ORCID Profile
0000-0002-3457-3320
Current Organisations
Universidade Federal de Minas Gerais
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INSERM
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Publisher: Mary Ann Liebert Inc
Date: 08-2008
Abstract: The inflammatory response of macrophages to infectious agents is a highly dynamic and orchestrated process involving the release of a variety of inflammatory mediators, including interleukin-12 (IL-12), as a consequence of the recognition of the pathogens. Regulation of IL-12 gene expression by the anti-inflammatory cytokine IL-10 represents a major homeostatic process underlying host-pathogen and host-self interactions. Our group first reported that the Th2-specific transcription factor c-Maf is expressed also in macrophages treated with lipopolysaccharide (LPS) and IL-10. When overexpressed, c-Maf can potently suppress IL-12 production. However, c-Maf does not appear to be a physiologic regulator of IL-12p40 gene transcription because p40 production is not dysregulated in c-Maf-deficient macrophages. In this study, we investigated the role of c-Maf in regulation of the transcription of the p35 gene, which encodes the chain that is rate limiting in the synthesis of the heterodimeric IL-12. We report that c-Maf is a physiologic modulator of IL-12p35 gene expression and IL-12p70 production. We identify a novel NF-kappaB element within the proximal p35 promoter and show that c-Maf inhibits p35 transcription by antagonizing the effects of NF-kappaB, especially c-Rel, on p35 activation. It does so not by directly interacting with the target DNA but by interfering with the nuclear localization of NF-kappaB c-Rel. This study contributes to our understanding of the molecular basis of the homeostatic regulation of IL-12 production by c-Maf, which plays a dual role both in the function of antigen-presenting cells (APCs) and in T helper cell differentiation.
Publisher: Mary Ann Liebert Inc
Date: 04-2008
Abstract: Synthetic small interfering RNAs (siRNAs) can trigger a strong innate immune response in mammalian cells. This nonspecific side effect may hinder the application of siRNAs as tools in gene silencing. Chemically synthesized siRNAs, including traditional 19-mers with 2-nt 3' overhangs, longer duplexes with blunt or 3' overhangs, and asymmetric duplexes with a blunt end and a 2-nt 3' overhang, can evoke strong dose-dependent interferon-alpha (IFN-alpha) and tumor necrosis factor-alpha (TNF-alpha) release in human peripheral blood mononuclear cells (PBMCs). This response is independent of retinoic acid-inducible gene I but may involve endosomal toll-like receptors (TLRs). The immunostimulatory effect of the siRNAs is directly related to either or both of the strands of the duplex in a sequence-dependent manner. However, although some single-stranded RNAs and siRNAs potently evoked both IFN-alpha and TNF-alpha induction, these responses were not always coupled. In accordance with this, specific chemical modifications differentially altered cytokine production, suggesting recruitment of different TLRs in a sequence-dependent manner.
Publisher: Microbiology Society
Date: 11-2008
DOI: 10.1099/VIR.0.2008/003558-0
Abstract: Viral infection of mammalian cells prompts the innate immune system to initiate an antiviral response. The recognition of the virus triggers several antiviral signalling pathways, which among others include the family of 2′-5′ oligoadenylate synthetase (OAS) proteins. The p59 protein encoded by the OAS-like ( OASL ) gene is an atypical member of the OAS family in the sense that it lacks the characteristic 2′-5′ oligoadenylate synthetase activity. We decided to investigate the putative antiviral activity of p59 by ectopically expressing this protein in Vero cells and then infecting these cells with virus. We demonstrate that OASL has an antiviral effect against the single-stranded RNA virus picornavirus, encephalomyocarditis virus, but not against a large DNA virus, herpes simplex virus 1. Importantly, this antiviral activity was lost in a truncated version of p59 lacking the ubiquitin-like C-terminal domain of p59. Taken together our results indicate that p59 is indeed an antiviral protein that works through a novel mechanism distinct from other OAS proteins.
Publisher: Springer Science and Business Media LLC
Date: 05-01-2023
Publisher: American Society for Microbiology
Date: 09-2005
DOI: 10.1128/JVI.79.17.11105-11114.2005
Abstract: p53 has been well characterized as a tumor suppressor gene, but its role in antiviral defense remains unclear. A recent report has demonstrated that p53 can be induced by interferons and is activated after vesicular stomatitis virus (VSV) infection. We observed that different nononcogenic viruses, including encephalomyocarditis virus (EMCV) and human parainfluenza virus type 3 (HPIV3), induced down-regulation of p53 in infected cells. Double-stranded RNA (dsRNA) and a mutant vaccinia virus lacking the dsRNA binding protein E3L can also induce this effect, indicating that dsRNA formed during viral infection is likely the trigger for down-regulation of p53. The mechanism of down-regulation of p53 by dsRNA relies on translation inhibition mediated by the PKR and RNase L pathways. In the absence of p53, the replication of both EMCV and HPIV3 was retarded, whereas, conversely, VSV replication was enhanced. Cell cycle analysis indicated that wild-type (WT) but not p53 knockout (KO) fibroblasts undergo an early-G 1 arrest following dsRNA treatment. Moreover, in WT cells the onset of dsRNA-induced apoptosis begins after p53 levels are down-regulated, whereas p53 KO cells, which lack the early-G 1 arrest, rapidly undergo apoptosis. Hence, our data suggest that the down-regulation of p53 facilitates apoptosis, thereby limiting viral replication.
Publisher: Informa UK Limited
Date: 2006
DOI: 10.2147/NANO.2006.1.2.155
Abstract: Small interfering RNA molecules (siRNA) hold great promise to specifically target cytoprotective factors to enhance cancer therapy. Like antisense RNA strategies, however, the use of siRNA is limited because of in vivo instability. As a first step to overcome delivery issues, a series of graft copolymers of polyethylene glycol and polyethylenimine (PEI-g-PEG) were synthesized and investigated as nontoxic carriers for delivery of siRNA targeting the signaling peptide of secretory clusterin (sCLU), a prosurvival factor that protects cells from ionizing radiation (IR) injury, as well as chemotherapeutic agents. Three copolymers with different PEG grafting densities were tested for their abilities to bind and form nanocomplexes with siRNA. A copolymer composed of 10 PEG grafts (2 kDa each) per PEI polymer (2k10 copolymer) gave the highest binding affinity to siRNA by ethidium bromide exclusion assays, and had the smallest nanocomplex size (115 +/- 13 nm diameter). In human breast cancer MCF-7 cells, 2k10-siRNA-sCLU nanocomplexes suppressed both basal as well as IR-induced sCLU protein expression, which led to an over 3-fold increase in IR-induced lethality over 2k10-siRNA scrambled controls. In summary, this study demonstrates the proof-of-principle in using nanoparticle-mediated delivery of specific siRNAs to enhance the lethality of IR exposure in vitro, opening the door for siRNA-mediated knockdown of specific cytoprotective factors, such as DNA repair, anti-apoptotic, free radical scavenging, and many other proteins.
Publisher: Wiley
Date: 04-2010
Publisher: Springer Science and Business Media LLC
Date: 30-04-2006
DOI: 10.1038/NBT1205
Abstract: Nonspecific effects triggered by small interfering RNAs (siRNAs) complicate the use of RNA interference (RNAi) to specifically downregulate gene expression. To uncover the basis of these nonspecific activities, we analyzed the effect of chemically synthesized siRNAs on mammalian double-stranded RNA (dsRNA)-activated signaling pathways. siRNAs ranging from 21 to 27 nucleotides (nt) in length activated the interferon system when they lacked 2-nt 3' overhangs, a characteristic of Dicer products. We show that the recognition of siRNAs is mediated by the RNA helicase RIG-I and that the presence of 3' overhangs impairs its ability to unwind the dsRNA substrate and activate downstream signaling to the transcription factor IRF-3. These results suggest a structural basis for discrimination between microRNAs that are endogenous Dicer products, and nonself dsRNAs such as by-products of viral replication. These findings will enable the rational design of siRNAs that avoid nonspecific effects or, alternatively, that induce bystander effects to potentially increase the efficacy of siRNA-based treatments of viral infections or cancer.
Publisher: Cold Spring Harbor Laboratory
Date: 05-06-2021
DOI: 10.1101/2021.06.05.447047
Abstract: Aedes aegypti and Aedes albopictus are major mosquito vectors for arthropod-borne viruses (arboviruses) such as dengue (DENV) and Zika (ZIKV) viruses. Mosquitoes also carry insect-specific viruses (ISVs) that may affect the transmission of arboviruses. Here, we analyzed the global virome in urban Aedes mosquitoes and observed that two insect-specific viruses, Phasi Charoen-like virus (PCLV) and Humaita Tubiacanga virus (HTV), were the most prevalent in A. aegypti worldwide except for African cities, where transmission of arboviruses is low. Spatiotemporal analysis revealed that presence of HTV and PCLV led to a 200% increase in the chances of having DENV in wild mosquitoes. In the laboratory, we showed that HTV and PCLV prevented downregulation of histone H4, a previously unrecognized proviral host factor, and rendered mosquitoes more susceptible to DENV and ZIKV. Altogether, our data reveals a molecular basis for the regulation of A. aegypti vector competence by highly prevalent ISVs that may impact how we analyze the risk of arbovirus outbreaks.
Publisher: Springer Science and Business Media LLC
Date: 11-2005
DOI: 10.1038/NBT1161
Abstract: Inhibition of gene expression through RNA interference (RNAi) is emerging as a powerful experimental tool for gene function and target validation studies. The potential uses of this technology seem unlimited, extending to the prevention and therapy of human diseases. However, recent work demonstrating that there are unanticipated, different nonspecific effects associated with the use of small interfering RNAs in mammals has raised concerns about the safe use of RNAi in vivo. These nonspecific effects include activation of the immune system, potentially harming the in idual. The application of screening assays for nonspecific activation of both innate and acquired immunity will be necessary for further development of RNAi as a therapeutic tool.
Publisher: Elsevier BV
Date: 03-2006
DOI: 10.1016/J.BBAMEM.2006.01.003
Abstract: Small interfering RNA (siRNA) is widely recognized as a powerful tool for targeted gene silencing. However, siRNA gene silencing occurs during transfection, limiting its use is in kinetic studies, deciphering toxic and off-target effects and phenotypic assays requiring temporal, and/or spatial regulation. We developed a novel controllable siRNA (csiRNA) that is activated by light. A single photo removable group is coupled during oligonucleotide synthesis to the 5' end of the antisense strand of the siRNA, which blocks the siRNA's activity. A low dose of light activates the siRNA, independent of transfection resulting in knock down of specific target mRNAs and proteins (GAPDH, p53, survivin, hNuf2) without stimulating non-specific effects such as regulated protein kinase PKR and induction of the interferon response. We demonstrate survivin and hNuf2 csiRNAs temporally knockdown their mRNAs causing multinucleation and cell death by mitotic arrest, respectively. Furthermore, we demonstrate a dose-dependent light regulation of hNuf2 csiRNA activity and resulting phenotype. The light controllable siRNAs are introduced into cells using commercially available reagents including the MPG peptide based delivery system. The csiRNAs are comparable to standard siRNAs in their transfection efficiency and potency of gene silencing. This technology should be of interest for phenotypic assays such as cell survival, cell cycle regulation, and cell development.
No related grants have been discovered for João Trindade Marques.