Abstract
Some multifunctional cellular proteins, as the monocyte chemotactic protein-induced protein 1 (MCPIP1) and the cyclin-dependent kinase inhibitor p21, have also shown to be able to modulate the cellular susceptibility to the human immunodeficiency virus type 1 (HIV-1). Several studies described that p21 is expressed at high levels ex vivo in cells from individuals who naturally control HIV-1 replication (HIC). The expression level of MCPIP1 in HIC was never described before, but a recent study in a model of renal carcinoma cells showed that MCPIP1 overexpression was associated with an increase of both p21 transcripts and proteins levels. Here, we explored the potential associations between MCPIP1 and p21 expression, as well as with cellular activation in HIC, sustaining undetectable (elite controllers – EC) or low (viremic controllers – VC) viral loads. We found a selective upregulation of MCPIP1 and p21 mRNA levels in PBMC from HIC compared with both ART– suppressed and HIV–negative control groups (P ≤ 0.02) and a strong positive correlation (r ≥ 0.57; P ≤ 0.014) between expressions of both transcripts independently of the VL, treatment condition and HIV status. The mRNA levels of p21, but not of MCPIP1, were positively correlated with activated CD4+ T cells levels in HIC and EC (r ≥ 0.53; P ≤ 0.017). In relation to the monocyte activation, the mRNA levels of both p21 (r = 0.74; P = 0.005) and MCPIP1 (r = 0.58; P = 0.040) were positively correlated with plasmatic levels of sCD14 only in EC. Multivariate analysis confirmed the association between MCPIP1 and p21 mRNA levels, and between the latter with the frequency of activated CD4+ T cells. These data show for the first time the simultaneous overexpression and positive correlation of MCPIP1 and p21 transcripts in the setting of natural suppression of HIV-1 replication in vivo. The positive correlation between MCPIP1 and p21 transcripts supports a common regulatory pathway connecting these multifunctional host factors and a possible synergistic effect on HIV-1 replication control. Pharmacological manipulation of these cellular proteins may open novel therapeutic perspectives to prevent HIV-1 replication and disease progression.
1 Introduction
Among the individuals infected by the human immunodeficiency virus type 1 (HIV-1), a rare group called HIV controllers (HIC) suppress viral replication in absence of antiretroviral therapy, maintaining RNA viral loads (VL) below the limit of detection (LOD) (elite controllers, EC) or at low levels (> LOD and < 2,000 copies/ml; viremic controllers, VC). Natural control of HIV-1 replication is probably a multifactorial feature that involves different combinations of host and/or viral factors (1).
Some intrinsic host proteins, termed restriction factors (RF), are components of the innate immune response (2,3) that have the ability to cause a significant reduction in viral infectivity by interacting directly with the pathogen and are generally induced by interferon (IFN), hence being known as IFN-stimulated genes (ISGs) (4). Several RF has been shown to limit HIV replication in vitro at different stages of its life cycle (3), including some classical RF such the Apolipoprotein B mRNA-Editing enzyme, Catalytic polypeptide-like (APOBEC3G), the Bone Stromal Tumor protein 2 (BST2)/Tetherin, and the Sterile Alpha Motif domain and HD domain-containing protein 1 (SAMHD1) (2), and others more recently characterized like the Myxovirus resistance protein 2 (Mx2), the Interferon-inducible transmembrane family proteins (IFITM1-3 members) and Schlafen 11 (SLFN11) (3). The mRNA levels of some RF including SAMHD1, Theterin, IFITM1, Mx2 and SLFN11 have been described to be elevated in peripheral blood mononuclear cells (PBMC) or CD4+ T cells of HIC compared to antiretroviral (ART)-suppressed and/or HIV-uninfected individuals (5–9), although with contrasting findings across different HIC cohorts.
Others host multifunctional proteins, not recognized as classical RF, are also able to modulate the cellular susceptibility to HIV-1 infection. The cyclin-dependent kinase (CDK) inhibitor p21, encoded by the CDKN1A gene, modulates multiple relevant processes of the immune system, including proliferation of activated/memory T cells, macrophage activation and inflammation (10–17). This protein also indirectly limits the HIV-1 replication in vitro in various cellular systems by blocking the biosynthesis of dNTPs required for viral reverse transcription and by inhibiting the CDK9 activity required for HIV-1 mRNA transcription (18–23). Several studies described that p21 is expressed at high levels ex vivo in CD4+ T cells from HICs (21,24–26) and that p21 mRNA levels correlated with CD4+ T cell activation in EC, but not in other HIV-infected groups (5). These evidences suggest that the inducibility of p21 to immune activation is a singular characteristic of EC and may contribute to the natural control of HIV-1 replication in vivo.
The monocyte chemotactic protein–induced protein 1 (MCPIP1), encoded by ZC3H12A gene, is another newly discovered host multifunctional modulator of immune response with antiviral activity (27). MCPIP1 plays a critical role in the regulation of the inflammatory response and immune homeostasis and also blocks HIV-1 replication in vitro by promoting the viral mRNA degradation through its RNase activity, particularly in quiescent CD4+ T cells (27,28). In activated CD4+ T cells, MCPIP1 is rapidly degraded (28) after its cleavage by the mucosa-associated lymphoid-tissue lymphoma-translocation 1 (MALT1) protein (29,30). In activated macrophage cells, by contrast, MCPIP1 transcripts are induced by TLR ligands and pro-inflammatory cytokines (mainly, TNF-α, IL-1β and CCL2/MCP-1), and its expression stimulate a negative feedback loop that attenuates the inflammatory state by decreasing its fundamental mediators (27,31).
The expression level of MCPIP1 in HIC was never described before. Interestingly, a recent study in renal carcinoma cells (Caki-1 cells) revealed that MCPIP1 overexpression reduces the cellular growth by increasing the levels of p21 transcripts, along with other proteins involved in cell cycle progression/arrest, supporting a coordinate regulation of MCPIP1 and p21 transcripts in that cell-line (32). This evidence prompted us to ask whether the expression of MCPIP1 could be elevated and positively correlated with p21 in the setting of natural control of HIV-1 infection. To test this hypothesis, we quantified the in vivo expression of MCPIP1, p21 and several antiviral host RF mRNAs in PBMC from HIC, ART-suppressed and HIV-uninfected individuals. We further explored the potential relationship between MCPIP1/p21 expression and levels of systemic cellular activation in HIC.
2 Methods
2.1 Study Subjects
We analyzed a cohort of 21 HIC subjects followed-up at the Instituto Nacional de Infectologia Evandro Chagas (INI) in Rio de Janeiro, Brazil. All HIC maintained RNA VL of < 2,000 copies/ml without antiretroviral therapy for at least five years and were subdivided in two sub-groups: EC (n = 13) when most (≥ 70%) plasma VL determinations were below the limit of detection (LOD), and VC (n = 8) when most (≥ 70%) VL determinations were > LOD and < 2,000 copies/ml. The limit of detection of plasma VL determinations varied over the follow-up period in according to the Brazilian Ministry of Health guidelines, with methodologies being updated overtime to improve sensitivity: Nuclisens HIV-1 RNA QT assay (Organon Teknika, Durham, NC, limit of detection: 80 copies/mL) from 1999 to 2007; the Versant HIV-1 3.0 RNA assay (bDNA 3.0, Siemens, Tarrytown, NY, limit of detection: 50 copies/mL) from 2007 to 2013; and the Abbott RealTime HIV-1 assay (Abbott Laboratories, Wiesbaden, Germany, limit of detection: 40 copies/mL) from 2013 to until today. Virological and immunological characteristics of these subjects were described in detail in previous studies (33,34). Two groups of ART-suppressed subjects (ART, n = 8) and healthy HIV-1-uninfected subjects (NEG, n = 10) were used as controls.
2.2 mRNA gene-expression analysis
Total RNA was extracted from 1 × 107 PBMC using RNeasy mini kit (Qiagen, Hilden, North Rhine-Westphalia, Germany) in which buffer RLT was supplemented with β-mercaptoethanol and displaced on-column DNase treatment using a Qiagen RNase-Free DNase Set (Qiagen, Hilden, North Rhine-Westphalia, Germany) according to manufacturer’s instruction. Total RNA yield and quality were determined using NanoDrop® 8000 spectrophotometer and an Agilent® 2100 Bioanalyzer. Only samples with an RNA integrity number (RIN) greater than 8.0 were used. Purified RNA (1 μg) was reverse-transcribed to cDNA using RT2 First Strand Kit (Qiagen, Hilden, North Rhine-Westphalia, Germany). The cDNA was mixed with RT2SYBR Green/ROX qPCR Master Mix (Qiagen, Hilden, North Rhine-Westphalia, Germany) and the mixture was added into customized RT2RNA PCR Array (Qiagen, Hilden, North Rhine-Westphalia, Germany) to measure the mRNA expression of 10 cellular target genes (APOBEC3G, SAMHD1, Tetherin, Mx1, Mx2, SLFN11, IFITM1, IFITM3, MCPIP1, and p21) besides three housekeeping genes (GAPDH, β-actin, and RNase-P), according to manufacturer’s instructions. Values of the crossing point at the maximum of the second derivative of the four-parameters fitted sigmoid curve second derivative, Cp, was determined for each sample. The efficiency of each amplification reaction was calculated as the ratio between the fluorescence of the cycle of quantification and fluorescence of the cycle immediately preceding that. Genes used in the normalization among samples were selected by the geNorm method (35). Data were expressed as fold-changes in mRNA abundance calculated as the normalized gene expression in any test sample divided by the mean normalized gene expression in the control HIV-negative group.
2.3 T cell and monocyte activation analyses
We used data of T cell and monocyte activation obtained in a previous study conducted by our group including these patients (34), in which plasma levels of soluble CD14 (sCD14) were determined by ELISA-sCD14 Quantikine assay (R&D Systems Minneapolis, MN) according to the manufacturer’s protocol and surface expression of combined HLA-DR and CD38 on CD4+ and CD8+ T cells was analyzed by flow cytometry.
2.4 Data analyses
The comparisons of mean log-fold changes in mRNA abundance were performed by either t-tests or one-way ANOVA nonparametric permutation tests (B = 1,000 permutations), followed by pair-wise comparisons with Holm-Bonferroni adjustment (36), for two or more groups respectively. Spearman coefficient was used for correlation analyses. A first-order log-Normal multiple regression analysis was fitted to model p21 gene expression as a function of MCPIP1 gene expression, CD4+ T cell activation (HLA-DR+CD38+), and HIC groups (EC and VC). The threshold for statistical significance was set to P < 0.05. Data were analyzed with R software (version 3.5.2) (37).
3 Results
Twenty-nine HIV-1 positive (21 HIC and 8 ART-suppressed) and 10 HIV-negative individuals were included in this cross-sectional study. Most HIV-positive (59%) and HIV-negative (60%) individuals were females and all individuals displayed CD4+ T cells counts above 500 cells/μl (Table 1). Although the EC subgroup shows a higher proportion of females (77%), the difference was not significant (Supplementary Table 1).
Analysis of the expression of multifunctional genes revealed a significant upregulation of both MCPIP1 and p21 transcripts in PBMC from HIC (Figure 1). The MCPIP1 mRNA was upregulated in PBMC from HIC compared to cells from both ART-suppressed (1.68-fold increase; P = 0.003) and HIV-negative (1.37-fold increase; P = 0.02) individuals (Figure 1A). A similar overexpression of the p21 mRNA was observed in PBMC from HIC compared to ART-suppressed (1.63-fold increase; P = 0.003) and HIV-negative (1.55-fold increase; P = 0.003) individuals (Figure 1B). In contrast, we found no significant differences in the mRNA levels of antiretroviral RF between the HIC and control groups, with the only exception of IFITM1 that was significantly elevated (1.15-fold increase; P = 0.03) in HIC in comparison to the HIV-negative group (Supplementary Figure S1).
We observed a significant positive correlation between the mRNA expression of MCPIP1 and p21 (r ≥ 0.57; P ≤ 0.014) in our cohort independently of the VL, treatment condition and HIV status (Figure 2). This positive correlation was maintained when individuals were subdivided by sex (Supplementary Figure S2). No significant correlations were observed between the mRNA expression of multifunctional genes MCPIP1/p21 and RF, with the only exception of a significant, negative correlation between MCPIP1/p21 and APOBEC3G in HIC (Supplementary Figure S3) and EC (Supplementary Figure S4).
To explore the potential relationship of p21 or MCPIP1 expression with immune activation, we measured the frequency of phenotype HLA-DR+CD38+ on CD4+ and CD8+ T cells (T cell activation) and plasma levels of sCD14 (monocyte activation) in our cohort. Frequencies of activated CD4+ T cell populations in VC and ART-suppressed subjects were higher than in EC (P < 0.0001) and HIV-negative (P = 0.0002) individuals (Supplementary Figure S5A). The VC subgroup also had significantly higher frequencies of activated CD8+ T cell than EC (P = 0.0007) and control groups (P ≤ 0.0009) (Supplementary Figure S5B). The median concentration of sCD14 in plasma was not significantly different across the groups (Supplementary Figure S5C). No significant correlations between mRNA levels of MCPIP1 and CD4+ T cell (Figure 3A) or CD8+ T cell (data not shown) activation were observed for HIC or EC subsets. The mRNA levels of p21 were positively associated with activated CD4+ T cells levels in HIC (r = 0.53; P = 0.016) and EC (r = 0.68; P = 0.017) (Figure 3B); but not with activated CD8+ T cell levels (data not shown). Levels of sCD14 were positively correlated with both MCPIP1 (r = 0.58; P = 0.04) and p21 (r = 0.74; P = 0.005) mRNA levels only in the EC subset (Figure 3C and D). No significant correlations between mRNA levels of MCPIP1/p21 and CD4+/CD8+ T cell activation or sCD14 levels were observed when ART-suppressed and HIV-negative individuals were included (Supplementary Figures S6). Multivariate analysis showed that the upregulation of MCPIP1 was positively associated with the increase of p21 expression in HIC (1.44-fold increase; P = 0.0035) (Supplementary Figure S7A). The frequency of activated CD4+ T cells also was positively associated with the increase of p21 expression in both EC and VC (1.48-fold increase; P = 0.0116), although this increase of the p21 expression was down-regulated by the increase of activated CD4+ T cells in VC when compared to EC (1.30-fold decrease by an increase of 1% CD4+HLA-DR+CD38+ T cells; P = 0.0284) (Supplementary Figure S7B). Overall, the model was highly significant (P = 0.003) and could explain as much as 70% (R2 = 0.492) of p21 expression.
4 Discussion
In this study, we observed that MCPIP1 and p21 mRNA expression were significantly increased in PBMC of HIC compared to cells of HIV-negative and -positive/ART-suppressed individuals. While elevated expression of p21 in PBMC of HIC had already been previously described (5,21,24–26), this is the first study to show overexpression of MCPIP1 alongside with p21 in these individuals.
The mRNA levels of MCPIP1 and p21 were positively correlated in HIC as well as in HIV–positive and –negative individuals. This supports a coordinated expression of these cellular genes in different settings, consistent with what has been shown for a renal carcinoma cell line (32). According to this study, MCPIP1 expression triggers the activation of p21 by two mechanisms: 1) down-modulation of damage-specific DNA binding protein 1 (DDB1) which regulates degradation of p21; and 2) upregulation of the mRNA levels of chromatin licensing and DNA replication factor 1 (CDT1) which activates p21 (32). In addition, following HIV-1 infection, the cellular let-7c miRNA is upregulated and it downregulate p21, resulting in higher copy number of viral genome transcripts in infected cells (38). MCPIP1 acts as a broad suppressor of the biogenesis pathway of both cellular (39) and viral miRNA (40). The involvement of the MCPIP1 in the degradation of another precursor of let-7 family (pre-let-7g) was already described (41), reinforcing the hypothesis that MCPIP1 might enhance the antiviral responses triggered by HIV-1 entry and infection by downregulating the miRNAs that target p21.
Increased expression of some host RF, which are also ISGs (4), has been previously observed in CD4+ T cells (i.e., SAMHD1, SLFN11 and IFITM1) (5,7,8) and PBMC (i.e., Mx1, Mx2, Tetherin and SLFN11) from HIC (6,9). With the only exception of IFITM1, no other RF analyzed here were upregulated in PBMC from our HIC cohort. In the chronic phase of HIV-1 infection in viremic untreated patients, most ISGs are upregulated in CD4+ T cells (42–44) and their expression is positively correlated with the percentage of activated T cells and negatively correlated with CD4+ T cell counts (42–46). This suggests that residual or low-level viremia observed in our HIC might not be enough to induce a generalized upregulation of ISGs during chronic infection (44). In addition, MCPIP1 (47,48) and p21 (16) negatively regulate the NF-κB cascade and their overexpression may also contribute to limit the chronic overexpression of ISGs in HIC. While most RF are mainly induced by IFN type I, IFITM1 can also be induced by IFN type II (49), indicating that another pathway may have stimulated its expression in our HIC cohort.
Although we have failed to detect an overall up-regulation of host RF in our HIC cohort, it is interesting to note that a few individuals displayed mRNA levels of SAMHD1 and/or SLFN11 well above the normal range (Supplementary Figure S1). These observations suggest that there might not be a unique host RF expression signature common to all HIC, but that different combinations of host RF could be associated with natural control of HIV-1 replication in distinct individuals. Thus, the particular set of increased host RF may vary across different HIC cohorts and this might explain the apparently contrasting findings across studies (5–9,50). Additionally, even though we were able to identify statistically significant differences in expression levels of MCPIP1 and p21 in PBMC between HIC and control groups, these findings warrant validation using larger cohorts.
Our results confirm previous observations that levels of p21 mRNA are positively correlated with CD4+ T cell activation in EC and HIC groups (5) and further support a positive correlation with sCD14, a marker of monocyte activation, in EC. These correlations are fully consistent with the critical role of p21 as a negative regulator of the proliferation of activated/memory T cells (10,13,14) and of macrophage-mediated inflammatory responses (15–17). Although MCPIP1 expression is also essential for suppressing peripheral T cell (51) and macrophage (52,53) activation, we only found a positive correlation of MCPIP1 mRNA with sCD14 in EC. While induction of MCPIP1 mRNA in vitro in response to TLR as well as IL-1β stimulation in macrophages is rapid and long-lasting (≥ 24h) (52– 54), the corresponding induction upon T cell receptor stimulation in CD4+ T cell is more ephemeral (< 12 hours) (55), which could have hindered the observation of a direct correlation between these two parameters. Notably, increased expression of MCPIP1/p21 associated with T cell and/or monocyte activation seems to be a unique characteristic of HIC/EC, because similar correlations were not observed in our study for other HIV-infected or HIV-negative subjects and previous studies have shown that viremic progressors display reduced levels of p21 even though exhibit high levels of cellular activation and inflammation (21). These results suggest that MCPIP1/p21 overexpression may be a distinctive homoeostatic innate response of HIC to limit the deleterious effects of aberrant chronic immune activation and inflammation driven by HIV-1 infection.
Transcript levels of RF here analyzed were not significantly correlated with T cell activation or sCD14, with the only exception of a negative correlation between APOBEC3G mRNA and sCD14 levels in EC (r = - 0.73. P = 0.006; data not shown). Surprisingly, transcripts levels of APOBEC3G were also negatively correlated with MCPIP1 and p21 mRNA levels in both HIC and EC. One possible explanation for these negative correlations lies in the interaction of APOBEC3G, MCPIP1, and p21 with the product of an important monocyte differentiation gene, the Kruppel-like factor 4 (KLF4). The expression of KLF4 in human macrophages is induced after IFN-γ, LPS, or TNF-α stimulus (56), mediating the proinflammatory signaling and the direct transcriptional regulation of CD14 in vitro (57). Interestingly, KLF4 is also able to induce expression of both MCPIP1 (58) and p21 (59,60), whereas APOBEC3G binds to the 3’-UTR of KLF4 mRNA and results in the reduction of its expression (61). Thus, lower levels of APOBEC3G mRNA may be associated with an upregulation of KLF4 that in turn induce higher levels of sCD14 and MCPIP1/p21 mRNA.
Selective upregulation of MCPIP1 and p21 in CD4+ T, macrophages and/or dendritic cells may directly limit HIV-1 replication by 1) reducing the reverse transcription and chromosomal integration of HIV-1 in quiescent cells and thus limiting the size of the latent proviral reservoir (18–20,62–64); 2) restricting HIV-1 LTR transcription (47,48,65,66); and, 3) degrading viral mRNA and miRNA (28,39,40,67). Upregulation of p21 and MCPIP1 may also indirectly limit HIV-1 replication and further prevent CD4+ T cells loss by reducing chronic IFN-I signaling, generalized inflammation and over-activation of the immune system (10,14–17,52,53,68–70), without affecting the activation of antiviral cellular responses. Although the enhanced antiviral and anti-inflammatory state may not be enough to fully restrict HIV-1 replication (71), it could act in concert with other innate and adaptive immune mechanisms to control HIV replication in HIC.
The enhanced expression of a few select host genes, including p21, was strongly associated with reduced CD4+ T cell-associated HIV RNA during ART, indicating that the p21 may contribute to the control of viral expression and ongoing replication during ART (72). Another study demonstrates that atorvastatin, a lipid-lowering medication, exert a broad spectrum of anti-inflammatory functions and further reduced HIV infection in both rested and activated CD4+ T cells in vitro via p21 upregulation (22). Interestingly, atorvastatin was found to up-regulates p21 through a p53 independent pathway, which is consistent with a potential role of MCPIP1 in that antiviral mechanism. These observations suggest that pharmacological manipulation of p21 and MCPIP1 may open novel therapeutic perspectives to prevent HIV-1 replication and to attenuate HIV-associated inflammation and immune activation during ART.
An important limitation of our study is the impossibility of assigning which cell(s) population(s) has increased expression of p21 and MCPIP1 in HIC. The expression profile of many RF and ISGs may be different between CD4+ T cells and monocytes (8), suggesting that the individualization of these cell types might better decipher the mechanisms of host factors regulation in the setting of natural control of HIV-1 infection. Another potential limitation is that only mRNA levels were analyzed. Previous studies showed that p21 mRNA levels mirror p21 protein levels in CD4+ T cells from HIC (21) and that MCPIP1 mRNA levels reflect MCPIP1 protein levels in HCV-infected hepatoma cells (73). Although this evidence indicates a close match between transcripts and protein expression levels, measuring the levels/activity of p21 and MCPIP1 proteins in cells from HIC should also help to elucidate the relevance of these RF for HIV control.
In summary, our data confirm the high levels of p21 mRNA expression and shows for the first-time the concurrent overexpression of MCPIP1 mRNA in HIC. Moreover, we found a positive correlation between p21 and MCPIP1 transcripts in HIC, indicating a possible synergistic effect of both innate host RF on natural suppression of HIV-1 replication in vivo. Further studies are needed to better understand the role of p21 and MCPIP1 in the natural control of HIV-1 replication and disease progression in HIC. These findings may also have important implications for the development of new immune-based therapeutic strategies for a functional cure of HIV-1 infection.
6 Ethics Statement
This study was carried out in accordance with the recommendations of the ethical committee of Instituto Nacional de Infectologia Evandro Chagas (INI-Fiocruz) that approved the study protocol (CAAE 1717.0.000.009-07). All subjects gave written informed consent in accordance with the Declaration of Helsinki.
7 Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
8 Author Contributions
GB and TMLS conceived and designed the study and supervised the experiments. SSDA conducted experiments and analyzed the data together with MR-A and GB. FH performed the CD4+ T cell and monocyte activation assays. ED collaborated with mRNA gene-expression analysis. BH, BG, and VGV conducted patient recruitment and follow-up. FH, ED and MGM provided intellectual input for results interpretations. SSDA, GB and MR-A wrote the first draft and all authors assisted with the writing and approved the final manuscript.
9 Acknowledgments
The authors thank the patients, who participated in the study, as well as all the technical staff involved in the clinical follow-up of these patients. We also thank Ms Marilia Alves Figueira de Melo for the excellent technical support in RNA quantification and integrity analyses and the Plataforma de PCR em Tempo Real – RJ (RPT09A) – FIOCRUZ and Plataforma de Sequenciamento de Ácidos Nucléicos de Nova Geração – RJ (RPT01J) – FIOCRUZ.
10 Funding
This work was supported by the Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro – FAPERJ (grant number E-26/110.123/2014) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (Grant Number 401220/2016-8). SSDA was supported by funding from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro – FAPERJ. ED was financed by a Postdoctoral fellowship from the “Programa Nacional de Pós-Doutorado (PNPD)” by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.