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The autophagy pathway is an essential component of the innate immune response, capable of rapidly targeting intracellular bacteria, which are subsequently degraded by lysosomal enzymes. Recent work has begun to elucidate the regulatory signalling for autophagy induction in response to pathogenic bacteria. However, the initial signalling regulating autophagy induction in response to the detection of pathogens remains largely unclear. Here we report that AMPK, an important upstream activator of the autophagy pathway, is rapidly stimulated upon detection of pathogenic bacteria, prior to bacterial invasion. Bacterial recognition is initially achieved through detection of outer membrane vesicles (OMVs). Additionally, we show that AMPK signalling relieves mTORC1-mediated repression of the autophagy pathway in response to Salmonella infection, positioning the cell for a rapid induction of autophagy. Surprisingly, we found that the activation of AMPK and inhibition of mTORC1 in response to extracellular Salmonella are not accompanied by an induction of bulk autophagy. However, upon Salmonella invasion AMPK signalling is required for efficient and selective targeting of bacteria-containing vesicles by the autophagy pathway through activation of pro-autophagic kinase complexes. Collectively, these results demonstrate a key role for AMPK signalling in coordinating the rapid autophagic response prior to invasion of pathogenic bacteria.

The dynamics of cell signalling are critical to cell fate decisions. Human Epidermal growth factor Receptors (HERs)-mediated Ras/Raf/MEK/ERK and PI3K/Akt signalling cascades relay extracellular signals from the plasma membrane to targets in the nucleus and cytoplasm and play pivotal roles in cell fate determination including proliferation, differentiation and cell survival. Both pathways, once activated, are further regulated by complex feedback loops which may exert either positive or negative effects on cascade components and can result in signalling oscillation. In this study, heregulin (HRG) - and epidermal growth factor (EGF)- stimulated oscillation of both p-ERK1/2 and p-Akt expression in breast cancer cell lines was demonstrated. The oscillation was cell line dependent and was observed in MCF-7 and MCF-7/HER2-18 cells but not in BT474 cells. The oscillation was augmented by cycloheximide implicating transcriptional involvement. Gene expression analysis identified 29 genes as possible candidates involved in the transcriptional feedback regulation. Apart from the feedback regulation, feedforward regulation was also observed. To expedite the analyses In-cell Western and Reverse Phase Protein Array (RPPA) assays were developed. A scheme of transcriptional feedback loops regulating the oscillation in the ERK1/2 pathway is proposed, including negative feedback loops to ERK1/2 from DUSPs, early positive and late negative feedback loops to MEK1/2 and positive feedback loops to HER-3 from AREG, HB-EGF, CYR61 and CTGF. Two HER-2-targeted inhibitory monoclonal antibodies were investigated – trastuzumab and pertuzumab. Trastuzumab not only inhibited the growth of HER-2 over-expressing MCF-7/HER2- 18 cells and BT474 cells but also that of EGF-driven MCF-7 cells which expressed low/moderate HER-2 levels. Pertuzumab blocked the growth of both MCF-7 and MCF-7/HER2-18 driven by either EGF or HRG. When used in combination with trastuzumab, pertuzumab had much more potent activity in inhibiting cell growth and signalling than either single drug. Trastuzumab and pertuzumab had opposing effects on immediate p-ERK1/2 signalling and trastuzumab’s effects on signalling could be mimicked by the PI3K inhibitor LY294002. PTPN13, a non-receptor type tyrosine protein phosphatase, is a proposed tumour suppressor in breast cancer. This was investigated as a candidate regulator of the signalling oscillation and although not observed as a transcriptional modulator of the oscillation, its high expression level was observed to be associated with cell growth inhibition in MCF-7/HER2-18 cells by trastuzumab. Moreover, immunohistochemical analysis of 121 clinical tumours which had received trastuzumab treatment revealed the correlation between the expression level of PTPN13 and the mutation status of PIK3CA. In conclusion, the observed oscillation may contribute to the elucidation of the complex regulation of signalling pathways, which is vital to the different cell fate decision made through the same core pathway. The synergy between trastuzumab and pertuzumab supports the clinical use of the combination treatment and suggested PI3K/Akt pathway as the major pathway in controlling tumour growth.

Die Fähigkeit, mit anderen Zellen zu kommunizieren, ist eine grundlegende Eigenschaft aller lebenden Zellen und essentiell für die normale Funktionsweise vielzelliger Organismen. Die Speicheldrüsen der Schmeißfliege Calliphora vicina bilden ein ausgezeichnetes physiologisches Modellsystem um zelluläre Signaltransduktionsprozesse an einem intakten Organ zu untersuchen. Die Speichelsekretion wird dabei hormonell durch das biogene Amin Serotonin (5-Hydroxytryptamin; 5-HT) reguliert. 5-HT aktiviert in den sekretorischen Zellen der Drüsen über die Bindung an mindestens zwei membranständige G-Protein gekoppelte Rezeptoren (GPCR) zwei separate Signalwege, den IP3/Ca2+- und den cAMP-Signalweg. Zur Identifizierung und Charakterisierung der 5-HT-Rezeptoren in den Speicheldrüsen von Calliphora wurden unter Anwendung verschiedener Klonierungsstrategien zwei cDNAs (Cv5-ht2α und Cv5-ht7) isoliert, die große Ähnlichkeit zu 5-HT2- und 5-HT7-Rezeptoren aus Säugetieren aufweisen. Die Hydropathieprofile der abgeleiteten Aminosäuresequenzen postulieren die für GPCRs charakteristische heptahelikale Architektur. Alle Aminosäuremotive, die für die Ligandenbindung, die Rezeptoraktivierung und die Kopplung an G-Proteine essentiell sind, liegen konserviert vor. Interessanterweise wurde für den Cv5-HT7-Rezeptor eine zusätzliche hydrophobe Domäne im N Terminus vorhergesagt. Die Cv5-HT2α-mRNA liegt in zwei alternativ gespleißten Varianten vor. Mittels RT-PCR-Experimenten konnte die Expression beider Rezeptoren in Gehirn und Speicheldrüsen adulter Fliegen nachgewiesen werden. Ein Antiserum gegen den Cv5-HT7 Rezeptor markiert in den Speicheldrüsen die basolaterale Plasmamembran. Die Expression der Rezeptoren in einem heterologen System (HEK 293-Zellen) bestätigte diese als funktionelle 5-HT Rezeptoren. So führte die Stimulation mit Serotonin für den Cv5-HT2α zu einer dosis-abhängigen Erhöhung der intrazellulären Ca2+ Konzentration ([Ca2+]i, EC50 = 24 nM). In Cv5-HT7-exprimierenden Zellen löste 5-HT dosisabhängig (EC50 = 4,1 nM) einen Anstieg der intrazellulären cAMP Konzentration ([cAMP]i) aus. Für beide heterolog exprimierten Rezeptoren wurden pharmakologische Profile erstellt. Liganden, die eine Rezeptorsubtyp-spezifische Wirkung vermuten ließen, wurden daraufhin auf ihre Wirkung auf das transepitheliale Potential (TEP) intakter Speicheldrüsenpräparate getestet. Drei 5-HT-Rezeptoragonisten: AS 19, R-(+)-Lisurid und 5-Carboxamidotryptamin führten zu einer cAMP-abhängigen Positivierung des TEP durch eine selektive Aktivierung der 5 HT7-Rezeptoren. Eine selektive Aktivierung des Ca2+-Signalweges durch den Cv5-HT2 Rezeptor ist mit Hilfe von 5-Methoxytryptamin möglich. Dagegen konnte Clozapin im TEP als selektiver Cv5-HT7-Rezeptorantagonist bestätigt werden. Die Kombination eines molekularen Ansatzes mit physiologischen Messungen ermöglichte somit die Identifikation selektiver Liganden für 5-HT2- bzw. 5-HT7-Rezeptoren aus Calliphora vicina. Dies ermöglicht zukünftig eine separate Aktivierung der 5-HT-gesteuerten Signalwege und erleichtert dadurch die weitere Erforschung der intrazellulären Signalwege und ihrer Wechselwirkungen. / Cellular communication is a fundamental property of living cells and essential for normal functioning of multicellular organisms. The salivary glands of the blowfly Calliphora vicina are a well established physiological model system to study cellular signaling in an intact organ. Fluid secretion in this gland is hormonally regulated by the biogenic amine serotonin (5-hydroxytryptamine, 5-HT). In the secretory cells, 5-HT causes a parallel activation of the InsP3/Ca2+- and the cAMP-signaling pathways through binding and stimulation of at least two G protein coupled receptors (GPCR). In order to characterize the respective 5-HT receptors on the secretory cells, we have cloned two cDNAs (Cv5-ht2α, Cv5-ht7) that share high similarity with mammalian 5-HT2 and 5-HT7 receptor classes. Analysis of the deduced amino acid sequences postulates the typical heptahelical architecture of GPCRs for both receptors. Sequence motifs that are essential for ligand binding, receptor activation and coupling to G-proteins are well conserved. Interestingly, a computer-based structural analysis of Cv5-HT7 predicts an additional eighth hydrophobic region in the N-terminus of the receptor. We also found an alternative splice variant of the Cv5-HT2α mRNA. Using RT-PCR experiments, transcripts of both receptor mRNAs could be detected in brain and salivary gland tissue. An antiserum raised against the Cv5 HT7 receptor stained the basolateral region of the salivary glands. Heterologous receptor expression in HEK 293 cells leads to a dose-dependent increase in the intracellular Ca2+-concentration ([Ca2+]i) for Cv5-HT2α (EC50 = 24 nM) and cAMP-concentration for Cv5-HT7 (EC50 = 4,1 nM) upon application of 5-HT. A pharmacological profile was established for both receptors. Ligands that appeared to act as specific ligands of either Cv5-HT2α or Cv5-HT7 in this approach, were then tested for their effect on the transepithelial potential (TEP) of intact blowfly salivary gland preparations. Three 5-HT receptor agonists: AS 19, R-(+)-lisuride and 5-carboxamidotryptamine showed a cAMP dependent positivation of the TEP, caused by a selective activation of the Cv5-HT7 receptor. 5-methoxytryptamine exclusively activates the Ca2+ pathway via Cv5-HT2α. Clozapine antagonizes the effects of 5-HT in blowfly salivary glands and was confirmed as a Cv5-HT7 antagonist. The combination of a molecular approach with physiological measurements enabled us to identify selective ligands for 5-HT2 and 5-HT7 receptors of Calliphora vicina. These results facilitate a selective activation of the intracellular signaling pathways activated by 5-HT and will facilitate future research on different aspects of intracellular signaling and crosstalk mechanisms.

Background and purpose: 5-Hydroxytryptamine (5-HT) has been shown to control and modulate many physiological and behavioural functions in insects. In this study, we report the cloning and pharmacological properties of a 5-HT1 receptor of an insect model for neurobiology, physiology and pharmacology. Experimental approach: A cDNA encoding for the Periplaneta americana 5-HT1 receptor was amplified from brain cDNA. The receptor was stably expressed in HEK 293 cells, and the functional and pharmacological properties were determined in cAMP assays. Receptor distribution was investigated by RT-PCR and by immunocytochemistry using an affinity-purified polyclonal antiserum. Key results: The P. americana 5-HT1 receptor (Pea5-HT1) shares pronounced sequence and functional similarity with mammalian 5-HT1 receptors. Activation with 5-HT reduced adenylyl cyclase activity in a dose-dependent manner. Pea5-HT1 was expressed as a constitutively active receptor with methiothepin acting as a neutral antagonist, and WAY 100635 as an inverse agonist. Receptor mRNA was present in various tissues including brain, salivary glands and midgut. Receptor-specific antibodies showed that the native protein was expressed in a glycosylated form in membrane samples of brain and salivary glands. Conclusions and implications: This study marks the first pharmacological identification of an inverse agonist and a neutral antagonist at an insect 5-HT1 receptor. The results presented here should facilitate further analyses of 5-HT1 receptors in mediating central and peripheral effects of 5-HT in insects.

The biogenic amine serotonin (5-HT) plays a key role in the regulation and modulation of many physiological and behavioural processes in both vertebrates and invertebrates. These functions are mediated through the binding of serotonin to its receptors, of which 13 subtypes have been characterized in vertebrates. We have isolated a cDNA from the honeybee Apis mellifera (Am5-ht7) sharing high similarity to members of the 5-HT7 receptor family. Expression of the Am5-HT7 receptor in HEK293 cells results in an increase in basal cAMP levels, suggesting that Am5-HT7 is expressed as a constitutively active receptor. Serotonin application to Am5-ht7-transfected cells elevates cyclic adenosine 3',5'-monophosphate (cAMP) levels in a dose-dependent manner (EC50 = 1.1-1.8 nM). The Am5-HT7 receptor is also activated by 5-carboxamidotryptamine, whereas methiothepin acts as an inverse agonist. Receptor expression has been investigated by RT-PCR, in situ hybridization, and western blotting experiments. Receptor mRNA is expressed in the perikarya of various brain neuropils, including intrinsic mushroom body neurons, and in peripheral organs. This study marks the first comprehensive characterization of a serotonin receptor in the honeybee and should facilitate further analysis of the role(s) of the receptor in mediating the various central and peripheral effects of 5-HT.

Orientador: Antonio Carlos Boschero / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T00:36:09Z (GMT). No. of bitstreams: 1 Rezende_LuizFernandode_D.pdf: 9509933 bytes, checksum: fc8e235ac3d4242d8e48c28911285e25 (MD5) Previous issue date: 2009 / Resumo: Introdução: O CNTF pertence à família da IL-6, e sendo assim, sinaliza pelo complexo receptor gp130, ativando diversas vias de sinalização dependendo do tipo celular, principalmente as vias STAT3, MAPK e PI3K. Seus efeitos incluem diferenciação e/ou sobrevivência neuronal, e é diferencialmente expresso ao longo da vida do animal. As ilhotas pancreáticas, por sua vez, são ricamente enervadas e expressam receptores para NGFs, podem apresentar respostas neurotípicas e expressam o CNTF. O objetivo desse estudo foi investigar os possíveis efeitos do CNTF sobre a diferenciação e/ou sobrevivência de ilhotas pancreáticas de ratos neonatos, qual(is) via(s) de sinalização ele ativa nessas ilhotas e como é expresso nelas ao longo da vida dos animais. Material e Métodos: Ilhotas pancreáticas de ratos neonatos (1-2 dias) foram isoladas pelo método de colagenase e cultivadas por 3 dias em meio RPMI com (CNTF) ou sem (CTL) 1nM de CNTF. Após isso, foram analisados a secreção de insulina estimulada por glicose (RIE), metabolismo (MTS, produção de NADPH), metabolismo de glicose (produção de 14CO2), expressão gênica (RTPCR), protéica (Western-Blot), atividade de caspase-3 (DEVD) e apoptose (fragmentação de DNA). Resultados: O CNTF reduziu a secreção de insulina estimulada por glicose e o metabolismo de ilhotas pancreáticas, não alterando o metabolismo de glicose e expressão de proteínas cruciais para a função das ilhotas. Por outro lado, o CNTF aumentou a expressão de proteínas relacionadas à sobrevivência das ilhotas pancreáticas, como Cx36, PAX4, e BCL-2, reduziu a atividade da caspase-3 e a apoptose das ilhotas. O CNTF também aumentou a fosforilação de STAT3, sua translocação ao núcleo e expressão de genes-alvo, como a SOCS-3, levando à redução da GSIS e sobrevivências observadas, apesar de não ativar as vias da MAPK e PI3K. Mais ainda, a expressão do CNTF é aumentada em ilhotas pancreáticas de ratos de 2 meses de idade, e assim permanecendo até os 20 meses de idade. Conclusão: O CNTF não promove a maturação de ilhotas pancreáticas, mas sim sua sobrevivência, e esses efeitos são mediados através da via JAK/STAT3, sem ativar as vias MAPK ou PI3K. Finalmente, o CNTF possui expressão diferenciada ao longo da vida do animal / Abstract: Introduction: CNTF belongs to the IL-6 cytokine family and as such, it signals through gp130 receptor complex, activating many pathways depending on the cell-type, mainly STAT3, MAPK and PI3K. Its effects include increased neuron differentiation and/or survival, and are differentially expressed throughout the animal life. Meanwhile, pancreatic islets are richly innervated and express receptors for NGFs, may undergo neurotypic responses, and express CNTF. The aim of this study was to investigate possible effects of CNTF on neonatal rat pancreatic islet differentiation and/or survival, which signalling pathway (s) it activates on pancreatic islets and how it is expressed in the pancreatic islets throughout the animal life. Methods: Pancreatic islets from neonatal rats (1-2 d old) were isolated by the collagenase method and cultured for 3 days in RPMI medium with (CNTF) or without (CTL) 1nM of CNTF. Thereafter, glucose-stimulated insulin secretion (RIA), general metabolism by (NAD(P)H production) (MTS), glucose metabolism (14CO2 production), gene (RT-PCR), protein expression (Western-Blot), caspase-3 activity (DEVD), and apoptosis (DNA fragmentation) were analysed. Results: CNTF reduced pancreatic islets GSIS and metabolism, whereas not affecting glucose metabolism and the expression of proteins crucial for the islets function. Conversely, CNTF significantly expression of proteins related pancreatic islets survival, such as Cx36, PAX4, and BCL-2, reduced caspase-3 activity and islet-cells apoptosis. CNTF also increases STAT3 fosforilation, translocation to the nuclei and expression of target genes, resulting in the reduced GSIS and survival observed, although not affecting MAPK and PI3K pathways. Moreover, CNTF expression is increased in rats pancreatic islets after 2 months of age, and it remains so until 20 months of age. Conclusion: CNTF has no effect over maturation of pancreatic islets function, whereas it improves pancreatic islets survival, and also that these effects are mediated through JAK/STAT3 but not through MAPK or PI3K pathways. Finally, CNTF is differentially expressed in rat pancreatic islets throughout the animal life / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

Made available in DSpace on 2014-06-11T19:32:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-12-22Bitstream added on 2014-06-13T20:44:20Z : No. of bitstreams: 1 miranda_et_dr_arafcf.pdf: 641942 bytes, checksum: 8d27d1b81ac16fe68d939f32e284f53f (MD5) / Universidade Estadual Paulista (UNESP) / Neste trabalho foi realizado um estudo comparativo entre isolados de Pb18 e Pb265, que têm capacidades distintas de adesão às células Hela e Vero, sob influência da temperatura e diversos fatores químicos. Foram também avaliados eventos em células de epitélio pulmonar expostas à gp 43 e células de Pb18, antes e após inoculação animal, representando, respectivamente, isolados menos e mais virulentos. Os resultados mostram que as adesinas fúngicas são termo-lábeis, sugerindo sua natureza protéica. Os açúcares aminados glucosamina e galactosamina foram os mais eficientes para inibir a adesão celular, em relação à manose, glicose e galactose, indicando que esta atividade envolve mecanismos específicos tipo lectina. O estudo com componente da matriz extracelular mostrou que a laminina, assim como seus derivados sintéticos, inibiu a adesão do isolado Pb18, fato não verificado com Pb265, tanto com células Hela, quanto com a linhagem Vero, mas variável sob influência de fatores químicos. As distintas vias de sinalização foram demonstradas pelos eventos Ras-Raf, Rho e AKT verificados, sendo que o isolado menos virulento causou menos estresse citotóxico, comprovado por menor sinal Ras- Raf e AKT de proliferação. Os sinais intensos de proliferação observados nos experimentos com o isolado mais virulento e exposição à gp 43 poderiam ser associados à maior sobrevida do fungo que se internaliza mais facilmente nas células do hospedeiro, dificultando o seu reconhecimento por células do sistema monocítico-fagocitário, antes de causar apoptose, evadir e disseminar-se. A ativação das vias de sobrevida, associada a sinais citosólicos quando o Pb entra na célula epitelial, foi demonstrada de modo inédito neste trabalho. / In this work it was accomplished a comparative study between isolates Pb18 and Pb265, which have distinct capacities of adhesion to the cells Hela and Vero under influence of temperature and several chemical factors. Events in pulmonary epithelial cells exposed to gp 43 and in Pb18 cells, before and after animal inoculation, representing, respectively, isolates less and more virulent were evaluated as well. The results show that the fungal adhesins are term-labels, suggesting its proteical nature. The aminated sugars glucosamine and galactosamine were the most efficient, in comparison with manose, glycose and galactose, in inhibiting the cellular adhesion, indicating that this activity involves specific mechanisms lectin-type. The study with extracellular matrix component indicated that the laminin, as well as its synthetic derivatives, inhibited the adhesion of the isolate Pb18 to the cells Vero and Hela, what did not occur with Pb265, but it was variable under influence of chemical factors. The distinct signaling pathways were demonstrated by the Ras-Raf, Rho and AKT events, considering that the less virulent isolate caused minor cytotoxic stress, proved by minor Ras-Raf and AKT proliferation signals. The intense proliferation signals observed in the experiments with more virulent isolate and exposition to gp 43 could be associated to larger time of survival of the fungus that more easily goes inside the host cells, making it difficult to the monocyte-macrophage system cells to recognize it before the apoptosis, the evasion and the dissemination. The Ras-Raf and AKT pathways acted synergetically with the effects of the cell survival. The decrease of the AKT event implied partial loss of the survival signal that intensified, following and in a opposite way, with Ras-Raf decrease. The activation of the survival pathways, associated to citosolic signals when Pb goes inside the epithelial cell, was for the first time demonstrated in this work.

A range of biochemically diverse molecules located in the plasma membrane— such as proteins, glycans, and lipids—mediate cellular recognition events, initiation of signalling pathways, and the regulation of processes important for the normal development and function of multicellular organisms. Interactions mediated by cell surface receptors can be challenging to detect in biochemical assays, because they are often highly transient, and membrane-embedded receptors are difficult to solubilise in their native conformation. The biochemical features of low-affinity extracellular protein interactions have therefore necessitated the development of bespoke methods to detect them. Here, I develop a genome-scale cell-based genetic screening approach using CRISPR-Cas9 knockout technology that reveals cellular pathways required for specific cell surface recognition events. Using a panel of high-affinity monoclonal antibodies, I first establish a method from which I identify not only the direct receptor but also other required gene products, such as co-receptors, post-translational modi cations, and transcription factors contributing to antigen expression and subsequent antibody-antigen recognition on the surface of cells. I next adapt this method to identify cellular factors required for receptor interactions for a panel of recombinant proteins corresponding to the ectodomains of cell surface proteins to the endogenous surface receptors present on a range of cell lines. In addition to finding general cellular features recognised by many ectodomains, I also identify direct interaction partners of recombinant protein probes on cell surfaces together with intracellular genes required for such associations. Using this method, I identify IGF2R as a binding partner for the R2 subunit of GABAB receptors, providing a mechanism for the internalisation and regulation of GABAB receptor signalling. The results here demonstrate that this single approach can identify the molecular nature and cell biology of surface receptors without the need to make any prior assumptions regarding their biochemical properties.