Submitted by Alessandra Portugal (alessandradf@ioc.fiocruz.br) on 2013-10-01T21:53:22Z No. of bitstreams: 1 Carolina da Paz Zampier.pdf: 2134379 bytes, checksum: d822f378a88e2cf44b02c5ea60f52ea0 (MD5) / Made available in DSpace on 2013-10-01T21:53:22Z (GMT). No. of bitstreams: 1 Carolina da Paz Zampier.pdf: 2134379 bytes, checksum: d822f378a88e2cf44b02c5ea60f52ea0 (MD5) Previous issue date: 2012 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A caveolina-1 (Cav-1), uma proteína essencial para a formação de cavéolas, apresenta atividade na modulação da sinalização intracelular. Cav-1 é capaz de interagir com diversas proteínas através de seu domínio CSD (caveolin scaffolding domain) e, em geral, essa interação leva à inibição das proteínas associadas. O peptídeo CSD tem sido utilizado como um mimético de Cav-1 em relação à sua capacidade modulatória sobre a atividade de outras proteínas. Recentemente, tem sido mostrado que Cav-1 é capaz de modular a resposta inflamatória em diversos aspectos. Neste trabalho, examinamos o papel de Cav-1 na regulação da síntese de mediadores inflamatórios por macrófagos. O lipopolissacarídeo (LPS) de E.coli, um protótipo de estímulo inflamatório, foi capaz de induzir a expressão de Cav-1 e Cox-2 em macrófagos peritoneais in vitro. Estas proteínas são induzidas em um curso temporal semelhante, sendo detectadas por Western blot a partir de 3h com níveis de expressão crescentes até 18h. Por imunofluorescência, observamos que Cav-1 e Cox-2 apresentam um padrão de expressão mutualmente exclusivo em macrófagos estimulados com LPS. Mostramos por Western blot que a expressão de Cox-2 é induzida por LPS e que o tratamento com CSD leva à inibição da expressão de Cox- 2, mas não de Cox-1. Observamos, também, a redução parcial dos níveis de PGE2 no sobrenadante de macrófagos estimulados com LPS e tratados com CSD. O tratamento com o peptídeo CSD também foi capaz de reduzir os níveis de IL-1, IL- 6, e IL-12 induzidos por LPS. O LPS induz o aumento da expressão e fosforilação de STAT-1. A fosforilação de STAT-1 foi diminuída após o tratamento com CSD, indicando que Cav-1 modula negativamente a ativação de STAT-1. Estudos posteriores são necessários para complementar os dados obtidos até o momento para esclarecer os mecanismos de modulação da síntese de mediadores inflamatórios por Cav-1. Em conclusão, Cav-1 apresenta uma atividade inibitória sobre a expressão de Cox-2 e produção dos mediadores inflamatórios PGE2, IL1, IL-6, e IL-12 em macrófagos estimulados com LPS in vitro. O mecanismo de inibição possivelmente envolve inibição da ativação de STAT-1. / Caveolin-1 (Cav-1), a protein essential for the formation of caveolae, shows activity in the modulation of intracellular signaling. Cav-1 can interact with several proteins by its caveolin scaffolding domain (CSD) and, in general, this interaction leads to inhibition of associated proteins. The peptide CSD has been used as a Cav- 1 mimetic in relation to its capacity on the modulatory activity of other proteins. Recently, it has been shown that Cav-1 can modulate the inflammatory response in several respects. We examined the role of Cav-1 in regulating the synthesis of inflammatory mediators by macrophages. Lipopolysaccharide (LPS) from E. coli, a prototype of inflammatory stimulus, was able to induce the expression of Cav-1 and Cox-2 in peritoneal macrophages in vitro. These proteins are induced in a similar time course, being detected by Western blot at 3 hours with increasing levels of expression up to 18 hours. By immunofluorescence, we observed that Cav-1 and Cox-2 have a mutually exclusive pattern of expression in macrophages stimulated with LPS. Western blot analysis showed that the expression of Cox-2 is induced by LPS and that treatment with CSD leads to inhibition of Cox-2 but not Cox-1. We also observed the partial reduction of PGE2 levels in supernatants of macrophages stimulated with LPS and treated with CSD. Treatment with CSD peptide was also able to reduce the levels of IL1, IL-6 and IL-12 induced by LPS. LPS induces increased expression and phosphorylation of STAT-1. The phosphorylation of STAT- 1 was decreased after treatment with the CSD, indicating that a Cav-1 negatively modulates activation of STAT-1. Further studies are needed to supplement the data obtained so far to clarify the mechanisms of modulation of the synthesis of inflammatory mediators by Cav-1. In conclusion, Cav-1 shows an inhibitory activity on Cox-2 expression and production of the inflammatory mediators PGE2, IL1β, IL-6 and IL-12 in macrophages stimulated with LPS in vitro. The mechanism of inhibition possibly involves inhibition of STAT-1 activation.

Skeletal muscle associated disorders are correlated with significant morbidity, including frailty, fatigue, reduced mobility and poor resistance to treatments as well as mental health repercussions resulting from a loss of independence. Thus, conditions affecting skeletal muscle put considerable pressure on the health care system. In response to injury, skeletal muscle can regenerate and the molecular mechanisms underlying this unique process has been the subject of intense research with the goal of developing better treatment modalities for muscle-related diseases. Our laboratory has previously demonstrated that C/EBPβ is a negative regulator of postnatal myogenic differentiation. Expressed in muscle satellite cells (MuSCs), the primary source of regenerative potential in skeletal muscle, C/EBPβ inhibits entry into the myogenic differentiation program and is required for MuSC self-renewal after injury. Despite the important role of C/EBPβ in muscle homeostasis, little is known about the genes it regulates. To better understand how C/EBPβ regulates these processes, I used both a candidate-based approach to identify the inhibitor of DNA binding and differentiation protein ID3 as a C/EBPβ target gene that mediates inhibition of myogenic differentiation, and an unbiased approach using RNA-seq. I compared gene expression profiles from C2C12 myoblasts overexpressing C/EBPβ to control cells under growth and differentiation conditions. I observed that more than 20% of the molecular signature found in quiescent MuSCs is regulated by C/EBPβ. Caveolin- 1 was implicated as a direct target of C/EBPβ and part of the molecular mechanism by which C/EBPβ maintains MuSCs quiescence. Interestingly, the RNA-seq data identified numerous C/EBPβ-regulated secreted proteins including growth factors and cytokines. Co-culture experiments indicate that secreted proteins mediate the inhibition of cell differentiation and fusion, suggesting that C/EBPβ functions in an autocrine and paracrine fashion to influence activation of myoblasts in the absence of cell-to-cell contact. Given the role of C/EBPβ in regulating secretory proteins that inhibit myogenic differentiation, I examined the requirement of C/EBPβ in the expression of anti myogenic proteins secreted by cancer cells that affect MuSCs function and drive the development of cancer cachexia. Indeed, the expression of C/EBPβ in cancer cells was found to be required for the production of a cachexia-inducing secretome by tumours in vitro and in vivo. Furthermore, C/EBPβ was found to be sufficient to convert non-cachectic tumours into cachexia-inducing ones. In comparing differentially expressed C/EBPβ-regulated secreted protein transcripts from our RNA-seq data to that from 27 different types of human cancers revealed an ~18% similarity between C/EBPβ-regulated secreted proteins and those enriched in cachectic tumours including pancreatic, gastric and brain cancers. Three of these C/EBPβ-regulated secreted proteins (SERPINF1, TNFRSF11B and CD93) were tested further and found to be inducers of muscle atrophy. This work provides molecular insight into the role of C/EBPβ in the regulation of MuSC function and the regulation of cachexia-inducing factors by tumours, placing C/EBPβ as a novel therapeutic target for the treatment of cancer cachexia and other muscle-related diseases.

The most active form of Vitamin D, 1alpha,25(OH)2D3, modulates cells via receptor mediated mechanisms. While studies have elucidated the pathway via the classical nuclear Vitamin D Receptor (VDR), little is known about the membrane-associated Vitamin D Receptor (ERp60). Caveolae and its characteristic protein Caveolin-1 have been involved in many signaling pathways due to its specific structure and physical configuration. Other studies have shown that many components of the Vitamin D pathway have been found in caveolae. This study hypothesizes that caveolae and Caveolin-1 are important for the effects of 1,25 Vitamin D signaling via ERp60. Research up to date have shown that in rat and mouse growth zone chondrocytes, cells deprived of intact caveolae either through disruption through beta-Cyclodextrin or genetic knockout do not exhibit the characteristic responses to Vitamin D through ERp60 when compared to chondrocytes with functional caveolae. Studies using immunofluorescence co-localization and caveolae fractionation have shown that ERp60 is localized in the caveolae domains. Cellular fractionation was also performed to examine the localization of the ERp60 receptor in lipid rafts and caveolae. Histology and transmission electron microscopy were also used to examine the physiological importance of caveolae and Caveolin-1 in growth plate morphology and cellular characteristics.

(1) Background: Antibody testing is commonly used to assess a dog’s immune status. For detection of antibodies against canine adenoviruses (CAVs), one point-of-care (POC) test is available. This study assessed the POC test´s performance. (2) Methods: Sera of 198 privately owned dogs and 40 specific pathogen-free (SPF) dogs were included. The reference standard for detection of anti-CAV antibodies was virus neutralization (VN) using CAV-1 and CAV-2 antigens. Specificity, sensitivity, positive predictive value (PPV), negative predictive value (NPV), and overall accuracy (OA) of the POC test were assessed. Specificity was considered most important. (3) Results: Prevalence of CAV-1 neutralizing antibodies (≥10) was 76% (182/238) in all dogs, 92% (182/198) in the subgroup of privately owned dogs, and 0% (0/40) in SPF dogs. Prevalence of CAV-2 neutralizing antibodies (≥10) was 76% (181/238) in all dogs, 91% (181/198) in privately owned dogs, and 0% (0/40) in SPF dogs. Specificity for detection of CAV-1 antibodies was lower (overall dogs, 88%; privately owned dogs, 56%; SPF dogs, 100%) compared with specificity for detection of CAV-2 antibodies (overall dogs, 90%; privately owned dogs, 65%; SPF dogs, 100%). (4) Conclusions: Since false positive results will lead to potentially unprotected dogs not being vaccinated, specificity should be improved to reliably detect anti-CAV antibodies that prevent infectious canine hepatitis in dogs.