, and associates with several tyrosine-phosphorylated proteins including the Fc receptor γ-chain through interaction with Syk SH2 domains. GP Ib physically associates with the γ-chain in GST-Syk-SH2 precipitates from platelets stimulated through GP Ib, and 2 Src family kinases, Lyn and Fyn, also associate with this signaling complex. In addition, GP Ib stimulation couples to tyrosine phosphorylation of phospholipase Cγ2. The Src family-specific inhibitor PP1 dose-dependently inhibits phosphorylation of Syk, its association with tyrosine-phosphorylated γ-chain, phosphorylation of PLCγ2, platelet aggregation, and 5-HT release. The results indicate that, upon activation, GP Ib is physically associated with FcR γ-chain and members of the Src family kinases, leading to phosphorylation of the γ-chain, recruitment, and activation of Syk. Phosphorylation of PLCγ2 also lies downstream of Src kinase activation and may critically couple early signaling events to functional platelet responses.

The Orai1 channel is regulated by stromal interaction molecules STIM1 and STIM2 within endoplasmic reticulum (ER)-plasma membrane (PM) contact sites. Ca2+signals generated by Orai1 activate Ca2+-dependent gene expression. When compared with STIM1, STIM2 is a weak activator of Orai1, but it has been suggested to have a unique role in nuclear factor of activated T cells 1 (NFAT1) activation triggered by Orai1-mediated Ca2+entry. In this study, we examined the contribution of STIM2 in NFAT1 activation. We report that STIM2 recruitment of Orai1/STIM1 to ER-PM junctions in response to depletion of ER-Ca2+promotes assembly of the channel with AKAP79 to form a signaling complex that couples Orai1 channel function to the activation of NFAT1. Knockdown of STIM2 expression had relatively little effect on Orai1/STIM1 clustering or local and global [Ca2+]iincreases but significantly attenuated NFAT1 activation and assembly of Orai1 with AKAP79. STIM1ΔK, which lacks the PIP2-binding polybasic domain, was recruited to ER-PM junctions following ER-Ca2+depletion by binding to Orai1 and caused local and global [Ca2+]iincreases comparable to those induced by STIM1 activation of Orai1. However, in contrast to STIM1, STIM1ΔK induced less NFAT1 activation and attenuated the association of Orai1 with STIM2 and AKAP79. Orai1-AKAP79 interaction and NFAT1 activation were recovered by coexpressing STIM2 with STIM1ΔK. Replacing the PIP2-binding domain of STIM1 with that of STIM2 eliminated the requirement of STIM2 for NFAT1 activation. Together, these data demonstrate an important role for STIM2 in coupling Orai1-mediated Ca2+influx to NFAT1 activation.

Nerve growth factor (NGF) activates the mitogen-activated protein (MAP) kinase cascade through a p21ras-dependent signal transduction pathway in PC12 cells. The linkage between p21ras and MEK1 was investigated to identify those elements which participate in the regulation of MEK1 activity. We have screened for MEK activators using a coupled assay in which the MAP kinase cascade has been reconstituted in vitro. We report that we have detected a single NGF-stimulated MEK-activating activity which has been identified as B-Raf. PC12 cells express both B-Raf and c-Raf1; however, the MEK-activating activity was found only in fractions containing B-Raf. c-Raf1-containing fractions did not exhibit a MEK-activating activity. Gel filtration analysis revealed that the B-Raf eluted with an apparent M(r) of 250,000 to 300,000, indicating that it is present within a stable complex with other unidentified proteins. Immunoprecipitation with B-Raf-specific antisera quantitatively precipitated all MEK activator activity from these fractions. We also demonstrate that B-Raf, as well as c-Raf1, directly interacted with activated p21ras immobilized on silica beads. NGF treatment of the cells had no effect on the ability of B-Raf or c-Raf1 to bind to activated p21ras. These data indicate that this interaction was not dependent upon the activation state of these enzymes; however, MEK kinase activity was found to be associated with p21ras following incubation with NGF-treated samples at levels higher than those obtained from unstimulated cells. These data provide direct evidence that NGF-stimulated B-Raf is responsible for the activation of the MAP kinase cascade in PC12 cells, whereas c-Raf1 activity was not found to function within this pathway.

Nerve growth factor (NGF) activates the mitogen-activated protein (MAP) kinase cascade through a p21ras-dependent signal transduction pathway in PC12 cells. The linkage between p21ras and MEK1 was investigated to identify those elements which participate in the regulation of MEK1 activity. We have screened for MEK activators using a coupled assay in which the MAP kinase cascade has been reconstituted in vitro. We report that we have detected a single NGF-stimulated MEK-activating activity which has been identified as B-Raf. PC12 cells express both B-Raf and c-Raf1; however, the MEK-activating activity was found only in fractions containing B-Raf. c-Raf1-containing fractions did not exhibit a MEK-activating activity. Gel filtration analysis revealed that the B-Raf eluted with an apparent M(r) of 250,000 to 300,000, indicating that it is present within a stable complex with other unidentified proteins. Immunoprecipitation with B-Raf-specific antisera quantitatively precipitated all MEK activator activity from these fractions. We also demonstrate that B-Raf, as well as c-Raf1, directly interacted with activated p21ras immobilized on silica beads. NGF treatment of the cells had no effect on the ability of B-Raf or c-Raf1 to bind to activated p21ras. These data indicate that this interaction was not dependent upon the activation state of these enzymes; however, MEK kinase activity was found to be associated with p21ras following incubation with NGF-treated samples at levels higher than those obtained from unstimulated cells. These data provide direct evidence that NGF-stimulated B-Raf is responsible for the activation of the MAP kinase cascade in PC12 cells, whereas c-Raf1 activity was not found to function within this pathway.

A growing body of data supports the conclusion that G protein-coupled receptors can regulate cellular growth and differentiation by controlling the activity of MAP kinases. The activation of heterotrimeric G protein pools initiates a complex network of signals leading to MAP kinase activation that frequently involves cross-talk between G protein-coupled receptors and receptor tyrosine kinases or focal adhesions. The dominant mechanism of MAP kinase activation varies significantly between receptor and cell type. Moreover, the mechanism of MAP kinase activation has a substantial impact on MAP kinase function. Some signals lead to the targeting of activated MAP kinase to specific extranuclear locations, while others activate a MAP kinase pool that is free to translocate to the nucleus and contribute to a mitogenic response.

Дисертація на здобуття наукового ступеня кандидата технічних наук (доктора філософії) за спеціальністю 05.17.11 "Технологія тугоплавких неметалічних матеріалів". – Національний технічний університет "Харківський політехнічний інститут", Харків, 2017. Дисертацію присвячено розробці безпігментних одношарових хімічно та термічно стійких темнозабарвлених склоемалевих покриттів для захисту побутової техніки, зокрема кухонних газових та електричних плит, що отримуються за технологією POESTA. Синтезовано основи отримання покриттів вказаного типу, згідно із якими розробляється скломатриця із заданими фізико-хімічними властивостями, на основі якої отримується склоемалева фрита шляхом введення в оптимізований склад скла комплексного активатору зчеплення, одночасно виконуючий роль активного забарвлюючого комплексу, який поєднує задані міцнісні характеристики безпігментних одношарових темнозабарвлених склоемалевих покриттів. Встановлено межі значень структурних факторів, які забезпечують міцну структуру кремнекисневого каркасу скла в системі R₂O (Na₂O+K₂O+Li₂O) – RO (CaO+BaO+SrO+MgO) – TiO₂ – ZrO₂ – B₂O₃ – SiO₂ і заданий рівень її структурнозалежних експлуатаційних властивостей за рахунок встановлених співвідношень склоутворювачів і модифікаторів. Розроблено склад і співвідношення комплексного активатора зчеплення із урахуванням його впливу на характеристики міцності системи "склоемаль – сталь", корозійну здатність склорозплаву та експлуатаційні властивості покриттів, який одночасно виконує роль активного забарвлюючого комплексу. Обрано іонний механізм забарвлення та встановлено колірні координати в ККС XYZ, RGB, L*a*b. Проведено промислові та лабораторно-промислові випробування на підприємствах та розроблено практичні рекомендації щодо використання результатів.
The dissertation on competion of a scientific degree of the candidate of engineering science on a speciality 05.17.11 "Technology of refractory nonmetallic materials". – National Technical University "Kharkiv Polytechnical Institute", Kharkiv, 2017. The dissertation is devoted to the development of pigments free direct chemically and thermally resistant dark-colored glass-enamel coatings for the protection of household appliances, in particular kitchen gas and electric plates, obtained by the technology POESTA. The bases of obtaining the coatings of this type are synthesized, according to which the glass matrix is developed with the given physicochemical properties, on the basis of which glassmelee frit is obtained by introducing into the optimized composition of the MS complex agglomer activator, simultaneously performing the role of the active coloring complex which combines the specified strength characteristics of pigments free direct glass-enamel coatings with their dark coloring. The boundaries of the values of structural factors, which provide a solid structure of the silica-oxygen glass frame in the system R₂O (Na₂O+K₂O+Li₂O) – RO (CaO+BaO+SrO+MgO) – TiO₂ – ZrO₂ – B₂O₃ – SiO₂ and the specified level of its structurally dependent performance properties due to the established ratios of glass modifiers and modifiers. The composition and ratio of the complex coupler activator have been developed taking into account its influence on the strength characteristics of the glass-enamel-steel system, the corrosion capacity of the glass-fiber alloy and the operational properties of the coatings at the firing temperatures of 800 to 830 °C. Selected the ionic mechanism of color, which was realized by the components of the filling station, and the color coordinates are established in the XYZ, RGB, L*a*b, according to RAL. Industrial and laboratory-industrial tests were carried and practical recommendations for the use of development results are developed.

Дисертація на здобуття наукового ступеня кандидата технічних наук (доктора філософії) за спеціальністю 05.17.11 "Технологія тугоплавких неметалічних матеріалів". – Національний технічний університет "Харківський політехнічний інститут", Харків, 2017. Дисертацію присвячено розробці безпігментних одношарових хімічно та термічно стійких темнозабарвлених склоемалевих покриттів для захисту побутової техніки, зокрема кухонних газових та електричних плит, що отримуються за технологією POESTA. Синтезовано основи отримання покриттів вказаного типу, згідно із якими розробляється скломатриця із заданими фізико-хімічними властивостями, на основі якої отримується склоемалева фрита шляхом введення в оптимізований склад скла комплексного активатору зчеплення, одночасно виконуючий роль активного забарвлюючого комплексу, який поєднує задані міцнісні характеристики безпігментних одношарових темнозабарвлених склоемалевих покриттів. Встановлено межі значень структурних факторів, які забезпечують міцну структуру кремнекисневого каркасу скла в системі R₂O (Na₂O+K₂O+Li₂O) – RO (CaO+BaO+SrO+MgO) – TiO₂ – ZrO₂ – B₂O₃ – SiO₂ і заданий рівень її структурнозалежних експлуатаційних властивостей за рахунок встановлених співвідношень склоутворювачів і модифікаторів. Розроблено склад і співвідношення комплексного активатора зчеплення із урахуванням його впливу на характеристики міцності системи "склоемаль – сталь", корозійну здатність склорозплаву та експлуатаційні властивості покриттів, який одночасно виконує роль активного забарвлюючого комплексу. Обрано іонний механізм забарвлення та встановлено колірні координати в ККС XYZ, RGB, L*a*b. Проведено промислові та лабораторно-промислові випробування на підприємствах та розроблено практичні рекомендації щодо використання результатів.
The dissertation on competion of a scientific degree of the candidate of engineering science on a speciality 05.17.11 "Technology of refractory nonmetallic materials". – National Technical University "Kharkiv Polytechnical Institute", Kharkiv, 2017. The dissertation is devoted to the development of pigments free direct chemically and thermally resistant dark-colored glass-enamel coatings for the protection of household appliances, in particular kitchen gas and electric plates, obtained by the technology POESTA. The bases of obtaining the coatings of this type are synthesized, according to which the glass matrix is developed with the given physicochemical properties, on the basis of which glassmelee frit is obtained by introducing into the optimized composition of the MS complex agglomer activator, simultaneously performing the role of the active coloring complex which combines the specified strength characteristics of pigments free direct glass-enamel coatings with their dark coloring. The boundaries of the values of structural factors, which provide a solid structure of the silica-oxygen glass frame in the system R₂O (Na₂O+K₂O+Li₂O) – RO (CaO+BaO+SrO+MgO) – TiO₂ – ZrO₂ – B₂O₃ – SiO₂ and the specified level of its structurally dependent performance properties due to the established ratios of glass modifiers and modifiers. The composition and ratio of the complex coupler activator have been developed taking into account its influence on the strength characteristics of the glass-enamel-steel system, the corrosion capacity of the glass-fiber alloy and the operational properties of the coatings at the firing temperatures of 800 to 830 °C. Selected the ionic mechanism of color, which was realized by the components of the filling station, and the color coordinates are established in the XYZ, RGB, L*a*b, according to RAL. Industrial and laboratory-industrial tests were carried and practical recommendations for the use of development results are developed.

Les récepteurs couplés aux protéines G (RCPG) constituent la plus grande famille de récepteurs membranaires et sont la cible de plus de 25% des médicaments. La compréhension des mécanismes moléculaires de l'activation de ces complexes oligomériques est cruciale pour le développement de drogues plus efficaces. Notre modèle d'étude, le récepteur métabotrope de l'acide γ-amino-butyrique (GABA-B), le principal neurotransmetteur inhibiteur du système nerveux central, module la transmission synaptique et constitue une cible pharmacologique pour le traitement de nombreux troubles neurologiques et psychiatriques incluant l'anxiété, l'épilepsie ou l'addiction aux drogues. Le récepteur GABA-B est un hétérodimère obligatoire formé de deux sous-unités GB1 et GB2, composées chacune d'un domaine extracellulaire appelé Vénus flytrap (VFT) et d'un domaine à sept helices transmembranaires (7TM) commun à tous les RCPG. Le VFT de GB1 lie le GABA, tandis que le domaine 7TM de GB2 contient le site de liaison de modulateurs allostériques positifs et est responsable du couplage à la protéine G. Mon travail de thèse a eu deux objectifs : (i) au niveau fondamental, il a consisté à mieux comprendre le mécanisme moléculaire d'activation du récepteur GABA-B. Nous avons démontré l'importance du mouvement relatif des VFT de GB1 et GB2 pour l'activation du récepteur, en développant l'approche « glycan wedge scanning ». D'autre part, nous avons démontré que la transactivation directe entre les deux domaines 7TM de l'hétérodimère représente une étape clé dans l'activation du récepteur ; (ii) au niveau technologique, j'ai mis en place un système senseur de l'état d'activation du récepteur GABA-B exprimé à la surface de cellules vivantes en utilisant de nouvelles techniques de marquage en fluorescence, compatibles avec des mesures de FRET en temps résolu. Pour cela, j'ai développé une méthode de marquage orthogonal entre un ACP-tag inséré dans une sous-unité et un Snap-tag fusionné à l'autre sous-unité. La mise en place de ce senseur devrait conduire à un nouveau test de criblage de molécules spécifiques du GABA-B, à moyen ou haut débit
G-protein coupled receptors (GPCRs) constitute the largest family of membrane receptors, and the target of more than 25% of drugs on the market. Understanding the molecular mechanisms of the activation of such oligomeric complexes is crucial to develop more potent drugs. The metabotropic γ-aminobutyric acid receptor (GABA-B) is activated by the main inhibitory neurotransmitter of the central nervous system (GABA). It plays an important role in brain functions and as such, it is a potential therapeutic target for the treatment of various neurologic and psychiatric disorders (anxiety, epilepsy or drug addiction). The GABA-B receptor is an obligatory heterodimer composed of two subunits, GB1 and GB2, each of them possessing an extracellular domain called Venus flytrap (VFT) and a seven transmembrane domain (7TM) common to all GPCRs. The VFT of GB1 contains the GABA binding site whereas 7TM domain of GB2, where the positive allosteric modulators bind, is responsible for G-protein activation. My doctoral research project had two main objectives. The first one was to better understand the molecular mechanism underlying the activation of GABA-B receptor. We first demonstrated the importance of the relative movement of GB1 and GB2 VFT domains in the activation, using a « glycan wedge scanning » approach. In addition, we showed a direct transactivation between the two 7TM that is a key step in GABA-B activation. The second objective was the development of a sensor to monitor the GABA-B receptor activation at the cell surface of living cells. This sensor, based on GABA-B receptor conformational changes during activation used new fluorescent tools compatible with time-resolved FRET experiments. To this aim, we set up an orthogonal labelling between an ACP-tag inserted in a loop of one subunit and a Snap-tag fused to the other. This sensor of GABA-B activation should lead to the development of a medium or high throughput screening of specific GABA-B molecules

L'activation de petites molécules azotées telles que l'azote et l'ammoniac a été développé dans notre laboratoire via la chimie organométallique de surface (COMS). Les recherches effectuées durant cette thèse ont permis d'établir la réactivité de complexe de tantale imido amido supporté sur silice, [(SiO)2Ta(=NH)(NH2)] vis-à-vis de l'hydrogène et de l'ammoniac. Des étapes élémentaires de clivage hétérolytique de liaison H-H ou N-H ont été établies. En particulier, l'importance d'une molécule d'ammoniac dans la deuxième sphère de coordination (outer sphere assistance) du système s'est avérée cruciale pour la diminution des barrières d'énergie des états de transition pendant le transfert de protons. Les études ont été faites pour déterminer et expliquer le mécanisme de réduction de N2 par les complexes d'hydrures de tantale. La compréhension du mécanisme a été établie grâce aux études avec N2, N2H4 et N2H2 pour trouver les intermédiaires de cette réduction suivis par in-situ infrarouge, RMN et l'analyse élémentaire, et à l'aide de calcul DFT. Un mécanisme de clivage de N2 par des complexes dihydrogènes de Ta(V) est proposé. Enfin, la réactivité du complexe [(SiO)2Ta(=NH)(NH2)] vers l'activation de liaison C-H de C6H6, C6H5-CH3, t-Bu-Ethylène et CH4 a été étudié par la spectroscopie infrarouge
The activation of small molecules such as nitrogen and ammonia was already developed in our laboratory using the surface organometallic chemistry (SOMC) approach. This thesis focused on understanding the reactivity of tantalum imido amido complex [(SiO)2Ta(=NH)(NH2)], under hydrogen and/ or ammonia atmosphere. Heterolytic H-H and N-H cleavage across Ta-NH2 and Ta=NH bonds appeared crucial. The assistance of an additional ammonia molecule in the outer sphere of the d0 tantalum(v) imido amido ammonia model complex in order to reduce the energy barriers of the transition states during proton transfer was also shown. Studies were done to identify the mechanism of N2 reduction by tantalum hydride complexes. Studies with N2, N2H4 and N2H2 allowed identifying the intermediaries via in situ IR, NMR and elemental analysis. Combined with DFT calculations, these experiments led to the proposal of a novel mechanism for N2 cleavage based on the central role of Ta(H2) adducts. Finally, the reactivity of imido amido complex toward C-H bond activation was studied with C6H6, C6H5-CH3, t-Bu-Ethylene and CH4

The glycoprotein hormones, Luteinizing Hormone (LH), human Chorionic Gonadotropin (hCG), Follicle Stimulating Hormone (FSH) and Thyroid Stimulating Hormone (TSH) are heterodimeric proteins with an identical α-subunit associated non-covalently with the hormone specific β-subunit and play important roles in reproduction and overall physiology of the organism [1]. The receptors of these hormones belong to the family of G-protein coupled receptors (GPCR) and have a large extracellular domain (ECD) comprising of 9-10 leucine rich repeats (LRR) followed by a flexible hinge region, a seven helical transmembrane domain (TMD) and a C terminal cytoplasmic tail [2]. Despite significant sequence and structural homologies observed between the ECDs of the receptors and the specific β-subunits of the hormones, the hormone-receptor pairs exhibit exquisite specificity with very low cross-reactivity with other members of the family. The TSH receptor (TSHR) is an especially interesting member of this family as it not only recognizes is cognate ligand, i.e. TSH, but also binds to the non-cognate ligands such as autoantibodies. TSHR autoantibodies come in different flavors; inhibitory antibodies that compete with the hormone for receptor binding and block its action, stimulatory antibodies that activate the receptor in a hormone independent manner and neutral antibodies that bind to the receptor but do not directly influence its functions. The inhibitory autoantibodies cause hypothyroidism and are responsible for Hashimoto’s Thyroiditis, whereas the stimulatory autoantibodies cause Graves’ thyrotoxicosis characterized by hyperthyroid condition [3]. The exact epitopes of these autoantibodies are not well delineated although it has been hypothesized that the blocking type- and the stimulatory type- autoantibodies have predominant epitopes in the TSHR ECD that overlap with hormone binding regions [4]. Insights into the mode of hormone or autoantibody binding to the receptor was primarily derived from the crystal structure of FSHR leucine rich repeat domain (LRRD) bound to single chain analog of FSH, and the crystal structures of TSHR LRRD bound to the stimulatory type human monoclonal antibody M22 [5] and the inhibitory type- monoclonal antibody K1-70 [6]. Both these crystal structures propose LRRDs as the primary ligand binding site which interacts with the hormone through its determinant loop in a hand-clasp fashion [7] while the autoantibodies mimics the hormone binding to a large extent [8] . These structures, while providing detailed understanding of the molecular interactions of the LRRs with the hormone, shed little light on the mechanism by which the signal generated at the LRRD are transduced to the downstream effector regions at the distally situated TMD. Hence, while one understands the ligand binding to a large extent, the activation process is not well understood, one of the central objective of the present study. Ligand-receptor interactions are typically studied by perturbing ligand/receptor structure by mutagenesis or by mapping conformational changes by biophysical or computational approaches. In addition to the above-mentioned approaches, the present work also uses highly specific antibodies against different domains of the receptor as molecular probes due to the ability of antibodies to distinguish between conformations likely to arise during the activation process. Use of antibodies to understand the receptor activation process is especially apt for TSHR due to the presence of physiologically relevant TSHR autoantibodies and their ability to influence hormone binding and receptor activation [9, 10]. Chapter 2 attempts to provide a comparison between the interactions of the hormone and the autoantibodies with TSHR. For this purpose, two assays were developed for identification of TSHR autoantibodies in the sera of patients suffering from autoimmune thyroid diseases (AITD), the first assay is based on the ability of TSHR autoantibodies to compete for radiolabeled hormone (The TSH binding inhibition (TBI), assay) and the second based on the capability of stimulatory antibody to produce cAMP in cells expressing TSHR (TSHR stimulatory immunoglobin (TSI) assay). A stable cell line expressing TSHR capable of recognizing both TSH and TSHR autoantibodies was thus created and used for prospective and retrospective analysis of AITD patients. Based on the TBI and TSI profiles of IgGs, purified from AITD patient's sera, it was recognized that TSHR stimulatory and TSH binding inhibitory effects of these antibodies correlated well, indicating overlap between hormone binding and IgG binding epitopes. It was also recognized that stimulatory IgGs are not affected by negative regulatory mechanism that governs TSH secretion substantiating the persistence of these antibodies in circulation. Kinetics of cAMP production by Graves’ stimulatory IgG was found to be fundamentally distinct, where the autoantibodies displayed pronounce hysteresis during the onset of the activation process when compared to the hormone. This could possibly be explained by the oligoclonality of the autoantibody population, a different mechanism of receptor activation or dissimilarity in autoantibody and hormone epitopes. To gain additional insights into the epitopes of TSHR autoantibodies and the regions that might be critical in the activation process, different overlapping fragments encompassing the entire TSH receptor ECD were cloned, expressed in E.coli as GST fusion proteins and purified: 1] the first three LRRs (TLRR 1-3, amino acid (aa) 21-127), 2] the first six LRRs (TLRR 1-6, aa 21-200), 3] the putative major hormone binding domain (TLRR 4-6, aa 128-200), and 4] the hinge region of TSH receptor along with LRR 7 to 9, (TLRR 7-HinR, aa 201-413). The receptor fragment TLRR 7-HinR was further subdivided into LRR 7-9 (TLRR 7-9, aa 201-161) and the hinge region (TSHR HinR, aa 261-413), expressed as N-terminal His-Tagged protein and purified using IMAC chromatography. Simultaneously, the full-length TSHR ECD was cloned, expressed and purified using the Pichia pastoris expression system. ELISA or immunoblot analysis of autoantibodies with the TSHR exodomain fragments suggested that Graves’ stimulatory antibody epitopes were distributed throughout the ECD with LRR 4-9 being the predominant site of binding. Interestingly, experiments involving neutralization of Graves’ IgG stimulated cAMP response by different receptor fragment indicated that fragments corresponding to the TSHR hinge region were better inhibitors of autoantibody stimulated receptor response than corresponding LRR fragments, suggesting that the hinge region might be an important component of the receptor activation process. This was in contrast to prevalent beliefs that considered the hinge region to be an inert linker connecting the LRRs to the TMD, a structural entity without any known functional significance. Mutagenesis in TSHR hinge region and agonistic antibodies against FSHR and LHR hinge regions, reported by the laboratory, recognized the importance of the hinge regions as critical for receptor activation and may not simply be a scaffold [11-13]. Unfortunately, the mechanism by which the hinge region regulates binding or response or both have not been well understood partially due to unavailability of structural information about this region. In addition poor sequence similarity within the GpHR family and within proteins of known structure, make this region difficult to model structurally. In chapter 3, effort is made to model the hinge regions of the three GpHR based on the knowledge driven and Ab initio protocols. An assembled structure comprising of the LRR domain (derived from the known structures of FSHR and TSHR LRR domains) and the modeled hinge region and transmembrane domain presents interesting differences between the three receptors, especially in the manner the hormone bound LRRD is oriented towards the TMD. These models also suggested that the α-subunit interactions in these three receptors are fundamentally different and this was verified by investigating the effects of two α-subunit specific MAbs C10/2A6 on hCG-LHR and hTSH-TSHR interactions. These two α-subunit MAbs had inverse effects on binding of hormone to the receptor. MAb C10 inhibited TSH binding to TSHR but not that of hCG, whereas MAb 2A6 inhibited binding of hCG to LHR but not of hTSH. Investigation into the accessibility of their epitopes in a preformed hormone receptor complex indicated that the α-subunit may become buried or undergo conformational change during the activation process and interaction may be different for LHR and TSHR. Fundamental differences in TSHR and LHR were further investigated in the next chapter (Chapter 4), especially with regards to the ligand independent receptor activation. Polyclonal antibodies were developed against LRR 1-6, TLRR 7-HinR and the TSHR HinR receptor fragments. The LRR 1-6 antibodies were potent inhibitor of receptor binding as well as response, similar to that observed with antibodies against the corresponding regions of LHR. Interestingly, the antibodies against the hinge region of TSHR were unable to inhibit hTSH binding, but were effective inhibitors of cAMP production suggesting that this region may be involved in a later stage of a multi-step activation process. This was also verified by studying the mechanism of inhibition of receptor response and their effect on ligand-receptor association and dissociation kinetics. Hinge region-specific antibodies immunopurified from TLRR 7-HinR antibodies behaved akin to those of the pure hinge region antibodies providing independent validation of the above results. This result was, however, in contrast to those observed with a similar antibody against LHR hinge region. As compared to the TSHR antibody, the LHR antibody inhibited both hormone binding and response. In addition, this antibody could dissociate a preformed hormone-receptor complex which was not observed for TSHR hinge region antibodies. Although unable to dissociate preformed hormone-receptor complex by itself, the TSHR HinR antibodies augmented hormone induced dissociation of the hormone-receptor complex suggesting that this region may be involved in modulation of negative cooperativity associated with TSHR. Molecular dissection of the role of hinge region of TSHR was further carried out by using monoclonal antibodies against LRR 1-3 (MAb 413.1.F7), LRR 7-9 (MAb 311.87), TSHR hinge region (MAb 311.62 and MAb PD1.37). MAb 311.62 which identifies the LRR/Cb-2 junction (aa 265-275), increased the affinity of TSHR for the hormone while concomitantly decreasing its efficacy, whereas MAb 311.87 recognizing LRR 7-9 (aa 201-259) acted as a non-competitive inhibitor of TSH binding. MAb 413.1.F7 did not affect hormone binding or response and was used as the control antibody for different experiments. Binding of MAbs was sensitive to the conformational changes caused by the activating and inactivating mutations and exhibited differential effects on hormone binding and response of these mutants. By studying the effects of these MAbs on truncation and chimeric mutants of thyroid stimulating hormone receptor (TSHR), this study confirms the tethered inverse agonistic role played by the hinge region and maps the interactions between TSHR hinge region [14] and exoloops responsible for maintenance of the receptor in its basal state. Mechanistic studies on the antibody-receptor interactions suggest that MAb 311.87 is an allosteric insurmountable antagonist and inhibits initiation of the hormone induced conformational changes in the hinge region, whereas MAb 311.62 acts as a partial agonist that recognizes a conformational epitope critical for coupling of hormone binding to receptor activation. Estimation of apparent affinities of the antibody to the receptor and the cooperativity factor suggests that epitope of MAb 311.87 (LRR 7-9) may act as a pivot involved in the initial events immediate to hormone binding at the LRRs. The anatgonsitic effect of MAB 311.62 on binding and response also suggested that binding of hormone is conformationally selective rather than an induced event. The hinge region, probably in close proximity with the α-subunit in the hormone-receptor complex, acts as a tunable switch between hormone binding and receptor activation. In contrast to the stimulatory nature of Cb-2 antibody such as MAb 311.62, MAb PD1.37, which identified residues aa 366–384 near Cb-3, was found to be inverse agonistic. Unlike other known inverse agonistic MAbs such as CS-17 [15] and 5C9 [16], MAb PD1.37 did not compete for TSH binding to TSHR, although it could inhibit hormone stimulated response. Moreover, unlike CS-17, MAb PD1.37 was able to decrease elevated basal cAMP of hinge region constitutively activated mutations only but not those in the extracellular loops. This is particularly important as interaction of hinge region residues with those of ECLs had been thought to be critical in maintenance of the basal level of receptor activation and are responsible for attenuating the constitutive basal activity of the mutant and wild-type receptors in the absence of the hormone. This was demonstrated by a marked increase in the basal constitutive activity of the receptor upon the complete removal of its extracellular domain, which returned to the wild-type levels upon reintroduction of the hinge region. However, careful comparison of the activities of the mutants (receptors harboring deletions and gain-of-function mutations) with maximally stimulated wild-type TSHR indicated that these mutations of the receptor resulted primarily in partial activation of the serpentine domain suggesting that only the ECD in complex with the hormone is the full agonist of the receptor. Confirmation of the above proposition has been difficult to verify primarily due to a highly transient conformational change in the tripartite interaction of the hinge region/hormone and the ECLs. The current approaches of using antibodies to probe the ECLs are difficult due to the conformational nature of the antigen as well as difficulty in obtaining a soluble protein. In chapter 5, the ligand induced conformational alterations in the hinge regions and inter-helical loops of LHR/FSHR/TSHR were mapped using the exoloop specific antibodies generated against a mini-Transmembrane domain (mini-TMD) protein. This mini-TMD protein, designed to mimic the native exoloop conformations, was created by joining the TSHR exoloops, constrained through the helical tethers and library derived linkers. The antibody against mini-TMD specifically recognized all three GpHRs and inhibited the basal and hormone stimulated cAMP production without affecting hormone binding. Interestingly, binding of the antibody to all three receptors was abolished by prior incubation of the receptors with the respective hormones suggesting that the exoloops are buried in the hormone-receptor complexes. The antibody also suppressed the high basal activities of gain-of-function mutations in the hinge regions, exoloops and TMDs such as those involved precocious puberty and thyroid toxic adenomas. Using the antibody and point/deletion/chimeric receptor mutants, dynamic changes in hinge region-exoloop interactions were mapped. The computational analysis suggests that mini-TMD antibodies act by conformationally locking the transmembrane helices by restraining the exoloops and juxta-membrane regions. This computational approach of generating synthetic TMDs bears promise in development of interesting antibodies with therapeutic potential, as well as, explains the role of exoloops during receptor activation. In conclusion (Chapter 6), the study provides a comprehensive outlook on the highly dynamic interaction of ligand and different subdomains of the TSHR (and to a certain extent of LHR and FSHR) and proposes a model of receptor activation where the receptor is in a dynamic equilibrium between the low affinities constrained state and the high affinity unconstrained state and bind to the hormone through the LRR 4-6. Upon binding the βL2 loop of the hormone contact LRR 8-10 that triggers a conformational change in the hinge region driving the α-subunit to contact the ECLs. Upon contact, the ECLs cooperatively causes helix movement in the TMH and ultimately in ICLs causing the inbuilt GTP-exchange function of a GPCR.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) couple extracellular receptor proteins to intracellular effector enzymes and ion channels. The observation that alterations in G protein-coupled signalling pathways can impact cellular function and proliferation, and cause human disease, has stimulated investigation into the molecular and pharmacological regulation of G protein expression and action. The most well characterized models for altered G protein expression defects have been based on naturally occurring mutations in GNAS, a complex gene at 20q13 which encodes the α‎ subunit of Gs, the G protein that stimulates adenylyl cyclase. Somatic mutations in GNAS (OMIM 139320) that activate Gα‎_s are present in a subset of endocrine tumours and in patients with the McCune–Albright syndrome (OMIM 174800), a sporadic disorder characterized by increased hormone production and/or cellular proliferation of many tissues. By contrast, germline mutations of the GNAS gene that decrease expression or function of Gα‎_s are present in subjects with Albright’s hereditary osteodystrophy (AHO), a heritable disorder associated with a constellation of developmental defects and, in many patients, reduced responsiveness to multiple hormones that signal through receptors that require Gα‎_s to activate adenylyl cyclase EC 4.6.1.1 (i.e. pseudohypoparathyroidism type 1a (OMIM 103580)). McCune–Albright syndrome (MAS) and AHO represent contrasting gain of function and loss of function mutations in the GNAS gene, respectively. Clinical and biochemical analyses of subjects with these syndromes have extended our understanding of the developmental and functional consequences of dysfunctional G protein action, and have provided unexpected insights into the importance of cAMP as a regulator of the growth and/or function of many tissues. This chapter will focus on the clinical implications of activating mutations of GNAS as the basis for MAS.

Masayuki Inoue of the University of Tokyo designed (Organic Lett. 2009, 11, 3630) a linker that specifically directed C-H hydroxylation, as illustrated by oxidation of 1 to 2. Phil S. Baran of Scripps/La Jolla detailed (Nature 2009, 459, 824; Angew. Chem. Int. Ed. 2009, 48, 9705) some of the factors that direct reactivity in intermolecular C-H hydroxylation. Ning Jiao of Peking University devised (Angew. Chem. Int. Ed. 2009, 48, 7094) a protocol for the direct oxidation of a methylated aromatic 3 to the nitrile 4. Armando J. L. Pombeiro of TU Lisbon developed (Adv. Synth. Cat. 2009, 351, 2936) a procedure for C-H carboxylation, converting 5 into 6. Maurizio Fagnoni of the University of Pavia showed (Chem. Commun. 2009, 7351) that sunlight was sufficient to promote the addition of cyclohexane 7 to methyl acrylate to give 8. Jin-Quan Yu, also of Scripps/La Jolla, established (J. Am. Chem. Soc. 2009, 131, 9886) conditions for the selective Pd-mediated coupling of 9 to iodobenzene to give 10. Alexei V. Novikov of the University of North Dakota demonstrated (Tetrahedron Lett. 2009, 50, 6963; Heterocycles 2009, 78, 2531) that both diazo sulfonates such 11 and the related diazo sulfones cyclized smoothly under Rh catalysis to give the six-membered ring products. In the course of a synthesis of the Psoralea corylifolia –derived bakuchiol, the adduct from the cyclization of 11 was converted into the vinylated product 12. C-H bonds can also be activated electronically by proximal functional groups. Yong-Min Liang of Lanzhou University observed (J. Org. Chem. 2009, 74, 7464) that an N-aryl cyclic amine 13 could be oxidized to the syn diacetoxylated product 14. Note that the α-acetoxy group of 14 is activated for ionization and further bond formation. Yuhong Zhang of Zhejiang University, Hangzhou, found (Organic Lett. 2009, 11, 3730) that the activated intermediate from the oxidation of 15 coupled with a silyl enol ether to deliver the coupled product 16. Bernd Plietker of the Universität Stuttgart devised (Angew. Chem. Int. Ed. 2009, 48, 5752) a Ru catalyst for the coupling of an alkyne 18 to an α,β-unsaturated ester 17 to give the diene 19. Both disubstituted alkynes and more complex α,β-unsaturated esters participated as well. Gregory K. Friestad of the University of Iowa observed (Organic Lett. 2009, 11, 819) that an N-vinyl amide 20 could be homologated to the ester 22.

AbstractSeveral elegant reactivities can be observed in reactions involving palladium(I) species, allowing access to molecular architectures that are often beyond the capabilities of popular diamagnetic palladium complexes. This review presents three main axes of research in this context, which have mostly emerged in the last decade. Reactions promoted by visible light enable synthetic methods that are unusual in their mild experimental conditions coupled with remarkably broad functional group tolerance. The use of discrete palladium(I) dimers as precatalysts allows one to perform a wide set of cross-coupling protocols, such as Kumada and Negishi reactions, and chalcogenation reactions, with a surgical precision on the carbon—halogen bond that is initially activated. The generation of alkyl radicals and palladium(I) species through a thermal strategy proves useful for the elaboration of substrates with several polyfluorinated fragments, which are otherwise elusive coupling partners for more common two-electron processes.

Günter Helmchen of the Ruprecht-Karls-Universität Heidelberg set (Organic Lett. 2010, 12, 1108) the absolute configuration of 3 by Ir*-mediated coupling of 1 with 2. Diastereoselective Pauson-Khand cyclization then led to (-)-α-kainic acid 5. Till Opatz, now at the Johannes Gutenberg-Universität Mainz, showed (Organic Lett. 2010, 12, 2140) that the product from the Dibal reduction of 6 could be condensed with the amine 7 without epimerization. Kim cyclization then directly delivered the pentacyclic alkaloid (+)-tylophorine 9. The interesting dimeric alkaloid lycoperine A 13 was recently isolated from the Japanese club moss Lycopodium hamiltonii. Scott D. Rychnovsky of the University of California, Irvine, prepared (Organic Lett. 2010, 12, 72) 12 by double alkylation of the bis-nitrile 11 with the enantiomerically pure allylic bromide 10. Although the projected reductive decyanation of 12 failed, hydrolysis followed by diastereoselective reductive amination successfully gave 13. Retrosynthetic analysis of fluvirucinine A2 16 could lead to an acyclic amino acid, which could be cyclized to the macrolactam. Young-Ger Suh of Seoul National University took (Organic Lett. 2010, 12, 2040) a different approach, building up the 14-membered ring system by two four-carbon ring expansions, beginning with an enantiomerically pure piperidine precursor. The second of these enolate-based aza-Claisen ring expansions is illustrated in the conversion of 14 to 15. Richmond Sarpong of the University of California, Berkeley, faced (J. Am. Chem. Soc. 2010, 132, 5926) a different sort of challenge in the synthesis of the dimeric Lycopodium alkaloid complanadine A 19. Even with established access to monomers such as 17 and its precursors, it was not clear how the 5-position of the pyridine ring could be selectively activated for bond formation. The solution to this dilemma was found in the work of Hartwig. Following that precedent, Ir-catalyzed activation of 17 converted it cleanly into the borinate 18, which could then be coupled with a pyridone triflate to complete the synthesis of 19.

The concept of solid phase peptide synthesis introduced by Merrifield in 1963 involves elongating a peptide chain on a polymeric support via a two-step repetitive process: removal of the Nα-protecting group and coupling of the next incoming amino acid. A second feature of the solid phase technique is that reagents are added in large excesses which can be removed by simple filtration and washing. Since these operations occur in a single reaction vessel, the entire process is amenable to automation. Essential requirements for a fully automatic synthesizer include a set of solvent and reagent reservoirs, as well as a suitable reaction vessel to contain the solid support and enable mixing with solvents and reagents. Additionally, a system is required for selection of specific solvents and reagents with accurate measurement for delivery to and removal from the reaction vessel, and a programmer to facilitate these automatic operations is necessary. The current commercially available instruments offer a variety of features in terms of their scale (15 mg to 5 kg of resin), chemical compatibility with 9-fluorenylmethyloxycarbonyl/tert-butyl (Fmoc/tBu) and tert-butyloxycarbonyl/ benzyl (Boc/Bzl)-based methods, software (reaction monitoring and feedback control), and flexibility (additional washing and multiple activation strategies). In addition, certain instruments are better suited for the synthesis of more complex peptides such as cyclic, phosphorylated, and glycosylated sequences while others possess the ability to assemble a large number of peptide sequences. The selection of an instrument is dependent on the requirements and demands of an individual laboratory. This chapter will describe the features of the currently available systems. As the field of solid phase synthesis evolved, manufacturers designed systems based on the synergy between chemistry and engineering. A key component to an instrument is the handling of amino acids and their subsequent activation to couple to a polymeric support. The goal of an automated system is to duplicate conditions that provide stability to reactive species that might decompose. Standard protocols for automated synthesis incorporate carbodiimide, phosphonium, and aminium/uronium reagents, preformed active esters, and acid fluorides. For further details on coupling methods, see Chapter 3. A second issue related to coupling chemistry is the time required to dissolve an amino acid and store this solution.

This report discusses the advantages and challenges in using direct psychological personality profile and psychodynamic assessments (corresponding to Otto Kernberg’s model of personally organization) of suicide bombers and lone actors. Two studies that administered various psychological instruments (i.e., self-report inventories, semi-structured interviews, and projective tests) were used to assess these subjects in a prison setting, before or after their trial. Main findings showed that suicide bombers displayed low levels of ego strength with dependent and/or avoidant personality styles, while most of the lone actors presented evidence of psychiatric histories. Also, the main methodological advantages and challenges of the assessment procedures and instruments utilized are discussed. Self-report inventories were found to be less valid. In contrast, semi-structured interviews assisted in identifying a more comprehensive theoretical understanding of both personality dynamics and the discerning of traumatic experiences in participants’ background related primarily to their family history. Projective tests had limited and restricted responses i.e., lacked the necessary complexity. This pattern likely reflected those participants with either limited mental resources, maladaptive personality styles, or hostile responses toward their assessors. Future directions are discussed in a psycho-cultural theoretical perspective regarding the development risk/threat assessment instruments to discern potential perpetrators who are victims of trauma in families living under specific cultural contexts. We assume that these victims’ manifest dissociation defences, present tendencies to activate mobilization, and immobilization energetic systems. These systems evoke complex behaviour patterns triggering suicidal tendencies coupled with rage tendencies aiming to end the lives of others, in this context, perceived political enemies.

The complex polycyclic structure of N-methylwelwitindolinone D isonitrile 3 was assigned in 1999. The welwitinines show an intriguing range of biological activity, including reversal of P-glycoprotein-mediated multidrug resistance in human carcinoma cells. Viresh H. Rawal of the University of Chicago described (J. Am. Chem. Soc. 2011, 133, 5798) the first synthesis of 3, using as a key step the Pd-catalyzed cyclization of 1 to 2. The ketone 1 was assembled by the convergent coupling of 7 with 11. The indole 7 was readily available by Batcho-Leimgruber cyclization of commercial 4 to 5. The expected 3-acylation followed by N -methylation delivered the stable ketone 6. The unstable 7 was prepared as needed. The anisole 8 was the starting material for the preparation of the alicyclic diene 11. Although this synthesis was carried out in the racemic series, enantiomerically enriched 9 could be prepared by Shi epoxidation of the β,γ-unsaturated ketone from Birch reduction The alcohol 7 was not stable to silica gel chromatography. The mixture of 11 with the crude alcohol 7 was therefore activated by the addition of TMSOTf, then added via cannula to aqueous HClO4 in THF to deliver the coupled product 1 as a single diastereomer. The remarkable cyclization of 1 to 2 required extensive screening. Eventually it was found that a combination of ( t -Bu)3 P with Pd(OAc)2 as the Pd source worked well. This concise convergent synthetic strategy makes the welwitinine core 2 available in gram quantities. There were two problems to be solved in the conversion of 2 to 3. The first was the installation of the oxy bridge. Indoles are notoriously sensitive to overoxidation. Nevertheless, addition of an acetone solution of dimethyl dioxirane to the bromo ketone 12 over 24 hours gave clean conversion to 13. The remaining challenge was the conversion of the aldehyde of 13 to the isonitrile. Kim had described the inversion of an oxime to the isothiocyanate. Optimization of this protocol led to the thiourea 14 as the best for this transformation. Mild desulfurization then delivered N -methylwelwitindolinone D isonitrile 3.

(−)-Leiodermatolide 4, isolated from the lithistid sponge Leiodermatium sp., showed 5.0-nM activity against PANC-1 pancreatic carcinoma cells, and reduced toxicity toward normal cells. Ian Paterson of the University of Cambridge established (Angew. Chem. Int. Ed. 2014, 53, 2692) a synthetic route to 4 based on sp2–sp2 coupling, as exemplified by the combination of 1 with 2 to give 3. Addition of the boron enolate of the enantiomerically-pure benzoate 5 to the iodoaldehyde 6 gave 7, that was silylated, reduced, and deprotected to give 1. Addition of the boron enolate of ent-5 to propanal gave 8. The α-acyloxy ketone of 8 served as a masked acylating agent. The addition of allyl magnesium bromide followed by oxidative cleavage led to the ketone 9. The preparation of 2 was com­pleted by diastereoselective Mukaiyama aldol condensation of 9 with the ketene silyl acetal 10. The intramolecular Heck coupling of 1 with 2 presumably proceeded by way of the organo-Pd intermediate 11. β-Hydride elimination could have given one or more of four possible dienes, but in fact the E,E product 3 dominated, as expected. The allylic H’s are activated for elimination, while the H’s β to the silyl ether are deacti­vated both electronically and sterically. The third component of 4 was the stannane 17. Applying the same strategy, the addition of ent-5 to the aldehyde 12 gave 13, that was protected and condensed with 14 to deliver, after oxidative cleavage, the alkynyl ketone 15. Conjugate addition of iodide proceeded with good geometric control to give 16, that was protected and stan­nylated to complete the preparation of 17. The diol 3 was oxidatively cleaved, and the resulting aldehyde was carried on to the iodide 18. This was coupled with the stannane 17 to give the diene 19. A sequence of deprotection, oxidation, and further deprotection yielded a tetraol, that was lac­tonized with high selectivity to give the 16-membered ring of (−)-leiodermatolide 4.

Recent research in autonomous robot construction and in computer graphics animation has found that a control architecture with networks of functional behaviors is far more successful for accomplishing real-world tasks than traditional methods. The high-level control and often the behaviors themselves are motivated lay the animal sciences, where the individual behaviors have the following properties: . . .• they are grounded in perception. . . . . . . • they normally participate in directing an agent’s effectors. . . . . . . • they may attempt to activate or deactivate one-auother. . . . . . . • each behavior by itself performs some task useful to the agent. . . . In both robotics and animation there is a desire to control agents in environments, though in graphics both are simulated, and in both cases the move to the animal sciences is out of discontent with traditional methods. Computer animation researchers are discontent with direct kinematic control and are increasingly willing to sacrifice complete control for realism. Robotics researchers are reacting against the traditional symbolic reasoning approaches to control such as automatic planning or expert systems. Symbolic reasoning approaches are brittle and incapable of adapting to unexpected situations (both advantageous and disastrous). The approach taken is, more or less, to tightly couple sensors and effectors and to rely on what Brooks [Bro90] calls emergent behavior, where independent behaviors interact to achieve a more complicated behavior. From autonomous robot research this approach has been proposed under a variety of names including: subsumption architecture by [Bro86], reactive planning by [GL90, Kae90], situated activity by [AC87], and others. Of particular interest to us, however, are those motivated explicitly by animal behavior: new AI by Brooks [Bro90], emergent reflexive behavior by Anderson and Donath [AD90], and computational neuro-ethology by Beer, Chiel, and Sterling [BCS90]. The motivating observation behind all of these is that even very simple animals with far less computational power than a calculator can solve real world problems in path planning, motion control, and survivalist goal attainment, whereas a mobile robot equipped with sonar sensors, laser-range finders, and a radio-Ethernet connection to a, Prolog-based hierarchical planner on a supercomputer is helpless when faced with the unexpected.

The coagulation cascade of blood may be initiated by flow induced platelet activation, which prompts clot formation in prosthetic cardiovascular devices and arterial disease processes. While platelet activation may be induced by biochemical agonists, shear stresses arising from pathological flow patterns enhance the propensity of platelets to activate and initiate the intrinsic pathway of coagulation, leading to thrombosis. Upon activation platelets undergo complex biochemical and morphological changes: organelles are centralized, membrane glycoproteins undergo conformational changes, and adhesive pseudopods are extended. Activated platelets polymerize fibrinogen into a fibrin network that enmeshes red blood cells. Activated platelets also cross-talk and aggregate to form thrombi. Current numerical simulations to model this complex process mostly treat blood as a continuum and solve the Navier-Stokes equations governing blood flow, coupled with diffusion-convection-reaction equations. It requires various complex constitutive relations or simplifying assumptions, and is limited to μm level scales. However, molecular mechanisms governing platelet shape change upon activation and their effect on rheological properties can be in the nm level scales. To address this challenge, a multiscale approach which departs from continuum approaches, may offer an effective means to bridge the gap between macroscopic flow and cellular scales. Molecular dynamics (MD) and dissipative particle dynamics (DPD) methods have been employed in recent years to simulate complex processes at the molecular scales, and various viscous fluids at low-to-high Reynolds numbers at mesoscopic scales. Such particle methods possess important properties at the mesoscopic scale: complex fluids with heterogeneous particles can be modeled, allowing the simulation of processes which are otherwise very difficult to solve by continuum approaches. It is becoming a powerful tool for simulating complex blood flow, red blood cells interactions, and platelet-mediated thrombosis involving platelet activation, aggregation, and adhesion.

The coagulation cascade of blood may be initiated by flow induced platelet activation, which prompts clot formation in prosthetic cardiovascular devices and arterial disease processes. While platelet activation may be induced by biochemical agonists, shear stresses arising from pathological flow patterns enhance the propensity of platelets to activate and initiate the intrinsic pathway of coagulation, leading to thrombosis. Upon activation platelets undergo complex biochemical and morphological changes: organelles are centralized, membrane glycoproteins undergo conformational changes, and adhesive pseudopods are extended. Activated platelets polymerize fibrinogen into a fibrin network that enmeshes red blood cells. Activated platelets also cross-talk and aggregate to form thrombi. Current numerical simulations to model this complex process mostly treat blood as a continuum and solve the Navier-Stokes equations governing blood flow, coupled with diffusion-convection-reaction equations. It requires various complex constitutive relations or simplifying assumptions, and is limited to μm level scales. However, molecular mechanisms governing platelet shape change upon activation and their effect on rheological properties can be in the nm level scales. To address this challenge, a multiscale approach which departs from continuum approaches, may offer an effective means to bridge the gap between macroscopic flow and cellular scales. Molecular dynamics (MD) and dissipative particle dynamics (DPD) methods have been employed in recent years to simulate complex processes at the molecular scales, and various viscous fluids at low-to-high Reynolds numbers at mesoscopic scales. Such particle methods possess important properties at the mesoscopic scale: complex fluids with heterogeneous particles can be modeled, allowing the simulation of processes which are otherwise very difficult to solve by continuum approaches. It is becoming a powerful tool for simulating complex blood flow, red blood cells interactions, and platelet-mediated thrombosis involving platelet activation, aggregation, and adhesion.

The coagulation cascade of blood may be initiated by flow induced platelet activation, which prompts clot formation in prosthetic cardiovascular devices and arterial disease processes. While platelet activation may be induced by biochemical agonists, shear stresses arising from pathological flow patterns enhance the propensity of platelets to activate and initiate the intrinsic pathway of coagulation, leading to thrombosis. Upon activation platelets undergo complex biochemical and morphological changes: organelles are centralized, membrane glycoproteins undergo conformational changes, and adhesive pseudopods are extended. Activated platelets polymerize fibrinogen into a fibrin network that enmeshes red blood cells. Activated platelets also cross-talk and aggregate to form thrombi. Current numerical simulations to model this complex process mostly treat blood as a continuum and solve the Navier-Stokes equations governing blood flow, coupled with diffusion-convection-reaction equations. It requires various complex constitutive relations or simplifying assumptions, and is limited to μm level scales. However, molecular mechanisms governing platelet shape change upon activation and their effect on rheological properties can be in the nm level scales. To address this challenge, a multiscale approach which departs from continuum approaches, may offer an effective means to bridge the gap between macroscopic flow and cellular scales. Coarse Grained Molecular dynamics (CGMD) and discrete/dissipative particle dynamics (DPD) methods have been employed in recent years to simulate complex processes at the molecular scales, and various viscous fluids at low-to-high Reynolds numbers at mesoscopic scales. Such particle methods possess important properties at the mesoscopic scale: complex fluids with heterogeneous particles can be modeled, allowing the simulation of processes which are otherwise very difficult to solve by continuum approaches. It is becoming a powerful tool for simulating complex blood flow, red blood cells interactions, and platelet-mediated thrombosis involving platelet activation, aggregation, and adhesion.

Previous reports have shown that f ibrin (Fn), plasminogen (Pig), and tissue plasminogen activator (tPA) can bind to bovine aortic endothelial cell (BAEC) respectively. The present studies are to examine the formation of the trimolecular complex of Fn, Pig, and tPA on the BAEC surface. BAEC monolayers were first incubated with one of the three components in buffer (pH 7.5, 25 C) and washed, and then incubated with the second component and washed. Finally, the BAEC monolayers after the first and second incubations were incubated with the third component in the presence of plasmin substrate S-2251. Plasminogen activation rates (PAR)(pM/min) were measured. The results (TABLE) show that Pig and tPA retain their respective activities after their binding to BAEC surface, either Pig or tPA as the first bound component. These results suggest that the formation of Plg-tPA complex can occur after their binding to the BAEC surface. With Fn as the second binding component, BAEC monolayers show higher PAR values (138 & 187, pM/imin) than those without Fn (34.5 & 59.1 pM/min), suggesting Fn potentiation of tPA-induced Pig activation on the BAEC surface. Since the reaction of Fn with the Plg-tPA complex is required for the expression of fibrin potentiation, the present results suggest that the formation of trimolecular complex can occur on the BAEC surface. Further, when Fn was the first bound component and Pig or tPA was the second or third bound component, the BAEC rmonolayers show the highest PAR values (353 & 331 nVrmin), suqqestinq that both Plq and tPA can bind not only to BAEC surface but also to Fn which was already first bound to BAEC surface. Thus, trimolecular complex may have multiple binding sites on BAEC surface and anyone of the three binding sites (Fn, Pig, and tPA) may act as the binding site for the trimolecular couplex on the BAEC surface.

Levels of factor XIIa- and kallikrein-Cl inhibitor (Cl-Inh) complexes in plasma reflect activation of the contact system in vivo. Here, we report the development of radioimmunoassays (RIAs) for these complexes using a monoclonal antibody (mAb K0K12) that reacts with a neodeterminant exposed on Cl-Inh after interaction with proteases. mAb K0K12 was obtained by a fusion experiment with spleen cells of a mouse hyperimmunized with Cl-Inh complexes.Experiments with purified Cl-Inh incubated with either Cls or elastase revealed that the determinant for mAb KOK12 is exposed on complexed as well as proteolytically inactivated (modified) Cl-Inh.Radioimmunoassays (RIAs) for the detection of factor Xlla-Cl-Inh and kallikrein-Cl-Inh complexes were performed as follows: mAb K0K12 was coupled to Sepharose and incubated with the sample to be tested. Binding of Cl-Inh complexes was detected by a subsequent incubation with 125I-antibodies against factor XII or (pre)kallikrein.With these RIAs, activation of 0.1% of factor XII or prekal-likrein in plasma is easily detected.Optimal conditions for blood sampling and processing were established, i.e. conditions that prevented any in vitro activation of factor XII and prekallikrein. Levels of factor XIIa-Cl-Inh and kallikrein-Cl-Inh complexes in plasma samples from normal donors were less than 0.1 U/ml (100 U/ml is the maximal amount of Cl-Inh complexes generated in pooled plasma by DXS). Considerably higher, and fluctuating levels were observed in patients with diseases such as septicaemia. These highly sensitive RIAs will facilitate studies concerning the role of the contact system in human pathophysiology.

Studies with calcium ionophores, permeabilised platelets, and platelets containing fluorescent calcium indicators quin2 and fura-2 have shown that elevation [Ca2+]i is an effective trigger for shape-change, aggregation, secretion and release of TxA2; and that elevation of [Ca2+]i is an important part of a complex “activation cascade” set up by natural agonists combining with their surface receptors. We have used calcium ionophores to impose [Ca2+]i changes, monitored by indicator dyes, to construct [Ca2+]i/function relationships for shape-change, secretion, aggregation, arachidonic acid release, TxA2 production, and myosin phosphorylation in intact platelets(e.g.1,2). Some of these functions can also be studied by analagous experiments using Ca2+-buffers to set known [Ca2+]i in permeabilised platelets. Our ability to monitor and modulate [Ca2+]i with fluorescent indicators has also allowed us to see what happens when [Ca2+]i changes are greately reduced or even absent and to investigate other pathways of intracellular activation. We think that formation of diacyl glycerol and activation of protein kinase-C can explain, some, but not all, of the cell activation that some agonists can apparently evoke at or near resting [Ca2+]i, and that combined or synergistic actions of Ca2+ and other intracellular mediators is the usual basis for physiological activation(3). Most agonists seem to promote both Ca2+ entry across the plasma membrahe and discharge from intracellular organelles, presumably the dense tubular system. The available evidence fits with the prevailing idea that Ins 1,4,5 P3 formed by agonist evoked hydrolysis of PIP2, is the internal messenger for Ca2+ release. Our kinetic measurements of [Ca2+]i transients require that optimal concentrations of InsP3 are formed within 250 milliseconds(4,5). The question of whether ADP receptors in human platelets are directly coupled with PIP2 breakdown remains contentious. Probably they do, weakly, and the differences from most other receptors are quantitative rather than qualitative. We do not understand the mechanisms of agonist-evoked Ca2+-entry; there is now plenty of evidence that argues against a role for membrane depolarisation and voltage-gated Ca channels, including some recent work with ionic substitution(5). Stopped-flow fluorescence analysis of [Ca2+]i rises in fura-2-loaded human platelets reveals some intriguing new insights(4,5). With thrombin, vasopressin and PAF at optimal concentrations, there is a highly reproducible delay before the signal starts to rise, which is approximately 250msec in the absence of external calcium compared to 190msec in the presence of external calcium. This suggests that Ca entry leads internal release, and gives ample time for complex coupling mechanisms for both processes.The delay with ADP, in the presence of external calcium, is much smaller suggesting a different coupling mechanism for Ca entry.

A large number of monoclonal antibodies to platelet membrane glycoprotein lb (GPIb) have been described but for most of these the position of the epitope is not known. Since many of these influence platelet function, a better understanding of struc-ture-function relationships requires this knowledge. The position of the epitopes for the monoclonal antibodies API (Dr. T.J. Kunicki), AN51 and SZ-2 (Dr. C-G. Ruan), WM23 (Dr. M.C. Berndt) and PI were determined by analysis of proteolytic cleavage fragments of glycocalicin via affinity chromatography on the monoclonal antibodies coupled to Sepharose, elution with diethyl ami ne solution, separation on SDS-gel electrophoresis and detection by silver-staining. First, intact glycocalicin was examined and was found to bind to all monoclonals with the exception of PI. All monoclonals bound intact GPIb. WM23 bound a 70 kDa glycopeptide from the highly-glycosylated 90 kDa tryptic fragment of glycocalicin. API, AN51 and SZ-2 all bound to 45 kDa and 40 kDa, poorly glycosylated tryptic fragments. The 40 kDa fragment is derived from the 45 kDa fragment and has been shown to be the N-terminal region of GPIb. All these monoclonals have been shown to inhibit von Willebrand factor induced platelet agglutination. Platelets were treated with either elastase or calcium activated protease and monoclonal binding checked by immunofluorescence. The immunofluorescence with API, AN51 and SZ-2 was minimal compared to control platelets whereas that of PI remained as strong as the controls. This indicates that the epitope for PI lies on GPIb in a region other than glycocalicin and its absence from glycocalicin is not simply due to conformational changes in that fragment. Since PI inhibits platelet activation by thrombin and ADP it must act via conformational effects and not by blocking the thrombin receptor which lies on the 45 kDa region of glycocalicin. These results support a more complex role for GPIb in platelet activation.

The intriguing finding that functions of t-PA coincide with structural domains and that these domains occur in related proteins, has been the basis to construct hybrid proteins by artificial exon shuffling to prove the conservation of functions in the shuffled domains. The heavy chain (Hch) of t-PA mediates both binding to fibrin and stimulation of plasminogen activator activity via its Finger- and Kringle-2 domain, whereas the light chain (Lch) contains the serine protease moiety of the protein. The Hch of u-PA is very homologous to the Lch of t-PA, but exhibits a higher plasminogen activator activity. This activity of u-PA is not stimulated by fibrin. We employed the ‘M13 in vitro outlooping’ technique to fuse the Hch of t-PA cDNA and the Hch of u-PA cDNA, to create two different hybrid cDNAs. On one hybrid cDNA, the t-PA and the u-PA sequences are coupled precisely at the exon-intron boundaries of the corresponding genes, while the other hybrid cDNA lacks a u-PA segment at the junction, encoding 13 amino acids of u-PA. The hybrid cDNAs were transiently expressed in mouse Ltk- cells and the recombinant proteins were characterized. The plasminogen activator activity of these proteins was determined in an indirect amidolytic assay, using plasminogen and the chromogenic substrate S2251. As anticipated, the activity of both t-PA/u-PA hybrid proteins is stimulated by fibrin, however, not to the same extent as t-PA. Remarkably, we found a decreased inhibition of the hybrid proteins by the endothelial plasminogen activator inhibitor (PAI-1) as compared to t-PA and u-PA, although stable complexes between the hybrid proteins and the inhibitor are formed. We conclude that functions of structural domains are maintained during exon shuffling

The reciproqual localization of VWF and t-PA in EC and MK was analysed in EM using immunocytochemical techniques. In parallel, PAGE electrophoresis, zymographic analysis and immuno-blotting were also performed in cell extracts using specific polyclonal and monoclonal antibodies against VWF and t-PA. By immunofluorescence, VWF showed a dense granular pattern in EC, MK and platelets, although no fluorescence was observed with specific antibodies to t-PA. In contrast, at optical level, with peroxidase or alcaline phosphatase conjugated antibodies, t-PA antigen in EC, MK and platelets appear lightly distributed in the cytoplasm. More precise localization of both antigens was studies in EM after incubation of ultrathin sections with specific colloidal gold coupled antibodies. VWF was detected by specific antibodies in storage granules : γ -granules in MK and Weibel-Palade bodies in EC only reacted with specific antibodies when the storage granules were opened by thin section ; VWF antigen was associated with the tubular structures of these granules. In EC gold or peroxidase coupled antibodies showed the presence of t-PA antigen in rough ergastoplasmic reticulum (RER) saccules and total absence of this antigen in granule structures. After thrombin stimulation, stained vesicles revealed t-PA in the cytoplasm, vesicles which rapidly disappeared after being released. In cultured MK, t-PA was detected by immunoperoxidase in perinuclear cisternae and in small vesicles near the Golgi complex, suggesting the synthesis of t-PA by MK. Concommitantly to appearance of the t-PA, fibrinolytic activity was detected. Immediatly after platelet production fibrinolytic activity disappeared probably by association with an inhibitor. MK and EC extracts after electrophoresis, immunoblotting and zymographic analysis revealed the presence of biologically active t-PA at the same position. Thus the cells have a basal level of active t-PA which can also be seen in intact cells when deposited on fibrin films. However the fibrinolytic activity of t-PA is inhibited in absence of VWF as it is the case in EC of patients with severe forms of VW disease although t-PA antigen is normally present.

Practical aero-engine fuel injection systems are highly complicated, combining complex fuel atomizer and air swirling elements to achieve good fuel-air mixing as well as long residence time in order to enhance both combustion efficiency and stability. While detailed understanding of the multiphase flow processes occurring in a realistic injector has been limited due to the complex geometries and the challenges in near-field measurements, high fidelity, first principles simulation offers, for the first time, the potential for a comprehensive physics-based understanding. In this work, such simulations have been performed to investigate the spray atomization and subsequent droplet transport in a swirling air stream generated by a complex multi-nozzle/swirler combination. A Coupled Level Set and Volume Of Fluid (CLSVOF) approach is used to directly capture the liquid-gas interface and an embedded boundary (EB) method is applied to flexibly handle the complex injector geometry. The ghost fluid (GF) method is also used to facilitate simulations at realistic fuel-air density ratio. Adaptive mesh refinement (AMR) and Lagrangian droplet models are used to efficiently resolve the multi-scale processes. To alleviate the global constraint on the time-step imposed by locally activated AMR near liquid jets, a separate AMR simulation focusing on jet atomization was performed for relatively short physical time and the resulting Lagrangian droplets are coupled into another simulation on a uniform grid at larger time-steps. The high cost simulations were performed at the U.S. Department of Defense high performance computing facilities using over 5000 processors. Experiments at the same flow conditions were conducted at UTRC. The simulation details of flow velocity and vorticity due to the interaction of the fuel jet and swirling air are presented. The velocity magnitude is compared with experimental measurement at two downstream planes. The two-phase spray spreading is compared with experimental images and the flow details are further analyzed to enhance understanding of the complex physics.

The proposed research was directed at determining the activation/binding domains and gene regulation of the PBAN-R’s thereby providing information for the design and screening of potential PBAN-R-blockers and to indicate possible ways of preventing the process from proceeding to its completion. Our specific aims included: (1) The identification of the PBAN-R binding domain by a combination of: (a) in silico modeling studies for identifying specific amino-acid side chains that are likely to be involved in binding PBAN with the receptor and; (b) bioassays to verify the modeling studies using mutant receptors, cell lines and pheromone glands (at tissue and organism levels) against selected, designed compounds to confirm if compounds are agonists or antagonists. (2) The elucidation ofthemolecular regulationmechanisms of PBAN-R by:(a) age-dependence of gene expression; (b) the effect of hormones and; (c) PBAN-R characterization in male hair-pencil complexes. Background to the topic Insects have several closely related G protein-coupled receptors (GPCRs) belonging to the pyrokinin/PBAN family, one with the ligand pheromone biosynthesis activating neuropeptide or pyrokinin-2 and another with diapause hormone or pyrokinin-1 as a ligand. We were unable to identify the diapause hormone receptor from Helicoverpa zea despite considerable effort. A third, related receptor is activated by a product of the capa gene, periviscerokinins. The pyrokinin/PBAN family of GPCRs and their ligands has been identified in various insects, such as Drosophila, several moth species, mosquitoes, Triboliumcastaneum, Apis mellifera, Nasoniavitripennis, and Acyrthosiphon pisum. Physiological functions of pyrokinin peptides include muscle contraction, whereas PBAN regulates pheromone production in moths plus other functions indicating the pleiotropic nature of these ligands. Based on the alignment of annotated genomic sequences, the primary and secondary structures of the pyrokinin/PBAN family of receptors have similarity with the corresponding structures of the capa or periviscerokinin receptors of insects and the neuromedin U receptors found in vertebrates. Major conclusions, solutions, achievements Evolutionary trace analysisof receptor extracellular domains exhibited several class-specific amino acid residues, which could indicate putative domains for activation of these receptors by ligand recognition and binding. Through site-directed point mutations, the 3rd extracellular domain of PBAN-R was shown to be critical for ligand selection. We identified three receptors that belong to the PBAN family of GPCRs and a partial sequence for the periviscerokinin receptor from the European corn borer, Ostrinianubilalis. Functional expression studies confirmed that only the C-variant of the PBAN-R is active. We identified a non-peptide agonist that will activate the PBAN-receptor from H. zea. We determined that there is transcriptional control of the PBAN-R in two moth species during the development of the pupa to adult, and we demonstrated that this transcriptional regulation is independent of juvenile hormone biosynthesis. This transcriptional control also occurs in male hair-pencil gland complexes of both moth species indicating a regulatory role for PBAN in males. Ultimate confirmation for PBAN's function in the male tissue was revealed through knockdown of the PBAN-R using RNAi-mediated gene-silencing. Implications, both scientific and agricultural The identification of a non-peptide agonist can be exploited in the future for the design of additional compounds that will activate the receptor and to elucidate the binding properties of this receptor. The increase in expression levels of the PBAN-R transcript was delineated to occur at a critical period of 5 hours post-eclosion and its regulation can now be studied. The mysterious role of PBAN in the males was elucidated by using a combination of physiological, biochemical and molecular genetics techniques.