Target of Rapamycin Complex 2 (TORC2) has conserved roles in regulating cytoskeleton dynamics and cell migration and has been linked to cancer metastasis. However, little is known about the mechanisms regulating TORC2 activity and function in any system. In Dictyostelium, TORC2 functions at the front of migrating cells downstream of the Ras protein RasC, controlling F-actin dynamics and cAMP production. Here, we report the identification of the small GTPase Rap1 as a conserved binding partner of the TORC2 component RIP3/SIN1, and that Rap1 positively regulates the RasC-mediated activation of TORC2 in Dictyostelium. Moreover, we show that active RasC binds to the catalytic domain of TOR, suggesting a mechanism of TORC2 activation that is similar to Rheb activation of TOR complex 1. Dual Ras/Rap1 regulation of TORC2 may allow for integration of Ras and Rap1 signaling pathways in directed cell migration.

The Interferon (IFN) family of cytokines are produced in direct response to virus infection and they constitute the first line of defence against virus infection by inducing hundreds of interferon stimulated genes (ISGs) which act in concert to establish the so-called “antiviral state”. Influenza A viruses and parainfluenza virus type 5 (PIV5) are both small negative strand RNA viruses that must circumvent their hosts’ interferon (IFN) response for replication. However, the ways in which these viruses interact with the IFN system are very different. Although PIV5 replication is initially severely impaired in cells in a pre-existing IFN-induced antiviral state, it manages to overcome the antiviral state by targeting an essential component of type I IFN signalling, STAT1, for degradation. Thus the cells cannot maintain the antiviral state indefinitely without continuous signalling. Consequently, the virus resumes its normal replication pattern after 24-48 hours post-infection. In clear contrast, influenza virus fails to establish its replication in the majority of infected cells (90-95%) with a pre-existing IFN-induced antiviral state, although a few cells are still able to produce viral antigens. To further investigate how influenza virus interacts with cells in a pre-existing IFN-induced antiviral state, I have used in situ hybridization to follow the fate of input and progeny genomes in cells that have, or have not, been treated with IFN prior to infection. Here I show for the first time that IFN pre-treatment blocks the nuclear import of influenza A virus genome, which prevents the establishment of virus replication, but this can be overcome by increasing multiplicities of infection. Of those IFN-induced antiviral molecules, human MxA is an essential component of the IFN-induced antiviral state in blocking influenza virus genome import, as this block can be abolished by lentivirus-mediated knockdown of MxA. I also show that in cells constitutively expressing MxA the viral genome still manages to be transported into the nucleus, indicating that MxA might require an unidentified IFN-induced factor to block nuclear import of the influenza virus genome. These results reveal that IFN exerts its action at an early stage of virus infection by inducing MxA to interfere with the transport of viral genome into the nucleus, which is the factory for viral RNA production.

Yeung, Barry Ho Sing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 137-154). / Abstracts in English and Chinese. / Abstract --- p.i / 論文摘要 --- p.iv / Acknowledgements --- p.vii / Publications based on work in this thesis. --- p.ix / List of abbreviations --- p.x / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Dorsal root ganglion cells --- p.1 / Chapter 1.1.1 --- Primary sensory neurons --- p.1 / Chapter 1.1.2 --- Non-neuronal cells --- p.3 / Chapter 1.1.2.1 --- Satellite glial cells --- p.3 / Chapter 1.1.2.2 --- Schwann cells --- p.6 / Chapter 1.2 --- Peripheral sensitization --- p.8 / Chapter 1.3 --- Neuron-glia interactions --- p.9 / Chapter 1.4 --- Aim of Thesis --- p.11 / Chapter Chapter 2 --- "Materials, media, buffers and solutions" --- p.13 / Chapter 2.1 --- Materials --- p.13 / Chapter 2.2 --- "Culture media, buffer and solutions" --- p.19 / Chapter 2.2.1 --- Culture media --- p.19 / Chapter 2.2.2 --- General culture buffers and culture plate coating reagents --- p.19 / Chapter 2.3 --- Antibodies used for identifying DRG cells --- p.23 / Chapter 2.3.1 --- Primary antibodies --- p.23 / Chapter 2.3.2 --- Secondary antibodies --- p.23 / Chapter Chapter 3 --- Methods --- p.24 / Chapter 3.1 --- Preparation of DRG cell cultures --- p.24 / Chapter 3.2 --- Preparation of neuron-enriched and glial cell cultures --- p.25 / Chapter 3.3 --- Immunocytochemistry --- p.26 / Chapter 3.4 --- Immunohistochemistry --- p.27 / Chapter 3.4 --- Determination of [3H]cAMP production in DRG cells --- p.28 / Chapter 3.4.1 --- Principle of assay --- p.28 / Chapter 3.4.2 --- Loading DRG cells with [3H]adenine --- p.28 / Chapter 3.4.3 --- Column preparation --- p.28 / Chapter 3.4.4 --- Measurement of [3H]cAMP production in DRG cells --- p.29 / Chapter 3.4.5 --- Data analysis --- p.30 / Chapter Chapter 4 --- Identification of DRG cells in dissociated cultures --- p.31 / Chapter 4.1 --- Introduction --- p.31 / Chapter 4.2 --- Aim of study --- p.34 / Chapter 4.3 --- Results --- p.35 / Chapter 4.3.1 --- Identification of DRG cells in isolated cultures --- p.35 / Chapter 4.3.2 --- Activation and proliferation of glial cells in isolated cell cultures --- p.36 / Chapter 4.3.3 --- Identification of glial cells in cultures --- p.38 / Chapter 4.3.4 --- Modification of staining methods --- p.40 / Chapter 4.3.5 --- Immunohistochemistry to identify DRG cells in DRG slices --- p.42 / Chapter 4.3.6 --- Comparison of antibody staining in whole DRG and isolated DRG cells --- p.44 / Chapter 4.4 --- Discussion --- p.44 / Chapter 4.5 --- Summary --- p.53 / Chapter Chapter 5 --- Characterization of GPCRs in isolated DRG cultures --- p.69 / Chapter 5.1 --- Introduction --- p.69 / Chapter 5.1.1 --- G-protein coupled receptors --- p.69 / Chapter 5.1.2 --- Pharmacological characterization of prostanoid receptors on DRG cells --- p.73 / Chapter 5.1.3 --- Gs- and Gi/o-coupled GPCRs in DRG cells --- p.75 / Chapter 5.1.3.1 --- Gs-coupled GPCR: β-adrenoceptors --- p.76 / Chapter 5.1.3.2 --- Gs-coupled GPCR: CGRP receptors --- p.79 / Chapter 5.1.3.3 --- Gi/o-coupled GPCR: α2-adrenoceptors --- p.82 / Chapter 5.1.3.4 --- Gi/o-coupled GPCR: Cannabinoid receptors --- p.85 / Chapter 5.1.3.5 --- Gi/o-coupled GPCR: 5-HT1Areceptors --- p.88 / Chapter 5.1.3.6 --- Gi/o-coupled GPCR: opioid and opioid-receptor-like 1 receptors --- p.90 / Chapter 5.2 --- Aims of study --- p.93 / Chapter 5.3 --- Results --- p.94 / Chapter 5.3.1 --- Characterization of prostanoid receptors in isolated DRG cells --- p.94 / Chapter 5.3.2 --- Characterization of CGRP receptors in isolated DRG cells --- p.96 / Chapter 5.3.3 --- Investigation of the effect of CGRP8.37 on CGRP responses --- p.97 / Chapter 5.3.4 --- Characterization of β1-adrenoceptors in isolated DRG cells --- p.97 / Chapter 5.3.5 --- Characterization of β2-adrenoceptors in isolated DRG cells --- p.98 / Chapter 5.3.6 --- Identification of β-adrenoceptor subtype mediating isoprenaline-stimulated responses.. --- p.99 / Chapter 5.3.7 --- Characterization of α2-adrenceptors in isolated DRG cells --- p.100 / Chapter 5.3.8 --- Characterization of cannabinoid 1 receptors in isolated DRG cells ... --- p.100 / Chapter 5.3.9 --- Characterization of cannabinoid 2 receptors in isolated DRG cells --- p.101 / Chapter 5.3.10 --- Characterization of 5-HT1A receptors in isolated DRG cells --- p.101 / Chapter 5.3.11 --- Characterization of μ-opioid receptors in isolated DRG cells --- p.102 / Chapter 5.3.12 --- Characterization of opioid-receptor-like 1 receptors in isolated DRG cells --- p.102 / Chapter 5.3.13 --- Effect of nerve growth factor on DRG cells --- p.103 / Chapter 5.4 --- Discussion --- p.106 / Chapter 5.5 --- Summary --- p.114 / Chapter Chapter 6 --- Conclusion and further studies --- p.134 / References --- p.137

The overall aim of this thesis is to investigate strategies to aid in the measurement of G-protein coupled receptor (GPCR) activity for high-throughput screening and sensing applications. GPCRs are cell surface receptors which have seven membrane spanning domains. They are the largest family of membrane proteins in the human genome and are involved in a number of physiological and pathophysiological pathways. They are the most widely targeted protein family for therapeutics being the target for over 30% of the currently available prescription drugs (Jacoby et al. 2006). For this reason commercial interest and investment into compound screening using these receptors as targets is of high importance in lead drug discovery. Additionally, the extensive ligand range of the GPCR superfamily, which includes light, odorants/ volatiles, neurotransmitters and hormones, make them an attractive biological recognition element in biosensor applications. This thesis demonstrates the functional expression of the H1-histamine, M2-muscarinic and α₂ₐ-adrenergic receptors of the G-protein coupled receptor family, along with their associated G-proteins (Gα, Gβ and Gγ). Expression was achieved using the Sf9/baculovirus expression system. The G-proteins were successfully incorporated into an assay system using time-resolved fluorescence resonance energy transfer (TRFRET). TR-FRET was used in order to create a homogeneous assay format capable of monitoring GPCR activation through the movement of the G-protein subunits. Fluorescence changes in the TR-FRET assay indicated a change in distance between the Gα subunit and Gβγ dimer. The separation of the Gα subunit and the Gβγ dimer after activation resulted in a significant decrease in TR-FRET measurement. The homogeneous set-up of the TR-FRET assay could potentially be adaptable to an array based format. This thesis describes the capture of vesicles containing functional GPCRs onto a solid substrate via the specific interaction between complementary oligonucleotides. GPCR presence and function within the immobilized vesicles, was demonstrated using fluorescent ligands. Further to this, alternative lipid hosts (to the vesicles), known as cubosomes, were introduced. When tagged with an oligonucleotide, these cubosome particles were also shown to immobilize site specifically onto a complementary oligonucleotide surface. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1369537 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

The overall aim of this thesis is to investigate strategies to aid in the measurement of G-protein coupled receptor (GPCR) activity for high-throughput screening and sensing applications. GPCRs are cell surface receptors which have seven membrane spanning domains. They are the largest family of membrane proteins in the human genome and are involved in a number of physiological and pathophysiological pathways. They are the most widely targeted protein family for therapeutics being the target for over 30% of the currently available prescription drugs (Jacoby et al. 2006). For this reason commercial interest and investment into compound screening using these receptors as targets is of high importance in lead drug discovery. Additionally, the extensive ligand range of the GPCR superfamily, which includes light, odorants/ volatiles, neurotransmitters and hormones, make them an attractive biological recognition element in biosensor applications. This thesis demonstrates the functional expression of the H1-histamine, M2-muscarinic and α₂ₐ-adrenergic receptors of the G-protein coupled receptor family, along with their associated G-proteins (Gα, Gβ and Gγ). Expression was achieved using the Sf9/baculovirus expression system. The G-proteins were successfully incorporated into an assay system using time-resolved fluorescence resonance energy transfer (TRFRET). TR-FRET was used in order to create a homogeneous assay format capable of monitoring GPCR activation through the movement of the G-protein subunits. Fluorescence changes in the TR-FRET assay indicated a change in distance between the Gα subunit and Gβγ dimer. The separation of the Gα subunit and the Gβγ dimer after activation resulted in a significant decrease in TR-FRET measurement. The homogeneous set-up of the TR-FRET assay could potentially be adaptable to an array based format. This thesis describes the capture of vesicles containing functional GPCRs onto a solid substrate via the specific interaction between complementary oligonucleotides. GPCR presence and function within the immobilized vesicles, was demonstrated using fluorescent ligands. Further to this, alternative lipid hosts (to the vesicles), known as cubosomes, were introduced. When tagged with an oligonucleotide, these cubosome particles were also shown to immobilize site specifically onto a complementary oligonucleotide surface. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1369537 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

[Truncated abstract] The calcium-sensing receptor (CaR) is a G protein-coupled receptor that can respond to changes in extracellular calcium and plays an integral role in calcium homeostasis. Later studies revealed that the CaR was stimulated by not just calcium, but a diverse range of stimuli and that activation of the receptor regulated a host of different biological processes. The CaR is linked to these cellular responses via the various signalling pathways initiated by the receptor. Recent yeast two-hybrid studies have identified a number of accessory proteins that, through their interaction with the intracellular tail of the CaR, are able to regulate important functional aspects of the receptor, including its signalling and degradation. We hypothesised that many more proteins that bind to the CaR-tail await identification, especially since most of the previous studies used the yeast two-hybrid system to screen cDNA libraries generated from tissues that are important to whole body calcium homeostasis, such as the parathyroid gland and kidney. In order to identify novel binding partners of the CaR, which may affect its function, particularly in biological processes that might be unrelated to calcium homeostasis, our laboratory performed a yeast two-hybrid screen of an EMLC.1 mouse pluripotent haemopoietic cell line library using the intracellular tail of the human CaR as bait. This screen revealed a large number of

The overall aim of this thesis is to investigate strategies to aid in the measurement of G-protein coupled receptor (GPCR) activity for high-throughput screening and sensing applications. GPCRs are cell surface receptors which have seven membrane spanning domains. They are the largest family of membrane proteins in the human genome and are involved in a number of physiological and pathophysiological pathways. They are the most widely targeted protein family for therapeutics being the target for over 30% of the currently available prescription drugs (Jacoby et al. 2006). For this reason commercial interest and investment into compound screening using these receptors as targets is of high importance in lead drug discovery. Additionally, the extensive ligand range of the GPCR superfamily, which includes light, odorants/ volatiles, neurotransmitters and hormones, make them an attractive biological recognition element in biosensor applications. This thesis demonstrates the functional expression of the H1-histamine, M2-muscarinic and α₂ₐ-adrenergic receptors of the G-protein coupled receptor family, along with their associated G-proteins (Gα, Gβ and Gγ). Expression was achieved using the Sf9/baculovirus expression system. The G-proteins were successfully incorporated into an assay system using time-resolved fluorescence resonance energy transfer (TRFRET). TR-FRET was used in order to create a homogeneous assay format capable of monitoring GPCR activation through the movement of the G-protein subunits. Fluorescence changes in the TR-FRET assay indicated a change in distance between the Gα subunit and Gβγ dimer. The separation of the Gα subunit and the Gβγ dimer after activation resulted in a significant decrease in TR-FRET measurement. The homogeneous set-up of the TR-FRET assay could potentially be adaptable to an array based format. This thesis describes the capture of vesicles containing functional GPCRs onto a solid substrate via the specific interaction between complementary oligonucleotides. GPCR presence and function within the immobilized vesicles, was demonstrated using fluorescent ligands. Further to this, alternative lipid hosts (to the vesicles), known as cubosomes, were introduced. When tagged with an oligonucleotide, these cubosome particles were also shown to immobilize site specifically onto a complementary oligonucleotide surface. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1369537 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

Thesis (Ph. D.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "June 2008" Includes bibliographical references.

Thesis (M.S.)--Mississippi State University. Department of Biological Sciences. / Title from title screen. Includes bibliographical references.

Les Anthracyclines (ex : Doxorubicine (Dox)) fréquemment utilisées en chimiothérapie anticancéreuse peuvent conduire à une cardiotoxicité aboutissant à de l’insuffisance cardiaque et à une cardiomyopathie dilatée. Au niveau cellulaire, la Dox est connue pour générer un stress oxydant fort, s’intercaler directement entre les brins d’ADN, inhiber les Topoisomérase II (TopII) ou encore provoquer une détresse énergétique conduisant à la mort aussi bien des cellules tumorales que des cardiomyocytes. Néanmoins, les voies de signalisations/mécanismes moléculaires complets ne sont pas identifiés à ce jour. L’objectif de ce travail de thèse consiste donc à mieux comprendre les mécanismes de la cardiotoxicité de la Dox et à identifier de nouvelles cibles cellulaires cardio-protectrices limitant les effets cardiaques délétères de cette Anthracycline. Dans ce but, nous focalisons nos recherches sur le rôle de la protéine EPAC1, un facteur d’échange pour les petites protéines G directement activé par l’AMPc, dans la réponse des cellules cardiaques à la Dox. EPAC1 est une protéine centrale de la voie de signalisation AMPc dans le cardiomyocyte en réponse à une stimulation β-adrénergique. Or, plusieurs études ont récemment montré l’implication de certains acteurs de cette voie (Rac, RhoA) dans la cardiotoxicité induite par la Dox faisant d’EPAC1 une cible thérapeutique potentielle. Nous avons donc étudié in vitro (cultures primaires de cardiomyocytes de rat nouveau-nés (Dox 1µM)) et in vivo (souris sauvages ou invalidées pour EPAC1 (Dox, iv, 12mg/kg total)) les effets de la Dox sur l’expression et l’activité d’EPAC1 et sur les voies de signalisation qu’il régule. In vivo, les souris sauvages traitées à la Dox développent une cardiomyopathie dilatée associée à une altération de l’homéostasie calcique 15 semaines après traitement. In vitro, la Dox induit des modifications de l’expression/activité d’EPAC1 et de l’homéostasie calcique, la formation de complexes TopIIβ/ADN conduisant à des dommages à l’ADN, une dérégulation de la biogénèse et de l’activité de la chaîne respiratoire mitochondriale et finalement à l’apoptose des cardiomyocytes. L’inhibition pharmacologique (Ce3F4, Esi09) ou génétique d’EPAC1 réduit l’ensemble des dommages cellulaires in vitro et empêche le développement de la cardiomyopathie dilatée in vivo. De manière importante, nous montrons que contrairement à ce qui est observé dans les cellules cardiaques, l’inhibition d’EPAC1 augmente la toxicité de la Dox envers les cellules tumorales et en particulier envers les cellules MCF-7 issues de cancer mammaire métastatique, principale indication de la Dox. Nos résultats suggèrent donc que l’inhibition d’EPAC1 semble être une stratégie thérapeutique prometteuse dans la prévention de la cardiomyopathie induite par les traitements anticancéreux à base d’Anthracyclines. / Doxorubicin (Dox) is an Anthracycline commonly used to treat many types of cancer; unfortunately this chemotherapeutic agent often induces side effects such as cardiotoxicity leading to cardiomyocyte death and dilated cardiomyopathy (DCM). This cardiotoxicity has been related to reactive oxygen species generation, DNA intercalation, topoisomerase II inhibition and bioenergetics alterations resulting in DNA damages and ultimately in cardiomyocyte death. Nevertheless, complete molecular mechanisms are not yet identified. Therefore, there is a need for new treatment options and strategies aiming at reducing Dox side effects in the heart. Among these mechanisms, EPAC1 (Exchange Protein directly Activated by cAMP) signaling could be worth investigating as EPAC1 indirectly activates small G proteins (Rac1 and Rho A), which are known to be involved in Dox-induced cardiotoxicity. Therefore, we have investigated the effect of Dox on EPAC1 signaling in both in vivo mice model (C57BL/6 vs EPAC1 KO mice, iv injections, 12mg/kg) and in vitro model (primary culture of neonatal rat cardiomyocytes (NRVM), Dox 1μM). In vivo, Dox-treated mice developed a DCM associated with Ca2+ homeostasis dysfunction. In vitro, Dox induced DNA damages and cell death associated with huge mitochondrial disorders, characterized by a decrease in mitochondrial biogenesis and respiratory chain activity. This cell death is associated with apoptotic features including mitochondrial membrane permeabilization, caspase activation, cell size reduction and relative plasma membrane integrity. We also observed that Dox led to a modification of the protein level and the activity of EPAC1 in the same manner to the cAMP level. By contrast, the inhibition of EPAC1, prevented DNA/TopIIβ complexes, decreased Dox-induced DNA damages, loss of mitochondrial membrane potential, apoptosis and finally cardiomyocyte death. Mitochondrial biogenesis and respiratory chain activity operated normally when EPAC1 was inhibited. These results were confirmed in vivo since Dox-induced cardiotoxicity was prevented in EPAC1 KO mice as evidenced by unaltered cardiac function (no DCM) at 15 weeks post-treatment. Interestingly, the protection conferred by EPAC1 inhibition was not transferred in human cancer cell lines treated by Dox. Inhibition of EPAC1 could thus be a valuable therapeutic strategy to limit Dox-induced cardiomyopathy during cancer chemotherapy.