Rozprawy doktorskie: „GPC1”

1

Blankenship, Elise. "Conserved solvent networks in GPCR activation". Case Western Reserve University School of Graduate Studies / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=case1458221506.

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2

Poudel, Sagar. "GPCR-Directed Libraries for High Throughput Screening". Thesis, University of Skövde, School of Humanities and Informatics, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-29.

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Guanine nucleotide binding protein (G-protein) coupled receptors (GPCRs), the largest receptor family, is enormously important for the pharmaceutical industry as they are the target of 50-60% of all existing medicines. Discovery of many new GPCR receptors by the “human genome project”, open up new opportunities for developing novel therapeutics. High throughput screening (HTS) of chemical libraries is a well established method for finding new lead compounds in drug discovery. Despite some success this approach has suffered from the near absence of more focused and specific targeted libraries. To improve the hit rates and to maximally exploit the full potential of current corporate screening collections, in this thesis work, identification and analysis of the critical drug-binding positions within the GPCRs were done, based on their overall sequence, their transmembrane regions and their drug binding fingerprints. A proper classification based on drug binding fingerprints on the basis for a successful pharmacophore modelling and virtual screening were done, which facilities in the development of more specific and focused targeted libraries for HTS.

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3

Majin, Wodu. "Mathematical modelling of GPCR-mediated calcium signalling". Thesis, University of Nottingham, 2012. http://eprints.nottingham.ac.uk/12451/.

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Ca2+ is an important messenger which mediates several physiological functions, including muscle contraction, fertilisation, heart regulation and gene transcription. One major way its cytosolic level is raised is via a G-protein coupled receptor (GPCR)- mediated release from intracellular stores. GPCR’s are the target of approximately 50% of all drugs in clinical use. Hence, understanding the underlying mechanisms of signalling in this pathway could lead to improved therapy in disease conditions associated with abnornmal Ca2+ signalling, and to the identification of new drug targets. To gain such insight, this thesis builds and analyses a detailed mathematical model of key processes leading to Ca2+ mobilisation. Ca2+ signalling is considered in the particular context of the M3 muscarinic receptor system. Guided by available data, the Ca2+ mobilisation model is assembled, first by analysing a base G-protein activation model, and subsequently extending it with downstream details. Computationally efficient designs of a global parameter sensitivity analysis method are used to identify the key controlling parameters with respect to the main features of the Ca2+ data. The underlying mechanism behind the experimentally observed, rapid, amplified Ca2+ response is shown to be a rapid rate of inositol trisphosphate (IP3) formation from Phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis. Using the same results, potential drug targets (apart fromthe GPCR) are identified, including the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and PIP2. Moreover, possible explanations for therapeutic failures were found when some parameters exerted a biphasic effect on the relative Ca2+ increase. The sensitivity analysis results are used to simplify the process of parameter estimation by a significant reduction of the parameter space of interest. An evolutionary algorithm is used to successfully fit the model to a significant portion of the Ca2+ data. Subsequent sensitivity analyses of the best-fitting parameter sets suggest that mechanistic modelling of kinase-mediated GPCR desensitisation, and SERCA dynamics may be required for a comprehensive representation of the data.

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4

Tang, Lisa Sarah. "GPCR expressions in Saccharomyces cerevisiae : engineering transductions". Thesis, University of Leeds, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.423190.

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5

Mishra, Satyakam. "Frequent Subgraph Mining Analysis of GPCR Activation". Case Western Reserve University School of Graduate Studies / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=case1613575702373053.

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6

Sladek, Barbara. "Structural studies of integral membrane GPCR accessory proteins". Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:09bf7ada-8e58-49f4-a979-bcd0cec95e8b.

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GPCR accessory proteins regulate the strength, efficiency and specificity of signal transfer upon receptor activation. Due to the inherent difficulties of studying membrane proteins in vitro and in vivo, little is known about the structure and topology of these small accessory proteins. Two examples of GPCR accessory proteins are the Melanocortin-2 receptor accessory protein (MRAP) and the Receptor-activity-modifying protein (RAMP) family. MRAP and RAMP1 are the main focus of this thesis in which they are thoroughly characterised by solution-state NMR and further biophysical techniques. The single-pass transmembrane domain protein MRAP regulates the class A GPCR melanocortin receptors. It is specifically required for trafficking the melanocortin-2-receptor from the endoplasmic reticulum to the cell surface and subsequent receptor activation. A remarkable characteristic of MRAP is its proposed native dual-topology, which leads to an antiparallel homodimeric conformation. Investigation of the biochemical and biophysical properties of MRAP revealed an α-helical transmembrane domain, and an α-helical N-terminal LD(Y/I)L-motif. Further efforts concentrated on establishing the homodimeric conformation of MRAP in vitro. RAMP1 facilitates receptor trafficking and alters the ligand specificity of the GPCR Class B receptors calcitonin receptors and calcitonin receptor-like receptors. Moreover, RAMP1 is required to act as a Calcitonin-gene-related peptide (CGRP) receptor (RAMP1). RAMP1 has been shown to form stable parallel homodimers in the absence of its cognate receptor. Its dimerisation and the possible dimerisation motif PxxxxP-motif were studied extensively. With the goal of understanding the mechanism of dimerisation and the role of GPCR accessory proteins I have used solution-state NMR in detergent micelles as my main technique. NMR provides unique possibilities for understanding the structure and dynamics of such small membrane proteins.

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7

Richardson, Kathryn. "Mechanisms of GPCR signal regulation in fission yeast". Thesis, University of Warwick, 2014. http://wrap.warwick.ac.uk/63554/.

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Cells communicate with each other and respond to environmental cues by sending and receiving signals. Many external signals (ligands) are detected through G protein-coupled receptors (GPCRs), a major class of transmembrane proteins. GPCRs transduce these external signals into appropriate intracellular responses, enabling the cell to adapt to its environment. Malfunctions in these signalling pathways can lead to a range of human diseases and hence GPCRs have become attractive candidates for pharmacological design. The activation of a single receptor has the ability to induce numerous intracellular responses. Coupling this with the great number of different GPCR-types expressed in human cells means that understanding the basic principles of signal transduction and termination in humans is complicated. This study utilises the more simplistic eukaryotic yeast Schizosaccharomyces pombe (S. pombe) to overcome this complexity, as it contains only two GPCR types and hence the cross-talk between pathways is greatly reduced, whilst the structure and signalling functions of GPCRs are often evolutionarily conserved between yeast and humans. Mathematical modelling was used to aid the understanding of GPCR signalling in S. pombe and to inform experimental design. Speci�cally, an ordinary differential equation model �rst developed by Croft et al. (2013) was extended to include all known downstream signal transduction, regulation and termination events. This model is the �rst of its kind to describe a whole GPCR signalling pathway within S. pombe. Although it accurately predicts the cellular response to GPCR signalling it could only reproduce the biological plateau in temporal response with the addition of a 'yet unknown mechanism' GPCR degradation term. This motivated the investigation of how GPCRs in S. pombe are internalised from the plasma membrane in response to ligand stimulation. The primary mechanism for signal termination is via internalisation of the GPCR. This study identi�ed three potential casein kinases (Cki1, Cki2 and Cki3) that promote internalisation of the S. pombe GPCR Mam2. Microscopy analyses in combination with quantitative transcriptional, cell growth and cell cycle position assays uncovered a novel role for these kinases: that Cki2 regulates cell size during vegetative growth, Cki1 and Cki3 regulate the GPCR-response pathway and that Cki3 is essential for completing cytokinesis in S. pombe that have already undergone formation of a conjugation tube in response to ligand. Confocal microscopy of uorescent labelled Mam2 indicated a role for Cki2 in the internalisation and hence termination of the GPCR-response pathway. These findings add to the growing body of evidence that casein kinases are implicated in GPCR desensitisation.

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8

Goddard, Alan David. "Functional analysis of GPCR signalling cascades in Schizosaccharomyces pombe". Thesis, University of Warwick, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.437696.

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9

Koyama, Hiroyuki. "Comprehensive Profiling of GPCR Expression in Ghrelin-producing Cells". Kyoto University, 2016. http://hdl.handle.net/2433/215953.

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10

Kiess, Alexandra. "Funktionelle Relevanz intrazellulärer Splicevarianten des Brain-specific Angiogenesis Inhibitor 2 (BAI2)". Doctoral thesis, Universitätsbibliothek Leipzig, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-156171.

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BAI2 gehört zu den Adhesion-G-Protein-gekoppelten Rezeptoren (aGPCR). Diese bisher wenig untersuchte Klasse von ca. 30 GPCR ist charakterisiert durch eine komplexe genomische Struktur, sehr große extrazelluläre Domänen und eine Vielzahl von Splicevarianten. Bisher ist bei den meisten aGPCR, wie auch bei BAI2, wenig über ihre Signaltransduktion und Funktion bekannt. Zum Verständnis der physiologischen Relevanz und zur Suche nach dem endogenen Agonist sind Kenntnisse über Proteinstruktur, Splicevarianten und Signaltransduktion essentiell. Ziel dieser Arbeit war es, mittels verschiedener in vitro-Methoden die Proteinstruktur des BAI2 in den transmembranären und intrazellulären Domänen näher zu untersuchen, sowie die natürlichen Splicevarianten in diesem Bereich, deren evolutionäre Konservierung, Gewebespezifität und Quantität zu erfassen. Für beide gefundenen Splicevarianten, eine im dritten intrazellulären Loop (ICL3) und eine im C-Terminus, konnte eine evolutionäre Konservierung auf Aminosäure- und genomischer Organisationsebene, sowie ihre Entstehung durch Exonskipping nachgewiesen werden. Nachfolgend wurden die Splicevarianten auf mögliche Interaktionen mit intrazellulären Komponenten untersucht. In dieser Arbeit konnte gezeigt werden, dass beide ICL3-Splicevarianten natürlicherweise in einem definierten Verhältnis auftreten. Außerdem konnte gezeigt werden, dass die lange ICL3-Variante des BAI2 nicht zu einer Änderung der Membrantopologie des Rezeptors, einer Homodimerisierung über die zusätzliche Aminosäuresequenz oder zu einer Interaktion mit dem C-Terminus führt. Die Splicevariante im humanen C-Terminus des BAI2 konnte als eine variable, durch Exonskipping entstandene Calcium-unabhängige Calmodulin-Bindungsstelle identifiziert werden. Diese Arbeit belegt die Existenz mehrerer BAI2-Isoformen in vivo. Die Struktur dieser Isoformen lässt unterschiedliche Funktionalitäten vermuten. Auch wenn erste Untersuchungen zwischen den beiden ICL3-Varianten keinen Unterschied ergaben, sind diese Erkenntnisse für die weitere Analyse der Signaltransduktion und Ligandensuche bedeutend. Es ist z.B. denkbar, dass sich die beiden ICL3-Varianten in der G-Protein-Kopplung oder bei der Rekrutierung von intrazellulären Interaktionspartnern unterscheiden oder dass die Splicevariante im C-Terminus zu einer Scaffold- Funktion des Calmodulins führt und/oder die Signaltransduktion durch eine permanente Bindung des Calmodulins an einer Isoform moduliert wird.

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11

Guerrero, Hernández Martina. "Targeting tumor microenvironment crosstalk through GPCR receptors and PI3K pathway". Doctoral thesis, Universitat de Barcelona, 2019. http://hdl.handle.net/10803/667975.

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The tumor microenvironment (TME) is gaining momentum due to its contribution to cancer progression and therapy resistance. This TME has a direct crosstalk with tumor cells that involves the activation of different pathways. Follicular lymphoma (FL) is the most common indolent non-Hodgkin lymphoma. Although FL is generally characterized by slow progression and high response rates to therapy, it is still considered incurable, because almost virtually all the patients relapse. FL is probably the NHL with the highest dependence on microenvironment. PI3K is a common denominator transducing the signaling from FL crosstalk with the TME and plays an important role in multiple cellular functions, and also contributes to cancer promoting aspects of the TME, such as angiogenesis and inflammatory cell recruitment. Idelalisib is a first-in-class δ isoform- specific PI3K inhibitor that receive regulatory approval for relapsed CLL, SLL and FL in 2014. Idelalisib blocks PI3K δ which is restricted to leukocytes. BCL2 deregulation is paramount in the pathogenesis of FL, as a consequence of the t(14;18), and therefore it is an attractive target for novel therapeutic approaches. Venetoclax (ABT-199, AbbVie) is a small BCL-2 inhibitors. Even though 85% of FL patients harbor the t(14;18), the results of the first clinical trial with venetoclax were not satisfactory (overall response 38%). From this first study we conclude that Idelalisib modulates key pathways in the germinal center and shapes the FL immune microenvironment by decreasing the recruitment of TFH and Treg to the tumor site leading to less immunesuppresive phenotype. Furthermore Idelalisib induces a moderate cytotoxic effect on FL cells in co-cultures. This co-culture decrease FL dependence on BCL-2 and consequently, venetoclax cytotoxicity, but Idelalisib sensitizes FL co-cultures to venetoclax. In summary, Idelalisib interferes with the crosstalk of FL and its immune microenvironment and potentiates the activity of venetoclax targeting the tumor cells, thus representing a promising combination therapy that may improve FL outcome. Colorectal cancer (CRC) is the third most common cancer in males and the second in females, and the fourth most common cause of cancer-related death worldwide. Patients with advanced and distal metastatic disease (stage IV), the survival rate drops to 10%, which accounts for approximately 18% of cases. The TME in CRC, is a complex structure composed by different type of cells, which are interacting each other’s and secreting a variety of growth factors and other molecules, such as cytokines and chemokines. Tumor development is based on the crosstalk between tumor cells and their surrounding microenvironment, and this crosstalk is mediated by the receptors and its ligand expression in both types of cells. G protein-coupled receptors (GPCRs) are an important family of membrane signaling receptors, which have an important role in cancer growth and development. Originally, GPCRs were considered as monomeric functional entities, nevertheless, in recent years has become evident that GPCRs form dimers and this dimers formation may modify the cellular response. In cancer, CXCR4 (has been studied extensively) plays an important role at different stages of cancer development, and is involved in the metastasis process of tumor cells. The up- regulation of CXCR4 in CRC correlates with a poor prognosis. Another GPCR, CB2 receptor modulates the downstream signaling and it is able to activate a wide range of signaling pathways, including extracellular signal-regulated kinases 1/2 (ERK1/2). In CRC, it has been described an up-regulation of CB2 receptor expression. GPCRs show differential expression in cancer cells and tissues, and they are highly druggable sites. From this second study we concluded that CXCR4 and CB2 expression is increased in primary colon tumor cells and in metastasis cells compared to normal epithelial cells from colon mucosa, and they formed heterodimers in colon tumoral cells and are associated with more aggressive phenotypes. Moreover, a bidirectional cross-antagonism crosstalk is established between these receptors. These heterodimers regulate in vitro CXCL12-induced migration, and in vivo, the simultaneous inhibition of both receptors shows superior anti-tumoral and anti-metastatic activities than the single agent inhibition. In summary, targeting the heterodimerization of CXCR4 and CB2 that are biologically relevant in cancer can be an effective way to reduce proliferation and dissemination in CRC.
El estudio del microambiente tumoral está ganando importancia en las últimas décadas debido a su contribución en la formación y desarrollo del cáncer, además de contribuir en la resistencia de las células tumorales a diferentes terapias. Este microambiente interactúa con las células tumorales y activa diferentes vías. El linfoma folicular (FL), es el linfoma no Hodgkin indolente más común y con mayor dependencia del microambiente tumoral, además es considerado incurable. PI3K desempeña un papel importante en la comunicación con el microambiente, y es importante en múltiples funciones celulares, además de contribuir en la angiogénesis, reclutamiento de células inflamatorias y promover el crecimiento tumoral. Idelalisib es un inhibidor de PI3K (específicamente de la isoforma δ), que se aprobó en 2014 por la FDA. Paralelamente la desregulación de BCL2 es primordial en la patogénesis de FL, como consecuencia de la t (14; 18), presente en un 85% de los pacientes, y por lo tanto es un objetivo atractivo para novedosos enfoques terapéuticos. Venetoclax (ABT-199, AbbVie) es un pequeño inhibidor de BCL2, que mostró unos resultados del primer ensayo clínico no satisfactorios (respuesta global del 38%). De este primer estudio concluimos que Idelalisib interfiere en la comunicación de FL y su microambiente inmune, además potencia la actividad de venetoclax atacando a las células tumorales, lo que representa una terapia de combinación prometedora que puede mejorar el resultado del tratamiento de FL.

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12

Hollier, Mark John. "Properties of the C-terminal tail of HIV-1 gp41". Thesis, University of Warwick, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.408463.

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Gardner, Jacob Andrew. "GPCR Signaling in the Genesis and Progression of Pancreatic Cancer". Diss., Temple University Libraries, 2009. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/35453.

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Molecular Biology and Genetics
Ph.D.
Ductal adenocarcinomas of the pancreas are the 4th most common cause of cancer death. The 1 and 5 year survival rates for all stages combined are currently 26% and 5% respectively. Median survival is less than 6 months. Despite remarkable progress in the fields of genetics, cancer biology, and advances in surgical techniques as well as chemotherapeutics, our ability to recognize and treat patients with pancreatic cancer remains poor. GPCR signaling modules have been increasingly implicated in the genesis and progression of pancreatic cancers. Aberrant agonist production, receptor expression and dysfunctional signaling resulting from genomic instability in a background of a heterotopic tumor-stromal microenvironment, contribute to the initiation, progression, and eventual metastasis of the disease. Numerous GPCR agonists, including lysophosphatidic acid (LPA), along with their cognate receptors have been implicated in this oncogenic process. LPA, one of the simplest bioactive lipids, has been shown to be a potent stimulant of metastatic behavior in in vitro models. It also acts as a mitogen by inducing proliferation and cell survival pathways in various normal and transformed cell lines. In patients with pancreatic cancer both the receptors and ligand have been found to be overexpressed. It has been noted that pancreatic cancer cell lines expressing higher levels of the LPA receptors present with greater motility. This has led to the hypothesis that LPA contributes to the progression of pancreatic cancer through the promotion of a metastatic phenotype. However, the underlying mechanisms have not been well described. LPA receptors have been shown to couple to the Gi, Gq, or G12 family of heterotrimeric G proteins. Consequently, signals transduced through these receptors have been shown to stimulate Gαi, Gαq, and Gα12/13 dependent pathways. While earlier studies have linked Gαi to LPA induced migration, there is recent evidence to suggest that Gα13 may provide a major signaling mechanism for LPA receptors stimulating migration in diverse cell types including cancer cell lines. Given the ominous nature of pancreatic cancers it is of critical importance to understand the mechanisms that promote more malignant phenotypes and to assess the role of Gα13 in this process. The goal of this thesis therefore is to define the role of Gα13 in LPA-mediated migration of pancreatic cancer cells. To assess the oncogenic potential of LPA and the role of Gα13 in stimulating the migration of pancreatic cancer cells, a panel of pancreatic cancer cell lines was assembled and characterized with regard to their expression of the LPA receptors as well as the Gα subunits of the heterotrimeric G proteins. These cell lines were further studied through a series of proliferation, wound healing, and transwell migration assays to assess the role of LPA in the induction of proliferation and migration in pancreatic cancer cells. The results demonstrated that LPA functions as a mitogen in certain pancreatic cancer cell lines, but is a potent stimulant of cell motility and invasive migration. Interestingly, these studies indicated that this response proceeds through routes that may not involve Gαi, as a potent migratory response was observed in MDAPanc28 cells which lack expression of the Gαi subunit. This was verified through the transwell assays conducted in the presence of PTX demonstrating that migration occurs independently of PTX sensitive mechanism and thus independently of Gαi.. Using a dominant negative mutant strategy, the studies presented in this thesis establishes the role of Gα13 in mediating LPA-LPAR stimulated migration of pancreatic cancer cells. Using pancreatic cancer cell lines that stably express the competitively inhibitory dominant negative mutant of Gα13, the ability of these mutants to inhibit a LPA mediated migratory response was monitored by wound-healing as well as transwell migration assays The results of these studies indicated a substantial attenuation of the migratory response and demonstrated for the first time the critical role of Gα13in LPA induced migration in a pancreatic cancer cell line.
Temple University--Theses

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14

Senarath, Kanishka D. "Interrogation of GPCR-G Protein Signaling using Novel Optogenetic Tools". University of Toledo / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=toledo155681039815937.

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15

Behrendt, Rayk. "Immunogene und immunsuppressive Eigenschaften des transmembranen Hüllproteins gp41 von HIV". Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2009. http://dx.doi.org/10.18452/15993.

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Die Entwicklung eines effektiven HIV-Impfstoffes ist bis heute nicht gelungen.Konventionelle Immunisierungsstrategien mit rekombinant hergestellten Hüllproteinen des Virus in verschiedensten Formen induzierten keine subtypenübergreifende, protektive Immunantwort gegen HIV. Die Gewinnung und Charakterisierung der gp41-spezifischen breit neutralisierenden monoklonalen Antikörper 2F5 und 4E10 bildete die Grundlage einer Reihe neuer epitopgerichteter Ansätze für die HIV-Impfstoffentwicklung. Bisherige Immunisierungsstudien basierten auf der Verwendung des linearen Hauptepitopes (E2) der beiden Antikörper aus dem C-terminalen Teil der Ektodomäne von gp41. Nach neueren Erkenntnissen, reicht für eine effektive Neutralisation durch 2F5 oder 4E10 die Bindung dieser Antikörper an ihr lineares Epitop in der membran proximalen externen Region (MPER) von gp41 allein nicht aus. Vielmehr wurde die Beteiligung einer N-terminalen Domäne (E1) von gp41 an der neutralisationsaktiven Bindung von 2F5 bzw. 4E10 postuliert. In dieser Arbeit wurden die beiden 2F5 und 4E10 spezifischen Epitopbereiche E1 und E2 des gp41 erstmals in das strukturell verwandte transmembrane Hüllprotein des Koala Retrovirus (KoRV) eingebracht. Die Applikation der hergestellten Antigene erfolgte sowohl in Form der codierenden DNA mittels ballistischer Immunisierung (GeneGun®) als auch durch bakteriell exprimierte Proteine. Mit beiden Strategien konnten für drei Hybridproteine in den ersten Studien eine HIV-1 gp41 spezifische, breit neutralisierende humorale Immunantwort induziert werden. Diese Ergebnisse konnten jedoch in späteren Studien nicht reproduziert werden. Die Analyse der induzierten Immunantworten zeigte eine Verlagerung der Hauptimmunantwort als deren Ursache eine bakterielle Fremdinfektion der Versuchtiere diskutiert wurde. Zur Evaluierung der Immunisierungsstudien wurde ein neuartiger real time PCR basierter in vitro Neutralisationstest um Kontrollen zur Virusspezifität und Cytotoxizität erweitert.
The development of an effective HIV vaccine is considered the to play a key role in controlling the HIV pandemic. Conventional immunisation strategies using recombinant envelope proteins of the virus did not lead to the induction of a broad range protective immunity. A new target sequence for the induction of a broadly neutralising humoral immune response has been discovered through the characterization of the gp41 specific broadly neutralising monoclonal antibodies 2F5 and 4E10. Until now all attempts to induce 2F5/4E10 like neutralising antibodies failed. So far only the linear main epitope (E2) of 2F5 and 4E10, located in the C-terminal part of the gp41 ectodomain was used as the target sequence. However, it was recently shown that an N-terminal domain (E1) of gp41 increases the avidity of 2F5 to its epitope. The E1 domain may therefore be involved in the mediation of a neutralisation active binding. For the first time immunisation strategies have been developed that target both previously identified domains (E1 and E2) of gp41. The sequences corresponding to E1 and E2 have been introduced at homologous positions in the structurally related transmembrane envelope protein p15E of the Koala Retrovirus (KoRV). These generated hybrid antigens have been used for immunisation of wistar rats. They were applied as recombinant proteins expressed in E.coli and as DNA using a ballistic immunisation (GeneGun®) approach. Although in first trials neutralising antibodies specific for gp41 of HIV-1 were induced, these results could not be reproduced. Analysis of the induced antibodies showed a shift of their binding specifity. A bacterial infection of the used animals was identified as the cause of the unexpected shift in the antigen specific humoral immune response. For evaluation of the immunisation studies a new neutralisation assay based on the measurement of provirus integration by duplex real time PCR has been extended for controls of virus specifity and cytotoxicity.

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BAUDET, SYLVIE. "Etude cristallographique du complexe barnase-d(gpc)". Paris 11, 1990. http://www.theses.fr/1990PA112016.

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La structure cristalline du complexe entre la ribonuclease externe de b. Amyloliquefaciens, la barnase, et un inhibiteur nucleotide, d(gpc) a ete resolue a 0,22 nm de resolution. La proteine a ete obtenue a partir du gene clone dans e. Coli. Apres purification et cocristallisation en sulfate d'ammonium 3;0 m avec le d(gpc), des cristaux ont ete obtenus dans le groupe d'espace p3#121, avec des parametres de maille a=b=0,58 nm et c=0,85 nm. Les donnees de diffraction ont ete enregistrees sur film a lure, orsay, et sur un detecteur bidimensionnel fast a l'embl, heidelberg. La structure de la barnase resolue en presence de zinc par mauguen et al. En 1982 (nature, 297, pp. 162-164) a ete utilisee comme modele pour resoudre le probleme des phases par la methode du remplacement moleculaire. Les fonctions de rotation et de translation ont donne la position de la molecule dans l'unite asymetrique du cristal de complexe. Apres affinement de cette position avec le programme d'affinement en blocs rigides, corels, les coordonnees atomiques ont ete affinees par dynamique moleculaire avec le programme gromos. Le facteur r a atteint 26% a 0,ee nm de resolution et l'analyse de la carte de densite resultante a permis de modeliser deux conformations alternatives du nucleotide. Ces deux positions du d(gpc) ne sont pas au site de reconnaissance de la barnase mais certainement dans un site de fixation secondaire. Par consequent, les interactions proteine-inhibiteur observees ne sont pas productives

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17

Adamson, Roslin Jane. "Probing GPCR-Gα interactions : a functional study by EM and SPR". Thesis, University of Oxford, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.669745.

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18

Mason, Vicki Louise. "Effects of different assay configurations on pharmacological profiling of GPCR targets". Thesis, University of Reading, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.494820.

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The effect of assay type and assay configuration on the pharmacological profile of a range of ligands acting at the D2 dopamine receptor was investigated in this thesis. The ligands explored have previously been shown to display a spectrum of efficacy at the D2 dopamine receptor. In competition ligand binding studies versus [³H]spiperone in HEK-293CREβlacD₂ˡ cell membranes, receptor-G protein coupling was increased in the absence of Na⁺, however the pK/ values for some agonists were higher in the presence of Na⁺, which suggested that different ligands are able to induce different receptor conformations.

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19

Valsechi, Marina Curado [UNESP]. "Análise funcional do gene GPC3 em carcinoma renal de células claras". Universidade Estadual Paulista (UNESP), 2013. http://hdl.handle.net/11449/127546.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
GPC3 (Glipican-3) é membro de uma família de proteoglicano de heparina sulfatada (HSPG). O GPC3 pode atuar controlando a migração celular, regulação negativa do crescimento celular e indução de apoptose. Esse gene é relatado por estar hipoexpresso em vários tipos de cânceres, o que pode resultar no crescimento celular descontrolado e contribuir para o fenótipo maligno de alguns tumores. O objetivo desse estudo foi analisar o mecanismo de ação do gene GPC3 em carcinoma renal de células claras (CCRCC). Primeiramente, foi construído o vetor de expressão e transfectado nas linhagens celulares de carcinoma renal ACHN e 786-O. A expressão de GPC3 foi analisada usando qRT-PCR e imunohistoquímica. A proliferação celular foi avaliada usando o MTT e o ensaio de formação de colônia. Análises de apoptose e ciclo celular foram avaliadas por citometria de fluxo. Foi observado que o gene GPC3 estava com baixa expressão em amostras de carcinoma renal de células claras e nas linhagens celulares quando comparado com amostras renais normais. Foi observado que a expressão de RNAm e os níveis de proteína GPC3 aumentaram após a transfecção com o vetor de expressão contendo a ORF de GPC3 nas linhagens celulares. A taxa de proliferação celular diminuiu nas células superexpressando GPC3 em ambas as linhagens, ACHN e 786-O (p<0,01). A apoptose não foi observada nas linhagens celulares de carcinoma renal superexpressando GPC3 (p > 0,05); entretanto, ocorreu um aumento na população de células na fase G1 do ciclo celular (p< 0,05). Esses resultados sugerem que o gene GPC3 reduz a taxa de proliferação celular por meio da parada do ciclo celular na fase G1 em carcinoma renal
Background: GPC3 (Glypican 3) is a member of the family of glypican heparan sulfate proteoglycans (HSPG). The GPC3 gene may play a role in controlling cell migration, negative regulation of cell growth and induction of apoptosis. This gene is reported to be downregulated in several cancers, which can result in uncontrolled cell growth and which can also contribute to the malignant phenotype of some tumors. The purpose of this study was to analyze the mechanism of action of the GPC3 gene in clear cell renal cell carcinoma (CCRCC). Methods: First, we constructed the expression vector and transfected renal carcinoma cell lines. GPC3 expression was analyzed using qRT-PCR and immunohistochemistry. We evaluated cell proliferation using MTT and colony formation assays. Apoptosis and cell cycle analyses were evaluated using flow cytometry. Results: We observed that the GPC3 gene was downexpressed in the clear cell renal cell carcinoma samples and in cell lines, which were both compared to normal renal samples. We observed that GPC3 mRNA expression and protein levels increased after the transfection into ACHN and 786-O cell lines. We found that the cell proliferation rate decreased in cells overexpressing GPC3 in both cell lines, ACHN and 786-O (p < 0.01). Also, apoptosis in the renal cell carcinoma cell line was not observed in cells overexpressing GPC3 (p > 0.05), but there was an increase in the cell population during the G1 phase in the cell cycle (p< 0.05). Conclusion: We suggest that the GPC3 gene reduced the cell proliferation rate through cell cycle arrest during the G1 phase in renal cell carcinoma

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20

Valsechi, Marina Curado. "Análise funcional do gene GPC3 em carcinoma renal de células claras /". São José do Rio Preto, 2013. http://hdl.handle.net/11449/127546.

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Orientador: Paula Rahal
Banca: Ana Carolina Gomes Jardim
Banca: Wilson Araújo da Silva Júnior
Banca: Ana Elizabete Silva
Banca: Sônia Maria Oliani
Resumo: GPC3 (Glipican-3) é membro de uma família de proteoglicano de heparina sulfatada (HSPG). O GPC3 pode atuar controlando a migração celular, regulação negativa do crescimento celular e indução de apoptose. Esse gene é relatado por estar hipoexpresso em vários tipos de cânceres, o que pode resultar no crescimento celular descontrolado e contribuir para o fenótipo maligno de alguns tumores. O objetivo desse estudo foi analisar o mecanismo de ação do gene GPC3 em carcinoma renal de células claras (CCRCC). Primeiramente, foi construído o vetor de expressão e transfectado nas linhagens celulares de carcinoma renal ACHN e 786-O. A expressão de GPC3 foi analisada usando qRT-PCR e imunohistoquímica. A proliferação celular foi avaliada usando o MTT e o ensaio de formação de colônia. Análises de apoptose e ciclo celular foram avaliadas por citometria de fluxo. Foi observado que o gene GPC3 estava com baixa expressão em amostras de carcinoma renal de células claras e nas linhagens celulares quando comparado com amostras renais normais. Foi observado que a expressão de RNAm e os níveis de proteína GPC3 aumentaram após a transfecção com o vetor de expressão contendo a ORF de GPC3 nas linhagens celulares. A taxa de proliferação celular diminuiu nas células superexpressando GPC3 em ambas as linhagens, ACHN e 786-O (p<0,01). A apoptose não foi observada nas linhagens celulares de carcinoma renal superexpressando GPC3 (p > 0,05); entretanto, ocorreu um aumento na população de células na fase G1 do ciclo celular (p< 0,05). Esses resultados sugerem que o gene GPC3 reduz a taxa de proliferação celular por meio da parada do ciclo celular na fase G1 em carcinoma renal
Abstract: Background: GPC3 (Glypican 3) is a member of the family of glypican heparan sulfate proteoglycans (HSPG). The GPC3 gene may play a role in controlling cell migration, negative regulation of cell growth and induction of apoptosis. This gene is reported to be downregulated in several cancers, which can result in uncontrolled cell growth and which can also contribute to the malignant phenotype of some tumors. The purpose of this study was to analyze the mechanism of action of the GPC3 gene in clear cell renal cell carcinoma (CCRCC). Methods: First, we constructed the expression vector and transfected renal carcinoma cell lines. GPC3 expression was analyzed using qRT-PCR and immunohistochemistry. We evaluated cell proliferation using MTT and colony formation assays. Apoptosis and cell cycle analyses were evaluated using flow cytometry. Results: We observed that the GPC3 gene was downexpressed in the clear cell renal cell carcinoma samples and in cell lines, which were both compared to normal renal samples. We observed that GPC3 mRNA expression and protein levels increased after the transfection into ACHN and 786-O cell lines. We found that the cell proliferation rate decreased in cells overexpressing GPC3 in both cell lines, ACHN and 786-O (p < 0.01). Also, apoptosis in the renal cell carcinoma cell line was not observed in cells overexpressing GPC3 (p > 0.05), but there was an increase in the cell population during the G1 phase in the cell cycle (p< 0.05). Conclusion: We suggest that the GPC3 gene reduced the cell proliferation rate through cell cycle arrest during the G1 phase in renal cell carcinoma
Doutor

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21

Watson, Douglas Stuart. "Lipopeptide immunogens targeting the membrane proximal region of HIV-1 gp41". Diss., Search in ProQuest Dissertations & Theses. UC Only, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3359565.

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Thesis (Ph. D.)--University of California, San Francisco with the University of California, Berkeley, 2009.
Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3649. Adviser: Francis C. Szoka.

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Schafranski, Luiz Erley. "O protótipo GPCP-1 :: jogo do planejamento e controle da produção /". Florianópolis, SC, 1998. http://repositorio.ufsc.br/xmlui/handle/123456789/77907.

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Dissertação (Mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico.
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Saunders, Charles William. "Characterization of cellulose esters via GPC/FT-IR". Diss., This resource online, 1990. http://scholar.lib.vt.edu/theses/available/etd-07282008-135448/.

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PIONTKUSKY, FILHO A. J. "Proposta de GPC Auto-Tunning Usando Algoritmo Genético". Universidade Federal do Espírito Santo, 2018. http://repositorio.ufes.br/handle/10/9564.

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Engemaier, Eva. "Die strukturelle und funktionelle Evolution des G-Protein-gekoppelten Rezeptors GPR34". Doctoral thesis, Universitätsbibliothek Leipzig, 2012. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-86270.

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Gegenstand der Arbeit ist eine umfassende Charakterisierung von Struktur und Funktion des G-Protein-gekoppelten Rezeptors GPR34 auf genomischer, mRNA und Proteinebene, die mögliche Rückschlüsse auf physiologische Funktionen oder den natürlichen Agonisten zulassen. Dazu wurde die genomische Organisation des GPR34 in Mensch, Maus und Ratte analysiert und festgestellt, dass neben der intronlosen kodierenden Sequenz auch der 5´-Bereich des GPR34 mit seiner nicht-kodierenden Intron-Exon-Struktur stark konserviert ist. Es wurden in der Ratte und der Maus mindestens zwei, beim Menschen ein putativer Transkriptionsstart identifiziert. Beim Menschen konnte ein kryptisches Intron innerhalb der kodierenden Sequenz des GPR34 gefunden werden, was zu einer Verkürzung des N-Terminus um 47 Aminosäuren führt. Auf der Transkriptionsebene wurde der GPR34 und der GPR34-like Rezeptor im Haushuhn (Gallus gallus) und die GPR34-Expression in der Ratte mittels quantitativer RT-PCR analysiert und die ubiquitäre Gewebeverteilung des Rezeptors bestätigt. Beim menschlichen GPR34 konnte festgestellt werden, dass die fünf putativen Translationsstarts innerhalb der kodierenden Sequenz auch als solche funktionstüchtig sind und der zweite Translationsstart bevorzugt genutzt wird. Die genetische Variabilität des GPR34 in der menschlichen Population ist sehr gering. Es konnte innerhalb einer weltweiten DNA-Probensammlung nur ein einziges Mal eine Mutation in der kodierenden Sequenz lokalisiert werden. Mithilfe des während dieser Arbeit entstandenen Mausmodelles ist eine weitere Charakterisierung der physiologischen Relevanz des GPR34 möglich.

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26

Thamm, Markus. "Charakterisierung der Serotonin-Rezeptoren der Honigbiene Apis mellifera : von den Genen zum Verhalten". Phd thesis, Universität Potsdam, 2009. http://opus.kobv.de/ubp/volltexte/2010/4073/.

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Das serotonerge System besitzt sowohl bei Invertebraten als auch bei Vertebraten eine große Bedeutung für die Kontrolle und Modulation vieler physiologischer Prozesse und Verhaltensleistungen. Bei der Honigbiene Apis mellifera spielt Serotonin (5-Hydroxytryptamin, 5-HT) eine wichtige Rolle bei der Arbeitsteilung und dem Lernen. Die 5-HT-Rezeptoren, die überwiegend zur Familie der G-Protein gekoppelten Rezeptoren (GPCRs) gehören, besitzen eine Schlüsselstellung für das Verständnis der molekularen Mechanismen der serotonergen Signalweiterleitung. Ziel dieser Arbeit war es, 5-HT-Rezeptoren der Honigbiene zu charakterisieren. Dazu zählt die Identifizierung der molekularen Struktur, die Ermittlung der intrazellulären Signalwege, die Erstellung von pharmakologischen Profilen, die Ermittlung der Expressionsmuster und die Ermittlung der physiologischen Funktionen der Rezeptoren. Mit Hilfe der Informationen aus dem Honey Bee Genome Project, konnten drei RezeptorcDNAs kloniert werden. Vergleiche der abgeleiteten Aminosäuresequenzen mit den Aminosäuresequenzen bereits charakterisierter Rezeptoren legten nahe, dass es sich dabei um einen 5-HT1- (Am5-HT1) und zwei 5-HT2-Rezeptoren (Am5-HT2α und Am5-HT2β) handelt. Die strukturelle Analyse der abgeleiteten Aminosäuresequenz dieser Rezeptoren postuliert das Vorhandensein der charakteristischen heptahelikalen Architektur von GPCRs und zeigt starkkonservierte Motive, die bedeutend für die Ligandenbindung, die Rezeptoraktivierung und die Kopplung an G-Proteine sind. Für die beiden 5 HT2-Rezeptoren konnte zudem alternatives Spleißen nachgewiesen werden. Mit den cDNAs des Am5-HT1- und des Am5-HT2α-Rezeptors wurden HEK293-Zellen stabil transfiziert und anschließend die Rezeptoren funktionell und pharmakologisch analysiert. Am5-HT1 hemmt bei Aktivierung abhängig von der 5-HT-Konzentration die cAMPProduktion.Die Substanzen 5-Methoxytryptamin (5-MT) und 5-Carboxamidotryptamin konnten als Agonisten identifiziert werden. Methiothepin dagegen blockiert die 5-HTWirkung vollständig. Prazosin und WAY100635 stellen partielle Antagonisten des Am5-HT1-Rezeptors dar. Der Am5-HT2_-Rezeptor stimuliert bei Aktivierung die Synthese des sekundären Botenstoffs Inositoltrisphosphat, was wiederum zu einer messbaren Erhöhung der intrazellulären Ca2+-Konzentration führt. 5-MT und 8-OH-DPAT zeigen eine deutliche agonistische Wirkung auf Am5-HT2α. Dagegen besitzen Clozapin, Methiothepin, Mianserin und Cyproheptadin die Fähigkeit, die 5-HT-Wirkung um 51-64 % zu vermindern. Die bereits erwähnte alternative Spleißvariante von Am5-HT2α wurde ebenfalls in HEK293-Zellen exprimiert und analysiert, scheint jedoch eigenständig nicht funktionell zu sein. Gegen die dritte cytoplasmatische Schleife (CPL3) wurde ein polyklonales Antiserum generiert. Dieses erkennt in Western-Blot-Analysen ein Protein mit einer Masse von ca. 50 kDa. Durch immunhistochemische Analysen am Bienengehirn wurde die Verteilung des Rezeptors genauer untersucht. Dabei zeigten die optischen Neuropile, besonders die Lamina und die Ocellarnerven, stets eine starke Markierung. Außerdem wird der Rezeptor in den α- und β-Loben sowie der Lippe, dem Basalring und dem Pedunculus der Pilzkörper exprimiert. Doppelmarkierungen zeigen stets eine enge Nachbarschaft von serotonergen Fasern und dem Am5-HT1-Rezeptor. Weiterhin konnte gezeigt werden, dass der Am5-HT1-Rezeptor sehr wahrscheinlich an der Regulation des phototaktischen Verhalten der Honigbiene beteiligt ist. Verfütterung von 5-HT hat eine deutlich negative Wirkung auf das phototaktischen Verhalten. Diese kann durch den Am5-HT1-Rezeptor-Agonisten 5-CT imitiert werden. Schließlich konnte gezeigt werden, dass der Am5-HT1-Antagonist Prazosin die 5-HT-Wirkung deutlich vermindern kann.
The serotonergic system plays an important role in the control and modulation of many physiological and behavioral processes in both vertebrates and invertebrates. In the honeybee Apis mellifera, serotonin (5-hydroxytryptamine, 5-HT) has been implicated in the control and regulation of division of labor as well as learning and memory. A key role in understanding the serotonergic system plays the molecular and functional characterization of 5-HT receptor subtypes. In most cases, serotonin receptors represent G protein-coupled receptors (GPCRs). This work describes the characterization of honeybee serotonin receptors. This comprises the identification of their molecular structure, intracellular second messenger pathways, pharmacological properties, expression profiles and functions. By screening the honeybee genome, we found three candidate genes encoding for putative serotonin receptors. The cDNAs of these genes were cloned and the deduced amino acid sequences were analysed. The sequence information was used to isolate the cDNAs encoding for these three receptors. Comparison of the deduced amino acid sequences with sequences of other known receptors suggests that one receptor belongs to the 5-HT1 (Am5-HT1) and the other two receptors to the 5-HT2 receptor class (Am5-HT2α and Am5-HT2β). Major characteristics common to all GPCRs (e.g. the heptahelical architecture) were confirmed by structural analyses of the deduced amino acid sequences. Furthermore, truncated receptor transcripts representing alternative splice variants of both 5-HT2 receptors could be detected. HEK293 cells were stably transfected with the cDNAs of Am5-HT1 or Am5-HT2_ and functionally and pharmacologically analysed. The activation of Am5-HT1 by 5-HT results in the dose dependent attenuation of adenylyl cyclase activity. 5-methoxytryptamine (5-MT) and 5-carboxamidotryptamine are able to imitate the 5-HT effect. In contrast, methiothepin is able to block the entire 5-HT effect, whereas prazosine and WAY100635 block the 5-HT effect only partially. The Am5-HT2α receptor stimulates the synthesis of the second messenger inositol trisphosphate which in turn mediates an increase in the intracellular Ca2+. The substances 5-MT and 8-OH-DPAT were identified as agonists of the Am5-HT2α receptor. In contrast, clozapine, methiothepine, mianserine, and cyproheptadine show strong antagonistic actions. A truncated alternative splice variant of the Am5-HT2α-receptor was also analysed but didn’t show any functional coupling by itself. An antiserum was raised against the third cytoplasmic loop (CPL3) of the Am5-HT1 receptor. This antiserum detects a protein with a molecular mass of 50 kDa in western blot analyses. The expression of the Am5-HT1 receptor was studied in detail using immunohistochemistry. Strong Am5-HT1-like immunofluorescence was observed in the ocellar nerve, in the three optic ganglia and in the α- and β-lobes, the pedunculi, the lip and the basal ring of the mushroom bodies. Furthermore, co-labeling with an antibody against 5-HT showed that this receptor is expressed in close vicinity to serotonergic neurons. Finally, behavioral experiments suggest a possible role of the Am5-HT1 receptor in phototactic behavior. Feeding of 5-HT to worker honeybees results in a decrease of phototactic behavior. This 5-HT action could be mimiced by feeding of the Am5-HT1 agonist 5-CT. In contrast, the Am5-HT1 antagonist prazosine prevents the 5-HT-induced decrease in phototaxis.

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Zhang, Boyang. "Functional and Structural Insights into the First and Second Intracellular Domains for D1-Class Dopaminergic Receptors". Thesis, Université d'Ottawa / University of Ottawa, 2017. http://hdl.handle.net/10393/35932.

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Previous studies have shown that the subtype-specific pharmacological properties of D1-class receptors (D1R and D5R) can be attributed to their third intracellular domain and C-terminal tail. However, the importance of their first and second intracellular domains (IC1 and IC2) has yet to be explored. Using mutagenesis and bioinformatics, we examine the functional and structural roles of Ser/Thr spanning IC1 and IC2—most of which are conserved not only among D1-class receptors but also among other GPCRs. Mutant receptors of human D1-class receptors (hD1R and hD5R) were constructed whereby all Ser and Thr were mutated to the respective Ala and Val in the IC1 region (termed ST1 mutant receptors) and in the IC2 region (termed ST2 mutant receptors). We found that hD1-ST2 and hD5-ST2 exhibited contrasting properties of agonist affinity, constitutive activity, and dopamine potency. On the other hand, both ST2 mutants underwent internalization as wild-type but displayed weakened desensitization abilities. Homology models, which have been refined under membrane simulations, illustrate that the conserved Ser3.55 and Thr3.65 utilize their side chains to anchor the loop regions of IC2 to cytoplasmic helices. We also found multiple functional alterations in the hD1-ST1 and hD5-ST2, but in a subtype-similar manner. Mutating the conserved Thr2.39 recapitulated the ablated basal activity and drastic decrease in dopamine potency previously witnessed in the hD1-ST1. Based on the recurring theme observed in crystal structures, the side-chain of Thr2.39 may help to position IC2 to have proper contacts with the G protein. Mutating the conserved Ser2.45 was found to be solely responsible for the elevated Emax (maximal response) of the hD1-ST1. Using single point mutagenesis, we further found that breaking the potential molecular interactions of Ser2.45 in hD1R (i.e. with Asn3.42 and Trp4.50) mimicked its elevated Emax. This elevated Emax was not found to be caused by altered abilities to undergo agonist-induced desensitization or internalization relative to hD1R. Overall, our work highlights the important functional and structural roles of IC1 and IC2 that needs to be accounted for in our current canonical models of GPCR signalling. Given the conserved nature of these Ser/Thr, our work may also be pertinent towards understanding the roles of IC1 and IC2 for other GPCRs.

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Gentsch, Marcus. "Funktionelle Analyse und Charakterisierung des Gpr1-Proteins in der Hefe Yarrowia lipolytica". Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2003. http://nbn-resolving.de/urn:nbn:de:swb:14-1071153552203-80428.

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In der Hefe Yarrowia lipolytica führen Mutationen im GPR1-Gen zu Essigsäuresensitivität. Die Deletion dieses Genes hat demgegenüber keinen Effekt auf den Phänotyp. In dieser Arbeit wurde das Gpr1-Protein näher charakterisiert. Es zeigte sich, dass GPR1-Mutantenstämme wesentlich schneller Acetat akkumulierten als der Wildtyp. Außerdem konnte bestetigt werden, dass Gpr1p ein integrales Membranprotein ist. Mittels Ortspezifischer Analyse wurden verschiedene funktionelle Bereiche untersucht. Das Protein unterliegt zudem einer Phosphorylierung/Dephosphorylierung. Auf der Grundlage der dargelegten Ergebnisse wurde ein Funktionsmodell für Gpr1p erstellt.

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Jaén, Cristina. "Differential coupling of RGS3s and RGS4 to GPCR-GIRK channel signaling complexes". [Tampa, Fla] : University of South Florida, 2006. http://purl.fcla.edu/usf/dc/et/SFE0001533.

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Altosaar, Katrin. "Dimer-dependent allosteric modulation within GPCR signalling complexes can influence signalling diversity". Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=114353.

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G protein-coupled receptors (GPCRs) comprise the largest group of cell surface receptors, translating environmental signals into cellular responses via cognate G protein partners. Contrary to our initial understanding, most GPCRs do not function in living cells as monomers, but most likely dimers, or even larger arrays of receptors. Standard drug design approaches rely on the notion that drugs binding the two receptors in a given dimer likely function independently of one another. However, this view has been challenged by recent work showing that ligand binding at both receptors can modulate dimeric receptors via allosteric communication. While one receptor may actually be needed to drive signalling, the other acts to control or modulate these signals, without a direct signalling outcome itself. Based on the notion of allosteric modulation within homo- and heterodimers, I tested and compared changes in signalling downstream as well as at the level of the receptor-G protein-effector (RGE) complex in response to different combinations of ligands at each protomer. Using a combination of calcium, cyclic adenosine monophosphate, and mitogen-activated protein kinase signalling assays, I have demonstrated functional interactions for a putative D2 dopamine receptor, oxytocin receptor heterodimer (D2R/OTR), in HEK 293 cells. Immunoprecipitation, bioluminescence resonance energy transfer (BRET) and confocal microscopy experiments reveal D2R and OTR do in fact form a heterodimer in vitro, which may explain the nature of these potential allosteric functional interactions. Using BRET, I assessed the RGE complex conformational dynamics in HEK 293 cells for two other heterodimers, β2-adrenergic receptor with cannabinoid CB1 receptor (β2AR/CB1R) and β2AR/OTR, in order to determine how they manifest in parallel to signalling events themselves. These studies reveal functional interactions can occur in terms of signalling complex conformation. Thus GPCR signalling can be modulated by its partner receptor at the level of downstream effector signalling or at the level of the signalling complex itself. With that said, putative heterodimers need to be reanalyzed in vivo for their allosteric properties, which may explain some of the side effects of so many drugs, and may have implications in drug design.
Les récepteurs couplés aux protéines G (RCPG) constituent le plus grand groupe de récepteurs de la surface cellulaire, qui traduisent les signaux environnementaux en réponses cellulaires via leurs protéines G associées. Contrairement à notre compréhension initiale, la majorité des RCPG ne fonctionnent pas en tant que monomères, mais possiblement en tant que dimères ou même oligomères. Les approches actuelles de conception de médicament estiment que lors de la liaison d'un médicament aux deux récepteurs d'un dimère quelconque, ces derniers fonctionnent potentiellement indépendamment l'un de l'autre. Cependant, cette notion a été reconsidérée par une étude récente montrant que la liaison d'un ligand aux deux récepteurs peut les altérer par voie de communication allostérique. Alors qu'un premier récepteur peut être requis pour initialiser la signalisation, un second peut contrôler ou modifier ces signaux, n'ayant pas nécessairement une signalisation directe comme résultante. Dans l'étude suivante, basée sur la notion de modulation allostérique au sein d'homodimère et d'hétérodimère, les changements de signalisation en aval ainsi qu'au niveau du complexe récepteur/protéine G/effecteur (RGE) ont été étudiés et comparés en réponse à différentes combinaisons de ligands pour chaque protomère. En utilisant une combinaison d'essais de signalisation de calcium, d'adénosine monophosphate cyclique (cAMP) et de protéine kinase activée par des agents mitogènes (MAPK), une interaction fonctionnelle entre le récepteur dopaminergique D2 et le récepteur de l'ocytocine (D2R/OTR) a été démontrée dans les cellules HEK 293. Des expériences d'immunoprécipitation, de transfert d'énergie de résonance par bioluminescence (BRET) et de microscopie confocale ont révélé la présence d'hétérodimère entre le D2R et l'OTR in vitro, ce qui pourrait expliquer la nature des interactions fonctionnelles allostériques. En utilisant la technique de BRET, la dynamique fonctionnelle du complexe RGE dans les cellules HEK 293 a été examinée chez deux autres hétérodimères, soit celui composé du récepteur adrénergique β2 et du récepteur cannabinoïde CB1 (β2AR/CB1R) et l'hétérodimère β2AR/OTR, afin de déterminer comment ils traduisent les évènements de signalisation. Ces études démontrent donc qu'une interaction fonctionnelle peut survenir sur le plan de la conformation du complexe de signalisation. Par conséquent, la signalisation d'un RCPG peut être modulée par son récepteur partenaire au niveau des effecteurs ou au niveau du complexe de signalisation lui-même. Pour cette raison, il serait impératif de réanalyser in vivo les propriétés allostériques d'hétérodimères putatifs, ce qui pourrait expliquer certains effets secondaires d'une multitude de médicaments et ce qui pourrait impliquer des changements majeurs dans la façon de concevoir de nouveaux médicaments.

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Hall, Caroline Jane. "The effect of GPCR crosstalk on intracellular Ca2+ responses and downstream signalling". Thesis, University of Leicester, 2011. http://hdl.handle.net/2381/9528.

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Jaén, Cristina. "Differential coupling of RGS3s and RGS4 to GPCR-GIRK channel signaling complexes". Scholar Commons, 2006. http://scholarcommons.usf.edu/etd/2571.

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'Regulators of G protein signaling' (RGS proteins) modulate the G proteincycle by enhancing the GTPase activity of Ga subunits. These changesaccelerate the kinetics of ion channel modulation by Gai/o-coupled receptors(GPCRs) such as the G protein-gated inward rectifier K+ (GIRK/Kir3) channel. Myexperiments indicate that a single cerebellar granule (CG) neuron, a cell type thatendogenously expresses GIRK channels is able to express a wide variety ofRGS proteins. I selected two of them, which are widely expressed andtranscriptionally regulated during pathophysiologic conditions, to compare theirfunctional properties. I originally described the differential modulatory effects oftwo RGS proteins, the RGS3 short isoform (RGS3s) and RGS4, on muscarinicm2 and serotonin 1A receptor-coupled Kir3.1/Kir3.2a channels expressed inChinese hamster ovary (CHO-K1) cells. Both RGS3s and RGS4 acceleratedGIRK activation and deactivation current kinetics in a similar way. However, onlyRGS3s si gnificantly decreased the maximal GIRK current (Imax) elicited by ACh(~45% inhibition) and significantly increased the EC50 for both GPCRs. Thehypothesis that emerged from this initial study was that the distinct RGS4 Nterminaldomain mediated a direct coupling of RGS4 to GPCR-GIRK channelsignaling complexes that was not shared by RGS3s. To test this hypothesis, Iepitope-tagged several GPCRs, the Kir3.1 subunit, RGS3s, RGS4, and severaldeletion mutants and chimeras for co-immunoprecipitation experiments. Using anepitope-tagged degradation resistant RGS4 mutant RGS4(C2V), I detected coprecipitationof different GPCR-GIRK channel complexes with RGS4 but notRGS3s.The functional impact of RGS4 coupling to the GPCR-Kir3 channelcomplex versus uncoupled RGS3s was not apparent in recordings from CHO-K1cells presumably due to a high degree of RGS collision-coupling. Controlledexpression in Xenopus oocytes revealed a 30-fold greater potency for RGS4 inthe accelerating GIRK channel gating kinetics. In summary, these findings demonstrate that one of the ways for the cellto achieve signaling pathway specificity may be through selective coupling of thedifferent GPCR-effector-RGS protein complexes.

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Ranganathan, Anirudh. "The impact of GPCR structures on understanding receptor function and ligand binding". Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-129879.

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G protein-coupled receptors (GPCRs) form the largest superfamily of eukaryotic membrane proteins and are responsible for the action of nearly 30% of all marketed drugs. For a long period, efforts to study these receptors were limited by the paucity of atomic-resolution structural information. Numerous receptors spread across the GPCR superfamily have recently been crystallized, revealing crucial clues about receptor function and ligand recognition. The work in this thesis has primarily focused on using computational techniques to capitalize on this increasing amount of structural information. In papers I, II, and III protocols were developed to identify novel ligands for pharmaceutically important targets from in silico screens of large chemical libraries. In these papers, the fragment-based lead discovery (FBLD) approach was evaluated for GPCR targets using molecular docking screens. The high hit-rates obtained in these studies indicate promise for the use of computational approaches for fragment screening. In paper IV, molecular dynamics was used to identify a possible role for a conserved ionizable residue (Asp792.50) as a protonation switch during the activation process of the β2 adrenergic receptor. Analyses from this paper indicated that this residue could also perform a similar function in other class A GPCRs. Papers V and VI detail the modeling strategy followed during the GPCR Dock 2013 assessment to blindly predict the structure of two serotonin receptor subtypes (5-HT1B and 5-HT2B) bound to ergotamine. The developed ligand-steered homology modeling protocol was largely successful resulting in the best-ranked predictions for the 5-HT1B subtype. It is hoped that the work described in this thesis has highlighted the potential for structure-based computational approaches to identify novel ligands for important pharmaceutical targets and improve understanding of GPCR function.

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

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Ebersold, Anne. "Production d'anticorps monoclonaux humains dirigés contre la gp41 du virus HIV-1". Lyon 1, 1992. http://www.theses.fr/1992LYO1T268.

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Bahena, Silvia. "Computational Methods for the structural and dynamical understanding of GPCR-RAMP interactions". Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-416790.

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Protein-protein interaction dominates all major biology processes in living cells. Recent studies suggestthat the surface expression and activity of G protein-coupled receptors (GPCRs), which are the largestfamily of receptors in human cells, can be modulated by receptor activity–modifying proteins (RAMPs). Computational tools are essential to complement experimental approaches for the understanding ofmolecular activity of living cells and molecular dynamics simulations are well suited to providemolecular details of proteins function and structure. The classical atom-level molecular modeling ofbiological systems is limited to small systems and short time scales. Therefore, its application iscomplicated for systems such as protein-protein interaction in cell-surface membrane. For this reason, coarse-grained (CG) models have become widely used and they represent an importantstep in the study of large biomolecular systems. CG models are computationally more effective becausethey simplify the complexity of the protein structure allowing simulations to have longer timescales. The aim of this degree project was to determine if the applications of coarse-grained molecularsimulations were suitable for the understanding of the dynamics and structural basis of the GPCRRAMP interactions in a membrane environment. Results indicate that the study of protein-proteininteractions using CG needs further improvement with a more accurate parameterization that will allowthe study of complex systems.

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Salcedo, Romero de Ávila José Vicente. "GPCs en espacio de estados para el control de sistemas no lineales". Doctoral thesis, Universitat Politècnica de València, 2008. http://hdl.handle.net/10251/1882.

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En esta tesis doctoral se aborda el control de sistemas no lineales mediante el empleo de controladores predictivos generalizados (GPCs) en espacio de estados. En primer lugar se realiza una revisión de la metodología de diseño del GPC en la versión entrada/salida (E/S). Partiendo de esta revisión se propone un modelo CARIMA en espacio de estados para el GPC que permite diseñar al mismo utilizando una menor cantidad de memoria y un menor tiempo de cómputo, así como de reducir la complejidad asociada la formulación E/S. Para la estimación de los estados del modelo CARIMA se propone el uso de un observador de rango completo que se diseña por asignación de polos, estableciéndose un importante resultado: los polos de este observador coinciden con las raíces de los polinomios de filtrado utilizados en la formulación E/S. Posteriormente se analizan las propiedades de observabilidad y controlabilidad del modelo CARIMA propuesto en espacio de estados, llegándose a la conclusión de que se trata de una realización mínima bajo condiciones no demasiado restrictivas, lo cual supone que la predicción se basa en un modelo con el mínimo orden posible. Tras esto, se presenta una metodología de análisis y diseño estable para el GPC mediante el uso del índice de coste como función de Lyapunov, y para el caso con restricciones de la teoría de conjuntos invariantes aplicada al GPC. Seguidamente, se presenta una metodología de diseño robusto para el GPC mediante el empleo de las desigualdades lineales matriciales (LMIs) y de algoritmos genéticos. En concreto, se analiza el caso de sistemas con incertidumbre invariante y variante con el tiempo de tipo lineal fraccional, una de las más complejas y generales utilizadas en la literatura analizada. Finalmente se presenta el controlador GPC-LPV una extensión del GPC en espacio de estados. Se trata de un controlador variante con el tiempo que presenta dependencia lineal fraccional con respecto de las señales de salida medibles. Su diseño es
Salcedo Romero De Ávila, JV. (2005). GPCs en espacio de estados para el control de sistemas no lineales [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/1882
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Scarlin, Hugh. "Studies on the synthesis of novel PP2A inhibitors and a GPCR detergent". Thesis, University of Glasgow, 2016. http://theses.gla.ac.uk/7370/.

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Chapter 1 While targeting kinases in oncology research has been explored extensively, targeting protein phosphatases is currently in its infancy. However, a number of pharmaceutical companies are currently looking to expand their research efforts in this area. PP2A has been shown to down-regulate ERK5, a mitogen-activated protein kinase (MAPK) that has been shown to be important in driving the invasive phenotype of prostate cancer. Fostriecin and its related structural analogues PD 113,270 and 113,271 have been shown to inhibit a mitotic entry checkpoint in cell growth through the potent and selective inhibition of protein phosphatases PP1, PP2A, and PP4 (IC50 of 45 μM, 1.5 nM, and 3 nM respectively). Fostriecin is one of the most selective protein phosphatase inhibitors disclosed to date with a 104 fold selectivity for PP2A/PP4 versus PP1. Unfortunately, fostriecin and its analogues are very unstable, and this instability has effectively prevented them from being used as effective therapeutic leads. The microcystins and nodularins on the other hand, exhibit significant inhibitory activity against PP1 and PP2A (IC50 = 26 pM and 1.8 nM respectively), but their high toxicity has prevented any therapeutic application. Truncation of the ADDA chain from these polypeptides completely attenuates PP inhibitory activity. Simpler analogues incorporating the N-acylated ADDA chain and D-Ala retain moderate activity against PP1 and PP2A (IC50 = 1.0 μM and 0.17 μM respectively). The generation of a new series of fostriecin analogues to further expand its structure-activity relationship is envisaged with a view to creating new more stable PP2A inhibitors. It was hoped that by incorporating some of the more stable structural features of ADDA into fostriecin that stability and activity could be reconciled. With that in mind a series of PP2A inhibitors were synthesised and biologically evaluated. Chapter 2 GPCRs are an important area of research and are the targets of a quarter of the drugs on the market (2005). As a result, GPCRs continue to be at the forefront of research in both small and large drug companies. However one of the difficulties in studying this diverse class of membrane proteins is their tendency to denature in aqueous solution. As a result there is a pressing need to develop new detergents to solubilise, stabilise and crystallise GPCRs in their native form for further study. Cholesterol analogues have been shown to be important for stabilising membrane proteins and preventing their thermal inactivation. In addition the β2-adrenergic receptor, a GPCR membrane protein, has been crystallised in the active state with two cholesterol molecules bound between the I, II, III and IV helices of the protein. This appears to represent a distinct cholesterol binding pocket on the membrane protein that is speculated to be conserved across up to 44% of the rhodopsin class of GPCRs. CHOBIMALT is a cholesterol-based detergent that has been shown to exhibit promising GPCR-stabilising properties. When benchmarked against other cholesterol based detergents it was found to be superior to all others tested except for cholesteryl hemisuccinate.1 CHOBIMALT has an aggregation number of roughly 200 and forms 210 ± 30 kDa micelles, which are significantly larger than those of most detergents used for biological systems which is likely due to the packing constraints associated with CHOBMALT’s large polar headgroup.2 As a result, CHOBIMALT is used mostly as an additive to other commercially available detergents in order to decrease micelle size. A branched dimaltoside motif is common in recently synthesised detergents by Chae and co-workers. These detergents have shown promising detergent properties, for example the maltose neopentyl glycol (MNG) detergent synthesised by Chae. This branched dimaltoside detergent was shown to be able to solubilise and stabilise the very labile light harvesting complex I (LHI) from Rhodopsin capsulatus in its active form for 20 days with little loss of protein conformation.3 A cholesterol-based detergent was envisaged that combines the cholesterol framework of CHOBIMALT but replaces its linear tetrasaccharide with a branched dimaltoside. This detergent would then be investigated to assess its ability to solubilise, stabilise and crystallise GPCR proteins. This cholesterol-based detergent (shown below) was eventually synthesised in 9 linear steps from cholesterol.

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Elenko, Eric. "Localization and fate of GAIP following G[alpha]i̳ linked GPCR stimulation /". Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2001. http://wwwlib.umi.com/cr/ucsd/fullcit?p3026385.

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Cornélio, Filho Plínio. "O modelo de simulação do GPCP-1 : planejamento e controle da produção". reponame:Repositório Institucional da UFSC, 1998. https://repositorio.ufsc.br/handle/123456789/111375.

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Jha, Ankita. "Quantitative control of GPCR organization and signaling by endocytosis in epithelial morphogenesis". Thesis, Aix-Marseille, 2017. http://www.theses.fr/2017AIXM0393/document.

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Au cours de la gastrulation de l’embryon de Drosophile, l’activation apicale du cytosquelette d’acto-myosine orchestre la constriction apicale dans le mésoderme en invagination ainsi que l’intercalation cellulaire dans l’ectoderme en extension. Un contrôle quantitatif de l’activité des GPCRs et, par conséquent, de l’activation de Rho1 est à l’origine des différences de déformation des cellules du mésoderme et de l’ectoderme mais ces mécanismes demeurent incompris. L’activité du GPCR Smog se concentre respectivement en deux compartiments distincts à la surface de la membrane plasmique (PM) et dans ses invaginations (PMI). Au moyen de la FCS, nous avons étudié la surface de la PM et pu montrer que Smog oligomérise en homo-clusters en réponse à son activation par le ligand Fog. L’endocytose de Smog est facilitée par la kinase Gprk2 et sa protéine adaptatrice la β-Arrestine-2 qui retire Smog actif de la PM. Lorsque que la concentration de Fog est élevée ou que l’endocytose est réduite, Smog s’organise en homo-clusters et s’accumule au niveau des PMI qui agissent comme des centres d’activation de Rho1. Une concentration plus importante d’homo-clusters de Smog et un nombre plus important PMI dans le mésoderme par comparaison avec l’ectoderme. Répartition dynamique de Smog actif à la surface de la PM ou dans ses invaginations impacte directement sur la signalisation Rho1. Les PMI accumulent de hauts niveaux de Rho1-GTP suggérant qu’elles forment des centres de signalisation. La concentration de Fog et l’endocytose de Smog sont des processus régulateurs couplés qui contrôlent la différence quantitative d’activation de Rho1 dans le mésoderme et l’ectoderme de la Drosophile
During Drosophila gastrulation, apical activation of the actomyosin networks drives apical constriction in the invaginating mesoderm and cell-cell intercalation in the extending ectoderm. Here, we show that cell-surface G-protein coupled receptor, Smog activates G-proteins, Rho1 and Rho-kinase that is required for apical constriction and cell-cell intercalation. Quantitative control over GPCR activity and thereby Rho1 activation underlies differences in deformation of the mesoderm and ectoderm cells but the mechanisms remain elusive. We show that GPCR-Smog activity is concentrated on two different apical plasma membrane compartments i.e. the surface and the plasma membrane invaginations. Using FCS, we probe the surface of the plasma membrane (PM) and show that Smog homo-clusters in response to its activating ligand Fog. Endocytosis of Smog is facilitated by the kinase Gprk2 and the adaptor protein β-Arrestin-2 that clears active Smog from the surface of PM. When Fog concentration is high or endocytosis is low, Smog arranges in homo-clusters and accumulates in plasma membrane invaginations (PMI), that are hubs for Rho1 activation. Lastly, we find high Smog homo-cluster concentrations and numerous apical PMIs in the mesoderm compared to the ectoderm. We identify that dynamic partitioning of active Smog on the surface of the PM or PMI directly impact on Rho1 signaling. PMIs accumulate high Rho1-GTP suggesting they form signaling centers. Fog concentration and Smog endocytosis form coupled regulatory processes that regulate quantitative differential Rho1/MyoII activation in the Drosophila mesoderm and ectoderm

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Zanella, Júnior Aldo. "Experimentações práticas e simuladas de controle preditivo generalizado - GPC". Universidade do Estado de Santa Catarina, 2015. http://tede.udesc.br/handle/handle/2084.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
This work introduces the report of performed studies in order to evaluate the applicability of generalized predictive control (GPC) to several plants. The main goal is to analyze the GPC performance in processes with different features, analyzing the influence of its tuning parameters. The study is justified by the fact that GPC presents itself as a generalized solution for several classes of processes, which are becoming increasingly complex and demanding for traditional controllers to handle. For the purpose to prove this proposal of GPC, it was performed several tests with plants of different orders and response characteristics, real and simulated, varying controller tuning parameters and measuring some quality indices. It was evaluated the influence of tuning parameters and it was made a report of conclusions that was reached. Through obtained results, it is shown that GPC satisfies the proposal and presents favorable results.
Esta dissertação traz o relato do estudo realizado a fim de avaliar a aplicabilidade do controlador preditivo generalizado (GPC) em plantas diversas. O objetivo principal é analisar o desempenho do GPC em processos com diferentes características, analisando a influência dos seus parâmetros de sintonia. O estudo se justifica pelo fato de que o GPC apresenta-se como uma solução generalizada para diversos tipos de processos, os quais estão se tornando cada vez mais complexos e com maiores exigências para o controlador. A fim de comprovar essa proposta do GPC, realizou-se inúmeros ensaios com plantas com respostas e ordem diferentes, reais e simuladas, variando-se os parâmetros de sintonia do controlador e medindo-se alguns parâmetros de qualidade. Avaliou-se a influência dos parâmetros de sintonia e fez-se um relato das conclusões a que se chegou. Através dos resultados obtidos, mostra-se que o GPC corresponde ao que se propõe para as plantas testadas e apresenta resultados favoráveis.

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Cruz, Daniel Miranda. "Estruturas de controle preditivo repetitivo baseadas na formulação GPC". reponame:Repositório Institucional da UFSC, 2015. https://repositorio.ufsc.br/xmlui/handle/123456789/136484.

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Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia de Automação e Sistemas, Florianópolis, 2015.
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Este trabalho apresenta um estudo de diversas estruturas de controle baseadas no algoritmo de controle preditivo generalizado, Generalized Predictive Control (GPC), e no controle repetitivo, Repetitive Control (RC). Os controladores analisados possuem as vantagens do controle preditivo no que diz respeito à otimização e ao tratamento de restrições e as do controle repetitivo no tratamento de perturbações periódicas, caracterizando a estrutura de controle preditivo generalizado repetitivo, Repetitive Generalized Predictive Control (RGPC). A estrutura proposta usa ação de controle repetitiva, baseada em um modelo interno com filtro de robustez e faz o tratamento das restrições considerando a repetitividade da ação de controle. O estudo compara diversas possibilidades de implementação, uma clássica e outra com o projeto separado, apontando vantagens e desvantagens de cada uma, assim como a aplicação em um estudo de caso.

Abstract : This work presents a study of different control schemes based on the GPC and RC. These controllers have the advantages of online optimization and contraint hadling of the predictive control and periodic signals treatment of the repetitive control, defining the RGPC. The proposed structure uses a repetitive control action based on an internal model with robustness filter and deals with constraints considering the control action repetitiveness. The study compares various implementation possibilities, a classic and a separated design, pointing advantages and disadvantages of each one as well as their implementation.

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Augstein, Antje. "Molekularbiologische Charakterisierung und funktionelle Analyse des GPR1-Genproduktes in der Hefe Yarrowia lipolytica". Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2001. http://nbn-resolving.de/urn:nbn:de:swb:14-997694149203-01172.

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Brewer, Cynthia. "Use of PC12 cells to characterize PAFR-GPCR-mediated activity in neural precursors". Thesis, University of Ottawa (Canada), 2001. http://hdl.handle.net/10393/9170.

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Platelet activating factor (PAF: 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a biologically active phospholipid mediator produced by neurons and glial cells. In adult central nervous system (CNS), physiological concentrations of PAF have been shown to mediate long-term potentiation, the purported cellular basis of mammalian learning and memory. Elevated levels of PAF are implicated in the pathophysiology of a number of neurodegenerative diseases, ischemia-reperfusion injury, human immunodeficiency virus-1 (HIV-1) associated dementia, and the developmental brain disorder Miller-Dieker Syndrome (MDS). Three neuropathologies are associated with sustained PAF exposure. It is not clear how these PAF-mediated effects are initiated in CNS. A seven transmembrane G-protein coupled receptor (PAFR-GPCR) has been cloned and mRNA is expressed in CNS. In addition, distinct intracellular receptor isoforms (iPAFRs) in rodent cortical extracts have been characterized pharmacologically. It is not known which of these PAF binding proteins initiate PAF-mediated neuropathology. Our ability to detect PAF receptors is further hindered by a lack of commercial PAF antibodies. To address these issues, in this thesis, I sought to: (1) Identify a neural precursor cell line capable of differentiating between PAFR-GPCR and PPAFR-initiated biological activity, (2) Determine the effect of PAFR-GPCR activation on differentiation of neural precursors to a neuronal phenotype, and (3) Characterize a new PAFR-GPCR antibody to facilitate localization of protein in CNS and CNS cell lines. (Abstract shortened by UMI.)

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Heidari, Yasin. "Identification and characterisation of a novel isoform of gp91 subunit of NADPH oxidase". Thesis, King's College London (University of London), 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.407934.

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Jackson, Verity. "Mechanistic studies of the adhesion-GPCR latrophilin and its interactions in neural guidance". Thesis, University of Oxford, 2017. http://ora.ox.ac.uk/objects/uuid:ecc6187a-3e20-4753-ada6-dc009ad7f6e2.

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The adhesion GPCRs are a poorly understood and evolutionarily ancient family of cell surface receptors, several of which have emerging functions in the development of the nervous system. aGPCRs comprise a large extracellular domain, providing binding sites for a variety of ligands, alongside a seven transmembrane domain characteristic of GPCRs. It has been proposed that aGPCRs may function as "context-recognisers", using their large ectodomains to bind different combinations of ligands depending on the molecular make-up of the environment. However there is a lack of direct evidence for this at a molecular level. The ectodomain of one subfamily of aGPCRs, the Latrophilins (Lphns) has been shown to directly interact with several ligands with roles in synaptogenesis and neural guidance. The best-validated of these interactions are those with Fibronectin Leucine-Rich Transmembrane (FLRT) proteins and the Teneurins. In addition, FLRT proteins, also interact with Uncoordinated5 (Unc5) proteins, mediating cell repulsion. Here I reveal that the FLRT-binding site of Lphn is bifunctional, mediating both cell adhesion and repulsion, and that Unc5 is capable of influencing the functional outcome of this interaction. Biophysics and structural studies show that fragments of the Lphn, FLRT and Unc5 ectodomains interact in an unusual and homologue-dependent stoichiometry. Despite the fact that Teneurin interacts with Lphn at a distinct site, Teneurin seems incapable of interacting with the Lphn-FLRT-Unc5 complex, but can form a ternary complex with Lphn and FLRT in the absence of Unc5. Alongside this I present a crystal structure of a large portion of a Teneurin ectodomain, revealing the ancient evolutionary origins of this receptor. Together these data provide strong molecular evidence for a role of Lphns as context-recognisers, by their abilities to bind diverse ligands in distinct combinations and variable stoichiometries.

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47

Cutolo, Pasquale. "Etude de l'interaction structurelle et fonctionnelle entre la chimiokine CXCL12 et ses récepteurs : CXCR4 et ACKR3/CXCR7". Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS550/document.

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L'axe formé par la chimiokine CXCL12 et son récepteur CXCR4 est conservé chez les vertébrés où il joue un rôle important dans l'embryogenèse et la vie adulte, régule de nombreux processus des réponses immunitaires grâce à ses fonctions dans la migration cellulaire, la survie et la prolifération.En outre, cet axe est impliqué dans les processus pathologiques tels que les cancers (croissance et métastase) et immunodéficiences ainsi que des dysfonctionnements (par exemple l'expression dérégulée, polymorphismes ou mutations) et est également détourné par certains agents pathogènes (par exemple le virus de l'immunodéficience humaine, virus du papillome humain).Un grand groupe de travail est consacré à cette paire comme cible thérapeutique, mais seulement un composé (à savoir Plérixafor) a atteint l'approbation pour une utilisation clinique faisant le potentiel de cet axe comme cible de médicament encore inexploré.Bien que cet axe est l'objet d'un grand intérêt, des questions demeurent quant aux déterminants structurels impliqués dans l'interaction CXCL12/CXCR4.Cependant, la structure récemment résolue par diffraction de CXCR4 a donné quelque indice au sujet de ces questions, et au delà, la possible stoichiométrie entre CXCL12 et CXCR4.Plusieurs éléments de preuve appuient le concept que les formes CXCR4 homo- et hétéro- oligomères (qui peut contribuer à la diversité des fonctions de récepteur), telles que la structure de diffraction, le gain de fonction d'un récepteur CXCR4 mutant responsable du syndrome WHIM et la modulation allostérique des fonctions de CXCR4 par CXCR7 (ACKR3), le second récepteur de CXCL12. La possibilité de former des oligomères ouvre de nombreuses questions en matière de CXCL12 et ses interactions avec CXCR4 et CXCR7/ACKR3. La stoichiométrie de cette interaction reste une question ouverte, comme le récepteur est capable de former des oligomères avec le même récepteur ou autre récepteurs, en particulier CXCR7/ACKR3. Ce récepteur, connu comme scavenger, n'a pas de structure résolue et son mécanisme d'interaction avec CXCL12 reste inconnu.Afin d'étudier les interactions CXCL12/CXCR4/CXCR7, nous avons appliqué plusieurs techniques de modélisation moléculaire tels que peptid-peptide docking et simulations de dynamique moléculaire.Objets du projet ont étés : la résolution des possibles formes stoichiométriques de l'interaction CXCR4/CXCL12 (modélisation moléculaire, docking et dynamique); la modélisation de la structure du récepteur CXCR7/ACKR3 et son interaction avec CXCL12 (homology modeling), avec caractérisation des domaines et des résidus clef de l'activation des pathways de signalisation en aval du récepteur (mutants CXCR7/ACKR3); l'étude et la caractérisation de nouveaux outils innovants pour la détection de l'oligomerisation de ces récepteurs en conditions endogènes. (Nanobodies, HTRF)Les résultats du premier objectif ont été publiés en janvier 2016 : PMID 26813575.La modélisation de CXCR7/ACKR3 nous a permit de générer plusieurs mutants du récepteur pour tester nos hypothèses sur l’activation.Les nanobodies caractérisés pour CXCR4 seront utilisé dans une deuxième étude pour l’identification des formes oligomériques du récepteur sur tissus et cellules
The axis formed by the chemokine CXCL12 and its receptor CXCR4 is conserved in vertebrates where it plays an important role in embryogenesis and adult life, regulates many processes of immune responses through its functions in cell migration, survival and proliferation.In addition, this axis is involved in pathological processes such as cancers (growth and metastasis) and immune deficiencies and malfunctions (eg deregulated expression, mutations or polymorphisms) and is also hijacked by certain pathogens (eg HIV, human papilloma virus).A large working group is dedicated to this pair as a therapeutic target, but only a compound (ie Plerixafor) achieved approval for clinical use by the potential of this area as a drug target unexplored.Although this axis is the subject of great interest, questions remain about the structural determinants involved in CXCL12 / CXCR4 interaction.However, the recently resolved diffraction structure of CXCR4 gave some clue about these questions, and beyond possible stoichiometry between CXCL12 and CXCR4.Several lines of evidence support the concept that forms CXCR4 homo- and hetero-oligomers (which can contribute to the diversity of the receptor functions), as shown in the diffraction structure, the gain function of a mutant CXCR4 receptor responsible for the syndrome WHIM and allosteric modulation of CXCR4 functions by CXCR7 (ACKR3), the second receptor of the chemokine CXCL12. The ability to form oligomers opens many issues of CXCL12 and its interaction with CXCR4 and CXCR7 / ACKR3.The stoichiometry of this interaction still remains an open question, as the receptor is capable to form oligomers with the same receptor or other receptors, particularly CXCR7 / ACKR3. This receptor, known as scavenger, has not solved structure and the mechanism of interaction with CXCL12 is unknown.To study the interactions CXCL12 / CXCR4 / CXCR7, we applied several molecular modeling techniques such as peptide-peptide docking and molecular dynamics simulations.Objectives of this project were: the resolution of the different stoichiometric forms for the interaction of CXCR4 and CXCL12 (molecular modeling, docking and dynamic); modeling the CXCR7 / ACKR3 receptor structure and its interaction with CXCL12 (homology modeling), with the characterization of domains and residues key in the activation of downstream signaling pathways of the receptor (CXCR7 / ACKR3 mutants); the study and characterization of new innovative tools for the detection of oligomerization of these receptors in endogenous conditions. (Nanobodies, HTRF)The results of the first objective were published in January 2016: PMID 26813575.Modeling of CXCR7 / ACKR3 allowed us to generate several mutants of the receptor to test our hypothesis about the activation pathways.Nanobodies were fully characterized for CXCR4 to be used in a second study to identify oligomeric forms of the receptor in tissues and cells

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Sintes-Zydowicz, Nathalie. "Polyimides - amides, microstructure : mécanismes réactionnels : étude par RMN et GPC". Lyon 1, 1991. http://www.theses.fr/1991LYO10165.

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Des polyimides-amides (pia) de basses masses sont synthetises par copolycondensation du diisocyanato 4-4 diphenylmethane, de l'anhydride trimellique et de l'acide benzoique qui joue le role d'agent limiteur de chaines en solution dans la n-methylpyrrolidone, a haute temperature. L'etude par rmn 1h et 13c permet de determiner la composition et la microstructure de ces pia, de mettre en evidence les produits de reactions secondaires, de quantifier les monomeres n'ayant pas reagi. A partir de ces donnees, il est possible de corriger la formulation initiale des pia. Cette nouvelle formulation permet de determiner la masse moyenne en nombre experimentale des pia. La relation entre cette masse et la masse moyenne en nombre theorique a ete etablie. Les pia sont examines en gpc; la masse absolue des premiers pics etant connue, la masse moyenne en nombre gpc a pu etre corrigee et la relation entre cette masse et la masse theorique a ete etablie. La mesure de la viscosite intrinseque permet la determination des parametres de mark houwink. La temperature de transition vitreuse des pia est determinee par analyse thermodifferentielle (dsc) et la loi de fox-flory est verifiee. L'etude par rmn de pia synthetises a des temperatures plus basses, montre la presence d'enchainements uree et d'une structure intermediaire de reaction, stable, issue de l'anhydride trimellique. Cette structure reagit avec les fonctions uree entre 140c et 180c pour former des groupements imide par elimination d'eau et de dioxyde de carbone. Les fonctions uree se forment au cours de la synthese, par reaction des acides sur les isocyanates, selon le mecanisme propose par d'olieslager et de aguirre

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Menezes, Marcos André Silveira. ""Implementação de um controlador GPC adaptativo aplicado a processos industriais"". reponame:Repositório Institucional da UFSC, 1993. https://repositorio.ufsc.br/handle/123456789/111637.

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Mazoco, Bruna Marques. "Proposta de um algoritmo GPC adaptativo com baixo custo computacional". reponame:Repositório Institucional da UFES, 2015. http://repositorio.ufes.br/handle/10/1423.

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Esta dissertação propõe um algoritmo do Controlador Preditivo Generalizado (GPC) com horizonte de controle igual a um para ser aplicado em plantas industriais com modelos variantes no tempo, simples o su ficiente para ser implementado em Controlador Lógico Programável (PLC). A solução explícita do controlador é obtida em função dos parâmetros do modelo e dos parâmetros de sintonia do GPC (horizonte nal de predição hp e o fator de supressão do sinal de controle ), além das entradas e saídas presentes e passadas. A sintonia do fator de supressão e do horizonte de previsão GPC é feita através do lugar das raízes da equação característica do sistema em malha fechada, sempre que os parâmetros do modelo da planta industrial (estável ou instável em malha aberta) forem modificados.
This dissertation proposes a new formulation of the Generalized Predictive Control (GPC) algorithm to be applied in industrial plants with time-varying models. The unitary control horizon premisse alows it to be simple enough to be implemented in a Programmable Logic Controller (PLC). The explicit solution of the control increment is obtained from the parameters of the model and the GPC tuning parameters (prediction horizon hp and supression weight ), in addition to past and present inputs and outputs. Supression weight tuning is done by Root Locus technique, constructed from the system closed loop characteristic polynomium, everytime the model parameters of the industrial plant (stable or not in open loop) su er modi fication.

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