Laboratório de indicadores de Governança Pública: uma proposta para mensurar a efetividade dos gastos na Segurança Pública Municipal

Anais do 35º Seminário de Extensão Universitária da Região Sul - Área temática: EducaçãoPressões por maior transparência e accountability tem sido o mote de muitas mudanças no setor público. No entanto, parece existir uma dificuldade de colocar tais conceitos em prática na área de segurança pública. Este trabalho apresenta algumas iniciativas do Laboratório de Indicadores de Governança Pública, do CESFI-UDESC, na criação de indicadores de efetividade dos gastos dos municípios do Estado de Santa Catarina, em segurança pública. São apresentados no trabalho o que foi feito até o momento e quais os desafios na mensuração das ações de políticas públicas para esta ár

The constitutive activity of the ghrelin receptor attenuates apoptosis via a protein kinase C-dependent pathway

The ghrelin receptor (GHS-R1a) displays a high level of constitutive signaling through a phospholipase C/protein kinase C-dependent pathway. Therefore, we have investigated the role of agonist-dependent and agonist-independent signaling of GHS-R1a in apoptosis using the seabream GHS-R1a stably expressed in human embryonic kidney 293 cells (HEK-sbGHS-R1a cells). Cadmium-induced activation of caspase-3 was significantly attenuated in HEK-sbGHS-R1a cells compared to wild-type HEK293 cells, while the apoptotic responses to the protein kinase C inhibitor staurosporine were similar. GHS-R1a ligands had no effect on caspase-3 activation or on cell proliferation. Concentrations of the inverse agonist [d-Arg1,d-Phe5,d-Trp7,9,Leu11]-substance P sufficient to inhibit constitutive inositol phosphate generation did not enhance caspase-3 activity, suggesting a possible role of phosphatidylcholine-specific phospholipase C in the anti-apoptotic activity of GHS-R1a. In conclusion, our data suggests that the constitutive activity of sbGHS-R1a may be sufficient alone to attenuate apoptosis via a protein kinase C-dependent pathway. © 2008 Elsevier Ireland Ltd. All rights reserved.Link_to_subscribed_fulltex

Over-expression of the truncated ghrelin receptor polypeptide attenuates the constitutive activation of phosphatidylinositol-specific phospholipase C by ghrelin receptors but has no effect on ghrelin-stimulated extracellular signal-regulated kinase 1/2 activity

In addition to regulating growth hormone release from the pituitary, ghrelin receptors also influence cell proliferation and apoptosis. By studying mitogen-activated protein kinase activity in human embryonic kidney 293 cells over-expressing ghrelin receptors, we aimed to identify the specific cell signalling pathways used by ghrelin receptors, and to determine if the truncated ghrelin receptor polypeptide had any influence on the functional activity of ghrelin receptors. We found that ghrelin activated extracellular signal-regulated kinases 1/2 with an EC50 value of 10 nM, and that this response was inhibited by the ghrelin receptor antagonists d-Lys(3)-GHRP-6 and [d-Arg1,d-Phe5,d-Trp7,9,Leu11 ]-substance P. Ghrelin had little or no effect on the activity of c-Jun N-terminal kinase, p38 kinase or Akt. Ghrelin appeared to activate extracellular signal-regulated kinases 1/2 through a calcium-independent novel protein kinase C isoform which may utilize diacylglycerol derived from hydrolysis of phosphatidylcholine rather than from phosphatidylinositol. Ghrelin-stimulated extracellular signal-regulated kinases 1/2 activity was independent of transactivation of epidermal growth factor receptors, and even when ghrelin receptor internalization was blocked by concanavalin A or a β-arrestin mutant, there was no decrease in phosphorylated extracellular signal-regulated kinases 1/2, suggesting this is a G protein-dependent process. The truncated ghrelin receptor polypeptide had no effect on ghrelin receptor signalling to extracellular signal-regulated kinases 1/2, but decreased the constitutive activation of phosphatidylinositol-specific phospholipase C by ghrelin receptors. In conclusion, our results suggest that any up-regulation of the truncated ghrelin receptor polypeptide might preferentially attenuate functional activity dependent on the constitutive activation of ghrelin receptors, while leaving ghrelin-dependent signalling unaffected. © 2006 Elsevier Ltd. All rights reserved.Link_to_subscribed_fulltex

The truncated ghrelin receptor polypeptide (GHS-R1b) acts as a dominant-negative mutant of the ghrelin receptor

The dimerization properties of the ghrelin receptor (GRLN-R) and its non-signalling, naturally occurring, truncated splice variant (GHS-R1b) have been investigated in human embryonic kidney 293 cells heterologously expressing these proteins. Using the techniques of bioluminescence resonance energy transfer and co-immunoprecipitation, we detected the formation of GRLN-R homodimers and GRLN-R/GHS-R1b heterodimers, but ghrelin-induced conformational changes were only detected in the GRLN-R homodimers. When the expression of GHS-R1b exceeded that of GRLN-R, there was a decrease in the cell surface expression of GRLN-R with a consequent decrease in constitutive activation of phosphatidylinositol-specific phospholipase C (PI-PLC). Furthermore, there was no change in ghrelin affinity, and the efficacy of cell signalling as measured by stimulation of PI-PLC and extracellular signal-regulated kinase 1/2 was unchanged. Cellular localization studies suggest that GRLN-R is normally distributed between the plasma membrane and cytosolic fractions, but in the presence of GHS-R1b, GRLN-R is localized to the nucleus. Therefore, we propose that the decrease in GRLN-R constitutive signalling was due to translocation of GRLN-R to the nucleus due to the formation of GRLN-R/GHS-R1b heterodimers. Therefore, GHS-R1b appears to act as a dominant-negative mutant of the full-length GRLN-R. © 2006 Elsevier Inc. All rights reserved.Link_to_subscribed_fulltex

Dynamic nucleolar localisation of miR-484.

<p>(A) ISH of miR-484 and U3 in HeLa cells 72 hrs after transfection with a scrambled siRNA. (B) Lung cancer cells A549 are infected with influenza A/Duck/HongKong/Y280/97 (H9N2) strain (MOI = 2) for 1 hr or 5 hrs, respectively. The successful infection of influenza A virus is indicated by viral NP staining. (C) LMB decreases cytoplasm miR-484. The HeLa cells are incubated with or without LMB (20 nM) for 6 hrs, and then fixed for ISH staining. SC35 is a nuclear speckle marker used to confirm the effect of LMB. Cell nuclei are stained with Hoechst 33258 (Blue). Scale bars: 10 µm.</p

Nucleolar miRNAs are a widespread phenomenon.

<p>(A) Localisation of miR-193b, miR-484 and miR-574-3p is examined in lung cancer cells H1299, liver cancer cells Huh7, RPE cells, human adult fibroblast AG06858 and primary mouse adult fibroblast. (B) Intracellular distribution of miR-484 (red) is detected in the HeLa cells 8 hours after exposure to UV (100 J/m²), or treated with ActD (5 µg/ml, 2 hrs), or DRB (25 µg/ml, 2 hrs). PSP1 (green) is used to confirm the effect of the treatments. The cell nuclei are counterstained with Hoechst 33258 (blue). Scale bars: 10 µm.</p

The intracellular dynamics of miRNAs.

<p>The intracellular dynamics of miRNAs.</p

Nucleolar location of mature miR-191, miR-193b, miR-484 and miR-574-3p in HeLa cells.

<p>(A) HeLa cells are co-hybridised with DIG-labeled miRNA probes (red) and an FITC labelled probe for U3 snoRNA (green). Four nucleolar microRNAs; miR-191, miR-193, miR-484, miR-574-3p, are examined for their nucleolar location. Cytoplasmic microRNA control miR-20a and a scrambled negative (f) control are also included. The nuclei are stained with Hoechst 33258 (blue). (B) Line profiles of the fluorescence signals of miR-484 (red) and U3 snoRNA (green) in the nucleolus. (C) Micronucleated RPE cells labelled with probes for miR-484 (red) and U3 (green). The micronuclei are stained with Hoechst 33258 (blue). The yellow box highlights a micronucleus without a nucleolus. The red box shows a micronucleus with a nucleolus, but with no miR-484 staining. Scale bars: 10 µm.</p