Shiga toxin-producing Escherichia coli (STEC) O113:H21 strains are associated with human diarrhea... more Shiga toxin-producing Escherichia coli (STEC) O113:H21 strains are associated with human diarrhea and some strains may cause hemolytic–uremic syndrome (HUS). In Brazil, these strains are commonly found in cattle but, so far, were not isolated from HUS patients. Here, a system biology approach was used to investigate the differential transcriptomic and phenotypic responses of enterocyte-like Caco-2 cells to two STEC O113:H21 strains with similar virulence factor profiles (i.e. expressing stx2, ehxA, epeA, espA, iha, saa, sab, and subA): EH41 (Caco-2/EH41), isolated from a HUS patient in Australia, and Ec472/01 (Caco-2/Ec472), isolated from bovine feces in Brazil, during a 3 h period of bacteria–enterocyte interaction. Gene co-expression network analysis for Caco-2/EH41 revealed a quite abrupt pattern of topological variation along 3 h of enterocyte–bacteria interaction when compared with networks obtained for Caco-2/Ec472. Transcriptional module characterization revealed that EH41 in...
The Down syndrome (DS) immune phenotype is characterized by thymus hypotrophy, higher propensity ... more The Down syndrome (DS) immune phenotype is characterized by thymus hypotrophy, higher propensity to organ-specific autoimmune disorders, and higher susceptibility to infections, among other features. Considering that AIRE (autoimmune regulator) is located on 21q22.3, we analyzed protein and gene expression in surgically removed thymuses from 14 DS patients with congenital heart defects, who were compared with 42 age-matched controls with heart anomaly as an isolated malformation. Immunohistochemistry revealed 70.48 ± 49.59 AIRE-positive cells/mm2 in DS versus 154.70 ± 61.16 AIRE-positive cells/mm2 in controls (p < 0.0001), and quantitative PCR as well as DNA microarray data confirmed those results. The number of FOXP3-positive cells/mm2 was equivalent in both groups. Thymus transcriptome analysis showed 407 genes significantly hypoexpressed in DS, most of which were related, according to network transcriptional analysis (FunNet), to cell division and to immunity. Immune response-...
The transcriptional response of human blood leukocytes to SARS-CoV-2 infection was investigated f... more The transcriptional response of human blood leukocytes to SARS-CoV-2 infection was investigated focusing on the differences between mild and severe cases and between age subgroups. Weighted gene co-expression network analysis and comparative gene expression analysis were used. Three transcriptional modules positively associated with the traits of interest and their respective high hierarchy genes were identified. Enrichment analyses showed that the yellow module, associated with severe cases and older patients, had an overrepresentation of genes involved in inflammatory and innate immune responses, and neutrophil activation. The magenta and black modules, associated with disease severity and younger patients, contained genes related to innate immunity and inflammation and genes that regulate those responses. Subnetworks for these modules were constructed using genes enriched for innate immunity, inflammation, immunoregulation and differentially expressed genes (severe vs. mild). The...
Systemic lupus erythematosus (SLE) is an autoimmune disease with a persistent systemic inflammati... more Systemic lupus erythematosus (SLE) is an autoimmune disease with a persistent systemic inflammation. Exercise induced inflammatory response in SLE remains to be fully elucidated. The aim of this study was to assess the effects of acuteexercise on leukocyte gene expression in active (SLEACTIVE) and inactive SLE (SLEINACTIVE) patients and healthy controls(HC). All subjects (n = 4 per group) performed a 30-min single bout of acute aerobic exercise (~70% of VO2peak) on a treadmill, and blood samples were collected for RNA extraction from circulating leukocyte at baseline, at the end of exercise, and after three hours of recovery. The expression of a panel of immune-related genes was evaluated by a quantitative PCR array assay. Moreover, network-based analyses were performed to interpret transcriptional changes occurring after the exercise challenge. In all groups, a single bout of acute exercise led to the down-regulation of the gene expression of innate and adaptive immunity at the end...
IPEX is one of the few Inborn Errors of Immunity that may manifest in the fetal period, and its i... more IPEX is one of the few Inborn Errors of Immunity that may manifest in the fetal period, and its intrauterine forms certainly represent the earliest human autoimmune diseases. Here, we review the clinical, histopathologic, and genetic findings from 21 individuals in 11 unrelated families, with nine different mutations, described as cases of intrauterine IPEX. Recurrent male fetal death (multigenerational in five families) due to hydrops in the midsemester of pregnancy was the commonest presentation (13/21). Noteworthy, in the affected families, there were only fetal- or perinatal-onset cases, with no affected individuals presenting milder forms with later-life manifestation. Most alive births were preterm (5/6). Skin desquamation and intrauterine growth restriction were observed in part of the cases. Fetal ultrasonography showed hyperechoic bowel or dilated bowel loops in the five cases with available imaging data. Histopathology showed multi-visceral infiltrates with T lymphocytes a...
The human thymus suffers a transient neonatal involution, recovers and then starts a process of d... more The human thymus suffers a transient neonatal involution, recovers and then starts a process of decline between the 1 st and 2 nd years of life. Age-related morphological changes in thymus were extensively investigated, but the genomic mechanisms underlying this process remain largely unknown. Through Weighted Gene Co-expression Network Analysis (WGCNA) and TF-miRNA-mRNA integrative analysis we studied the transcriptome of neonate and infant thymic tissues grouped by age: 0-30 days (A); 31days-6 months (B); 7-12 months (C); 13-18 months (D); 19-31months (E). Age-related transcriptional modules, hubs and high gene significance (HGS) genes were identified, as well as TF-miRNA-hub/HGS co-expression correlations. Three transcriptional modules were correlated with A and/or E groups. Hubs were mostly related to cellular/metabolic processes; few were differentially expressed (DE) or related to T-cell development. Inversely, HGS genes in groups A and E were mostly DE. In A (neonate) one third of the hyper-expressed HGS genes were related to T-cell development, against one-twentieth in E, what may correlate with the early neonatal depletion and recovery of thymic T-cell populations. This genomic mechanism is tightly regulated by TF-miRNA-hub/HGS interactions that differentially govern cellular and molecular processes involved in the functioning of the neonate thymus and in the beginning of thymic decline.
AIRE expression in thymus is downregulated by estrogen after puberty, what probably renders women... more AIRE expression in thymus is downregulated by estrogen after puberty, what probably renders women more susceptible to autoimmune disorders. Here we investigated the effects of minipuberty on male and female infant human thymic tissue in order to verify if this initial transient increase in sex hormones - along the first six months of life - could affect thymic transcriptional network regulation and AIRE expression. Gene co-expression network analysis for differentially expressed genes and miRNA-target analysis revealed sex differences in thymic tissue during minipuberty, but such differences were not detected in the thymic tissue of infants aged 7-18 months, i.e. the non-puberty group. AIRE expression was essentially the same in both sexes in minipuberty and in non-puberty groups, as assessed by genomic and immunohistochemical assays. However, AIRE-interactors networks showed several differences in all groups regarding gene-gene expression correlation. Therefore, minipuberty and gen...
Germline mutations in BRCA1, BRCA2 and TP53 genes have been identified as one of the most importa... more Germline mutations in BRCA1, BRCA2 and TP53 genes have been identified as one of the most important disease-causing issues in young breast cancer patients worldwide. The specific defective biological processes that trigger germline mutation-associated and-negative tumors remain unclear. To delineate an initial portrait of Brazilian early-onset breast cancer, we performed an investigation combining both germline and tumor analysis. Germline screening of the BRCA1, BRCA2, CHEK2 (c.1100delC) and TP53 genes was performed in 54 unrelated patients ,35 y; their tumors were investigated with respect to transcriptional and genomic profiles as well as hormonal receptors and HER2 expression/amplification. Germline mutations were detected in 12 out of 54 patients (22%) [7 in BRCA1 (13%), 4 in BRCA2 (7%) and one in TP53 (2%) gene]. A cancer familial history was present in 31.4% of the unrelated patients, from them 43.7% were carriers for germline mutation (37.5% in BRCA1 and in 6.2% in the BRCA2 genes). Fifty percent of the unrelated patients with hormone receptornegative tumors carried BRCA1 mutations, percentage increasing to 83% in cases with familial history of cancer. Overrepresentation of DNA damage-, cellular and cell cycle-related processes was detected in the up-regulated genes of BRCA1/ 2-associated tumors, whereas cell and embryo development-related processes were over-represented in the up-regulated genes of BRCA1/2-negative tumors, suggesting distinct mechanisms driving the tumorigenesis. An initial portrait of the early-onset breast cancer patients in Brazil was generated pointing out that hormone receptor-negative tumors and positive familial history are two major risk factors for detection of a BRCA1 germline mutation. Additionally, the data revealed molecular factors that potentially trigger the tumor development in young patients.
Genomic diversity among 34 strains of Escherichia coli belonging to different serotypes of the O2... more Genomic diversity among 34 strains of Escherichia coli belonging to different serotypes of the O26 serogroup^encompassing strains from different geographical origins and Shiga toxin-negative Brazilian strains^was evaluated through random amplified polymorphic DNA (RAPD) analysis. Our results indicate that Brazilian and non-Brazilian O26 strains fall under distinct but closely related differentiation clusters. RFLP-PCR analysis of the fliC gene sequence was done in order to identify the H 3 serotypes and served to confirm the clustering pattern obtained in the dendrogram generated from RAPD data. The epidemiological significance of these data is discussed.
Age at epilepsy onset has a broad impact on brain plasticity and epilepsy pathomechanisms. Prolon... more Age at epilepsy onset has a broad impact on brain plasticity and epilepsy pathomechanisms. Prolonged febrile seizures in early childhood (FS) constitute an initial precipitating insult (IPI) commonly associated with mesial temporal lobe epilepsy (MTLE). FS-MTLE patients may have early disease onset, i.e. just after the IPI, in early childhood, or lateonset, ranging from mid-adolescence to early adult life. The mechanisms governing early (E) or late (L) disease onset are largely unknown. In order to unveil the molecular pathways underlying E and L subtypes of FS-MTLE we investigated global gene expression in hippocampal CA3 explants of FS-MTLE patients submitted to hippocampectomy. Gene coexpression networks (GCNs) were obtained for the E and L patient groups. A networkbased approach for GCN analysis was employed allowing: i) the visualization and analysis of differentially expressed (DE) and complete (CO)-all valid GO annotated transcripts-GCNs for the E and L groups; ii) the study of interactions between all the system's constituents based on community detection and coarse-grained community structure methods. We found that the E-DE communities with strongest connection weights harbor highly connected genes mainly related to neural excitability and febrile seizures, whereas in L-DE communities these genes are not only involved in network excitability but also playing roles in other epilepsy-related processes. Inversely, in E-CO the strongly connected communities are related to compensatory pathways (seizure inhibition, neuronal survival and responses to stress conditions) while in L-CO these communities harbor several genes related to pro-epileptic effects, seizure-related mechanisms and vulnerability to epilepsy. These results fit the concept, based on fMRI and behavioral studies, that early PLOS ONE |
In this chapter we characterized trisomy 21-driven transcriptional alterations in human thymus th... more In this chapter we characterized trisomy 21-driven transcriptional alterations in human thymus through gene coexpression network (GCN) analysis. We used whole thymic tissue (corticomedullar sections)—obtained at heart surgery from Down syndrome (DS) and karyotipically normal individuals (CT)—and a network-based approach for GCN analysis allowing the study of interactions between all the system’s constituents based on community detection. Changes in the degree of connections observed for hierarchically important hubs in DS and CT gene networks corresponded to sub-network changes, i.e. module (communities) changes. Distinct communities of highly interconnected gene sets were topologically identified for DS and CT networks. Trisomy 21 gene dysregulation in thymus may therefore be viewed as the breakdown and altered reorganization of functional modules.
Stenotrophomonas ssp. has a wide environmental distribution and is also found as an opportunistic... more Stenotrophomonas ssp. has a wide environmental distribution and is also found as an opportunistic pathogen, causing nosocomial or community-acquired infections. One species, S. maltophilia, presents multidrug resistance and has been associated with serious infections in pediatric and immunocompromised patients. Therefore, it is relevant to conduct resistance profile and phylogenetic studies in clinical isolates for identifying infection origins and isolates with augmented pathogenic potential. Here, multilocus sequence typing was performed for phylogenetic analysis of nosocomial isolates of Stenotrophomonas spp. and, environmental and clinical strains of S. maltophilia. Biochemical and multidrug resistance profiles of nosocomial and clinical strains were determined. The inferred phylogenetic profile showed high clonal variability, what correlates with the adaptability process of Stenotrophomonas to different habitats. Two clinical isolates subgroups of S. maltophilia sharing high phylogenetic homogeneity presented intergroup recombination, thus indicating the high permittivity to horizontal gene transfer, a mechanism involved in the acquisition of antibiotic resistance and expression of virulence factors. For most of the clinical strains, phylogenetic inference was made using only partial ppsA gene sequence. Therefore, the sequencing of just one specific fragment of this gene would allow, in many cases, determining whether the infection with S. maltophilia was nosocomial or community-acquired.
Gliomas are aggressive primary brain tumors with high infiltrative potential. The expression of A... more Gliomas are aggressive primary brain tumors with high infiltrative potential. The expression of Angiotensin II (Ang II) receptors has been associated with poor prognosis in human astrocytomas, the most common type of glioma. In this study, we investigated the role of Angiotensin II in glioma malignancy through transcriptional profiling and network analysis of cultured C6 rat glioma cells exposed to Ang II and to inhibitors of its membrane receptor subtypes. C6 cells were treated with Ang II and specific antagonists of AT1 and AT2 receptors. Total RNA was isolated after three and six hours of Ang II treatment and analyzed by oligonucleotide microarray technology. Gene expression data was evaluated through transcriptional network modeling to identify how differentially expressed (DE) genes are connected to each other. Moreover, other genes co-expressing with the DE genes were considered in these analyses in order to support the identification of enriched functions and pathways. A hub-...
About 5-10% of breast and ovarian carcinomas are hereditary and most of these result from germlin... more About 5-10% of breast and ovarian carcinomas are hereditary and most of these result from germline mutations in the BRCA1 and BRCA2 genes. In women of Ashkenazi Jewish ascendance, up to 30% of breast and ovarian carcinomas may be attributable to mutations in these genes, where 3 founder mutations, c.68_69del (185delAG) and c.5266dup (5382insC) in BRCA1 and c.5946del (6174delT) in BRCA2, are commonly encountered. It has been suggested by some authors that screening for founder mutations should be undertaken in all Brazilian women with breast cancer. Thus, the goal of this study was to determine the prevalence of three founder mutations, commonly identified in Ashkenazi individuals in a sample of non-Ashkenazi cancer-affected Brazilian women with clearly defined risk factors for hereditary breast and ovarian cancer (HBOC) syndrome. Among 137 unrelated Brazilian women from HBOC families, the BRCA1 c.5266dup mutation was identified in seven individuals (5%). This prevalence is similar t...
PURPOSE: Screening for mutations in the entire Cystic Fibrosis gene (CFTR) of Brazilian infertile... more PURPOSE: Screening for mutations in the entire Cystic Fibrosis gene (CFTR) of Brazilian infertile men with congenital absence of vas deferens, in order to prevent transmission of CFTR mutations to offspring with the use of assisted reproductive technologies. METHOD: Specific polymerase chain reaction (PCR) primers were designed to each of the 27 exons and splicing sites of interest followed by single strand conformational polymorphism and Heteroduplex Analysis (SSCP-HA) in precast 12.5% polyacrylamide gels at 7 O C and 20 O C. Fragments with abnormal SSCP migration pattern were sequenced. RESULTS: Two novel missense mutations (S753R and G149W) were found in three patients (two brothers) together with the IVS8-5T allele in hetrozygosis. CONCLUSION: The available screenings for CF mutations do not include the atypical mutations associated to absence of vas deferens and thus, when these tests fail to find mutations, there is still a genetic risk of affected children with the help of assisted reproduction. We recommend the screening of the whole CFTR gene for these infertile couples, as part of the work-up before assisted reproduction.
A new Bordetella pertussis strain isolated from a whooping cough Brazilian patient was characteri... more A new Bordetella pertussis strain isolated from a whooping cough Brazilian patient was characterized under molecular and immunological aspects and compared with strains used in the production of whole-cell pertussis vaccine. The isolate, named 21A1, exhibited the bacteriological characteristics classically described for B. pertussis. RAPD and SDS-PAGE analysis showed similar DNA and proteic profiles between classical vaccinal strains and the new isolate. Comparative analysis about the efficacy of vaccines in protecting mice against the intracerebral challenge showed that the 21A1 vaccine was able to induce the highest mouse protection when compared with other B. pertussis vaccines. The results presented here indicate that the inclusion of selected new strain isolates in the composition of the B. pertussis vaccines can be an alternative to maintain or increase vaccine potency.
The low cost of sperm sexing methods combined with in vitro embryo production in genetic improvem... more The low cost of sperm sexing methods combined with in vitro embryo production in genetic improvement programs can increase the profitability of cattle production, in particular when it does not decrease reproductive efficiency. The aim of this work was to evaluate the sex ratio deviation of thawed bovine semen processed by density gradient centrifugation and swim-up. Semen doses were collected from ten bulls of different breeds, and each experimental group was replicated ten times.A Percoll™ gradient was prepared by mixing Dulbecco’s Modified Eagle Medium (DMEM; Sigma Aldrich, St. Louis, MO, USA) isotonic solutions with Percoll™ (GE Healthcare Bio-Science AB, Uppsala, Sweden) stock solution with 0.3% BSA, resulting in densities ranging from 1.110 to 1.123 g mL-1. The layers of discontinuous density gradient were disposed from the larger density (bottom of the tube) to the smaller into 15-mL conical centrifuge tubes. About 40 million sperm were overlaid on Percoll™ gradient and were ...
Ischemia/reperfusion injury (IRI) is a leading cause of acute renal failure. The definition of th... more Ischemia/reperfusion injury (IRI) is a leading cause of acute renal failure. The definition of the molecular mechanisms involved in renal IRI and counter protection promoted by ischemic pre-conditioning (IPC) or Hemin treatment is an important milestone that needs to be accomplished in this research area. We examined, through an oligonucleotide microarray protocol, the renal differential transcriptome profiles of mice submitted to IRI, IPC and Hemin treatment. After identifying the profiles of differentially expressed genes observed for each comparison, we carried out functional enrichment analysis to reveal transcripts putatively involved in potential relevant biological processes and signaling pathways. The most relevant processes found in these comparisons were stress, apoptosis, cell differentiation, angiogenesis, focal adhesion, ECM-receptor interaction, ion transport, angiogenesis, mitosis and cell cycle, inflammatory response, olfactory transduction and regulation of actin cytoskeleton. In addition, the most important overrepresented pathways were MAPK, ErbB, JAK/STAT, Toll and Nod like receptors, Angiotensin II, Arachidonic acid metabolism, Wnt and coagulation cascade. Also, new insights were gained about the underlying protection mechanisms against renal IRI promoted by IPC and Hemin treatment. Venn diagram analysis allowed us to uncover common and exclusively differentially expressed genes between these two protective maneuvers, underscoring potential common and exclusive biological functions regulated in each case. In summary, IPC exclusively regulated the expression of genes belonging to stress, protein modification and apoptosis, highlighting the role of IPC in controlling exacerbated stress response. Treatment with the Hmox1 inducer Hemin, in turn, exclusively regulated the expression of genes associated with cell differentiation, metabolic pathways, cell cycle, mitosis, development, regulation of actin cytoskeleton and arachidonic acid metabolism, suggesting a pleiotropic effect for Hemin. These findings improve the biological understanding of how the kidney behaves after IRI. They also illustrate some possible underlying molecular mechanisms involved in kidney protection observed with IPC or Hemin treatment maneuvers.
Shiga toxin-producing Escherichia coli (STEC) O113:H21 strains are associated with human diarrhea... more Shiga toxin-producing Escherichia coli (STEC) O113:H21 strains are associated with human diarrhea and some strains may cause hemolytic–uremic syndrome (HUS). In Brazil, these strains are commonly found in cattle but, so far, were not isolated from HUS patients. Here, a system biology approach was used to investigate the differential transcriptomic and phenotypic responses of enterocyte-like Caco-2 cells to two STEC O113:H21 strains with similar virulence factor profiles (i.e. expressing stx2, ehxA, epeA, espA, iha, saa, sab, and subA): EH41 (Caco-2/EH41), isolated from a HUS patient in Australia, and Ec472/01 (Caco-2/Ec472), isolated from bovine feces in Brazil, during a 3 h period of bacteria–enterocyte interaction. Gene co-expression network analysis for Caco-2/EH41 revealed a quite abrupt pattern of topological variation along 3 h of enterocyte–bacteria interaction when compared with networks obtained for Caco-2/Ec472. Transcriptional module characterization revealed that EH41 in...
The Down syndrome (DS) immune phenotype is characterized by thymus hypotrophy, higher propensity ... more The Down syndrome (DS) immune phenotype is characterized by thymus hypotrophy, higher propensity to organ-specific autoimmune disorders, and higher susceptibility to infections, among other features. Considering that AIRE (autoimmune regulator) is located on 21q22.3, we analyzed protein and gene expression in surgically removed thymuses from 14 DS patients with congenital heart defects, who were compared with 42 age-matched controls with heart anomaly as an isolated malformation. Immunohistochemistry revealed 70.48 ± 49.59 AIRE-positive cells/mm2 in DS versus 154.70 ± 61.16 AIRE-positive cells/mm2 in controls (p < 0.0001), and quantitative PCR as well as DNA microarray data confirmed those results. The number of FOXP3-positive cells/mm2 was equivalent in both groups. Thymus transcriptome analysis showed 407 genes significantly hypoexpressed in DS, most of which were related, according to network transcriptional analysis (FunNet), to cell division and to immunity. Immune response-...
The transcriptional response of human blood leukocytes to SARS-CoV-2 infection was investigated f... more The transcriptional response of human blood leukocytes to SARS-CoV-2 infection was investigated focusing on the differences between mild and severe cases and between age subgroups. Weighted gene co-expression network analysis and comparative gene expression analysis were used. Three transcriptional modules positively associated with the traits of interest and their respective high hierarchy genes were identified. Enrichment analyses showed that the yellow module, associated with severe cases and older patients, had an overrepresentation of genes involved in inflammatory and innate immune responses, and neutrophil activation. The magenta and black modules, associated with disease severity and younger patients, contained genes related to innate immunity and inflammation and genes that regulate those responses. Subnetworks for these modules were constructed using genes enriched for innate immunity, inflammation, immunoregulation and differentially expressed genes (severe vs. mild). The...
Systemic lupus erythematosus (SLE) is an autoimmune disease with a persistent systemic inflammati... more Systemic lupus erythematosus (SLE) is an autoimmune disease with a persistent systemic inflammation. Exercise induced inflammatory response in SLE remains to be fully elucidated. The aim of this study was to assess the effects of acuteexercise on leukocyte gene expression in active (SLEACTIVE) and inactive SLE (SLEINACTIVE) patients and healthy controls(HC). All subjects (n = 4 per group) performed a 30-min single bout of acute aerobic exercise (~70% of VO2peak) on a treadmill, and blood samples were collected for RNA extraction from circulating leukocyte at baseline, at the end of exercise, and after three hours of recovery. The expression of a panel of immune-related genes was evaluated by a quantitative PCR array assay. Moreover, network-based analyses were performed to interpret transcriptional changes occurring after the exercise challenge. In all groups, a single bout of acute exercise led to the down-regulation of the gene expression of innate and adaptive immunity at the end...
IPEX is one of the few Inborn Errors of Immunity that may manifest in the fetal period, and its i... more IPEX is one of the few Inborn Errors of Immunity that may manifest in the fetal period, and its intrauterine forms certainly represent the earliest human autoimmune diseases. Here, we review the clinical, histopathologic, and genetic findings from 21 individuals in 11 unrelated families, with nine different mutations, described as cases of intrauterine IPEX. Recurrent male fetal death (multigenerational in five families) due to hydrops in the midsemester of pregnancy was the commonest presentation (13/21). Noteworthy, in the affected families, there were only fetal- or perinatal-onset cases, with no affected individuals presenting milder forms with later-life manifestation. Most alive births were preterm (5/6). Skin desquamation and intrauterine growth restriction were observed in part of the cases. Fetal ultrasonography showed hyperechoic bowel or dilated bowel loops in the five cases with available imaging data. Histopathology showed multi-visceral infiltrates with T lymphocytes a...
The human thymus suffers a transient neonatal involution, recovers and then starts a process of d... more The human thymus suffers a transient neonatal involution, recovers and then starts a process of decline between the 1 st and 2 nd years of life. Age-related morphological changes in thymus were extensively investigated, but the genomic mechanisms underlying this process remain largely unknown. Through Weighted Gene Co-expression Network Analysis (WGCNA) and TF-miRNA-mRNA integrative analysis we studied the transcriptome of neonate and infant thymic tissues grouped by age: 0-30 days (A); 31days-6 months (B); 7-12 months (C); 13-18 months (D); 19-31months (E). Age-related transcriptional modules, hubs and high gene significance (HGS) genes were identified, as well as TF-miRNA-hub/HGS co-expression correlations. Three transcriptional modules were correlated with A and/or E groups. Hubs were mostly related to cellular/metabolic processes; few were differentially expressed (DE) or related to T-cell development. Inversely, HGS genes in groups A and E were mostly DE. In A (neonate) one third of the hyper-expressed HGS genes were related to T-cell development, against one-twentieth in E, what may correlate with the early neonatal depletion and recovery of thymic T-cell populations. This genomic mechanism is tightly regulated by TF-miRNA-hub/HGS interactions that differentially govern cellular and molecular processes involved in the functioning of the neonate thymus and in the beginning of thymic decline.
AIRE expression in thymus is downregulated by estrogen after puberty, what probably renders women... more AIRE expression in thymus is downregulated by estrogen after puberty, what probably renders women more susceptible to autoimmune disorders. Here we investigated the effects of minipuberty on male and female infant human thymic tissue in order to verify if this initial transient increase in sex hormones - along the first six months of life - could affect thymic transcriptional network regulation and AIRE expression. Gene co-expression network analysis for differentially expressed genes and miRNA-target analysis revealed sex differences in thymic tissue during minipuberty, but such differences were not detected in the thymic tissue of infants aged 7-18 months, i.e. the non-puberty group. AIRE expression was essentially the same in both sexes in minipuberty and in non-puberty groups, as assessed by genomic and immunohistochemical assays. However, AIRE-interactors networks showed several differences in all groups regarding gene-gene expression correlation. Therefore, minipuberty and gen...
Germline mutations in BRCA1, BRCA2 and TP53 genes have been identified as one of the most importa... more Germline mutations in BRCA1, BRCA2 and TP53 genes have been identified as one of the most important disease-causing issues in young breast cancer patients worldwide. The specific defective biological processes that trigger germline mutation-associated and-negative tumors remain unclear. To delineate an initial portrait of Brazilian early-onset breast cancer, we performed an investigation combining both germline and tumor analysis. Germline screening of the BRCA1, BRCA2, CHEK2 (c.1100delC) and TP53 genes was performed in 54 unrelated patients ,35 y; their tumors were investigated with respect to transcriptional and genomic profiles as well as hormonal receptors and HER2 expression/amplification. Germline mutations were detected in 12 out of 54 patients (22%) [7 in BRCA1 (13%), 4 in BRCA2 (7%) and one in TP53 (2%) gene]. A cancer familial history was present in 31.4% of the unrelated patients, from them 43.7% were carriers for germline mutation (37.5% in BRCA1 and in 6.2% in the BRCA2 genes). Fifty percent of the unrelated patients with hormone receptornegative tumors carried BRCA1 mutations, percentage increasing to 83% in cases with familial history of cancer. Overrepresentation of DNA damage-, cellular and cell cycle-related processes was detected in the up-regulated genes of BRCA1/ 2-associated tumors, whereas cell and embryo development-related processes were over-represented in the up-regulated genes of BRCA1/2-negative tumors, suggesting distinct mechanisms driving the tumorigenesis. An initial portrait of the early-onset breast cancer patients in Brazil was generated pointing out that hormone receptor-negative tumors and positive familial history are two major risk factors for detection of a BRCA1 germline mutation. Additionally, the data revealed molecular factors that potentially trigger the tumor development in young patients.
Genomic diversity among 34 strains of Escherichia coli belonging to different serotypes of the O2... more Genomic diversity among 34 strains of Escherichia coli belonging to different serotypes of the O26 serogroup^encompassing strains from different geographical origins and Shiga toxin-negative Brazilian strains^was evaluated through random amplified polymorphic DNA (RAPD) analysis. Our results indicate that Brazilian and non-Brazilian O26 strains fall under distinct but closely related differentiation clusters. RFLP-PCR analysis of the fliC gene sequence was done in order to identify the H 3 serotypes and served to confirm the clustering pattern obtained in the dendrogram generated from RAPD data. The epidemiological significance of these data is discussed.
Age at epilepsy onset has a broad impact on brain plasticity and epilepsy pathomechanisms. Prolon... more Age at epilepsy onset has a broad impact on brain plasticity and epilepsy pathomechanisms. Prolonged febrile seizures in early childhood (FS) constitute an initial precipitating insult (IPI) commonly associated with mesial temporal lobe epilepsy (MTLE). FS-MTLE patients may have early disease onset, i.e. just after the IPI, in early childhood, or lateonset, ranging from mid-adolescence to early adult life. The mechanisms governing early (E) or late (L) disease onset are largely unknown. In order to unveil the molecular pathways underlying E and L subtypes of FS-MTLE we investigated global gene expression in hippocampal CA3 explants of FS-MTLE patients submitted to hippocampectomy. Gene coexpression networks (GCNs) were obtained for the E and L patient groups. A networkbased approach for GCN analysis was employed allowing: i) the visualization and analysis of differentially expressed (DE) and complete (CO)-all valid GO annotated transcripts-GCNs for the E and L groups; ii) the study of interactions between all the system's constituents based on community detection and coarse-grained community structure methods. We found that the E-DE communities with strongest connection weights harbor highly connected genes mainly related to neural excitability and febrile seizures, whereas in L-DE communities these genes are not only involved in network excitability but also playing roles in other epilepsy-related processes. Inversely, in E-CO the strongly connected communities are related to compensatory pathways (seizure inhibition, neuronal survival and responses to stress conditions) while in L-CO these communities harbor several genes related to pro-epileptic effects, seizure-related mechanisms and vulnerability to epilepsy. These results fit the concept, based on fMRI and behavioral studies, that early PLOS ONE |
In this chapter we characterized trisomy 21-driven transcriptional alterations in human thymus th... more In this chapter we characterized trisomy 21-driven transcriptional alterations in human thymus through gene coexpression network (GCN) analysis. We used whole thymic tissue (corticomedullar sections)—obtained at heart surgery from Down syndrome (DS) and karyotipically normal individuals (CT)—and a network-based approach for GCN analysis allowing the study of interactions between all the system’s constituents based on community detection. Changes in the degree of connections observed for hierarchically important hubs in DS and CT gene networks corresponded to sub-network changes, i.e. module (communities) changes. Distinct communities of highly interconnected gene sets were topologically identified for DS and CT networks. Trisomy 21 gene dysregulation in thymus may therefore be viewed as the breakdown and altered reorganization of functional modules.
Stenotrophomonas ssp. has a wide environmental distribution and is also found as an opportunistic... more Stenotrophomonas ssp. has a wide environmental distribution and is also found as an opportunistic pathogen, causing nosocomial or community-acquired infections. One species, S. maltophilia, presents multidrug resistance and has been associated with serious infections in pediatric and immunocompromised patients. Therefore, it is relevant to conduct resistance profile and phylogenetic studies in clinical isolates for identifying infection origins and isolates with augmented pathogenic potential. Here, multilocus sequence typing was performed for phylogenetic analysis of nosocomial isolates of Stenotrophomonas spp. and, environmental and clinical strains of S. maltophilia. Biochemical and multidrug resistance profiles of nosocomial and clinical strains were determined. The inferred phylogenetic profile showed high clonal variability, what correlates with the adaptability process of Stenotrophomonas to different habitats. Two clinical isolates subgroups of S. maltophilia sharing high phylogenetic homogeneity presented intergroup recombination, thus indicating the high permittivity to horizontal gene transfer, a mechanism involved in the acquisition of antibiotic resistance and expression of virulence factors. For most of the clinical strains, phylogenetic inference was made using only partial ppsA gene sequence. Therefore, the sequencing of just one specific fragment of this gene would allow, in many cases, determining whether the infection with S. maltophilia was nosocomial or community-acquired.
Gliomas are aggressive primary brain tumors with high infiltrative potential. The expression of A... more Gliomas are aggressive primary brain tumors with high infiltrative potential. The expression of Angiotensin II (Ang II) receptors has been associated with poor prognosis in human astrocytomas, the most common type of glioma. In this study, we investigated the role of Angiotensin II in glioma malignancy through transcriptional profiling and network analysis of cultured C6 rat glioma cells exposed to Ang II and to inhibitors of its membrane receptor subtypes. C6 cells were treated with Ang II and specific antagonists of AT1 and AT2 receptors. Total RNA was isolated after three and six hours of Ang II treatment and analyzed by oligonucleotide microarray technology. Gene expression data was evaluated through transcriptional network modeling to identify how differentially expressed (DE) genes are connected to each other. Moreover, other genes co-expressing with the DE genes were considered in these analyses in order to support the identification of enriched functions and pathways. A hub-...
About 5-10% of breast and ovarian carcinomas are hereditary and most of these result from germlin... more About 5-10% of breast and ovarian carcinomas are hereditary and most of these result from germline mutations in the BRCA1 and BRCA2 genes. In women of Ashkenazi Jewish ascendance, up to 30% of breast and ovarian carcinomas may be attributable to mutations in these genes, where 3 founder mutations, c.68_69del (185delAG) and c.5266dup (5382insC) in BRCA1 and c.5946del (6174delT) in BRCA2, are commonly encountered. It has been suggested by some authors that screening for founder mutations should be undertaken in all Brazilian women with breast cancer. Thus, the goal of this study was to determine the prevalence of three founder mutations, commonly identified in Ashkenazi individuals in a sample of non-Ashkenazi cancer-affected Brazilian women with clearly defined risk factors for hereditary breast and ovarian cancer (HBOC) syndrome. Among 137 unrelated Brazilian women from HBOC families, the BRCA1 c.5266dup mutation was identified in seven individuals (5%). This prevalence is similar t...
PURPOSE: Screening for mutations in the entire Cystic Fibrosis gene (CFTR) of Brazilian infertile... more PURPOSE: Screening for mutations in the entire Cystic Fibrosis gene (CFTR) of Brazilian infertile men with congenital absence of vas deferens, in order to prevent transmission of CFTR mutations to offspring with the use of assisted reproductive technologies. METHOD: Specific polymerase chain reaction (PCR) primers were designed to each of the 27 exons and splicing sites of interest followed by single strand conformational polymorphism and Heteroduplex Analysis (SSCP-HA) in precast 12.5% polyacrylamide gels at 7 O C and 20 O C. Fragments with abnormal SSCP migration pattern were sequenced. RESULTS: Two novel missense mutations (S753R and G149W) were found in three patients (two brothers) together with the IVS8-5T allele in hetrozygosis. CONCLUSION: The available screenings for CF mutations do not include the atypical mutations associated to absence of vas deferens and thus, when these tests fail to find mutations, there is still a genetic risk of affected children with the help of assisted reproduction. We recommend the screening of the whole CFTR gene for these infertile couples, as part of the work-up before assisted reproduction.
A new Bordetella pertussis strain isolated from a whooping cough Brazilian patient was characteri... more A new Bordetella pertussis strain isolated from a whooping cough Brazilian patient was characterized under molecular and immunological aspects and compared with strains used in the production of whole-cell pertussis vaccine. The isolate, named 21A1, exhibited the bacteriological characteristics classically described for B. pertussis. RAPD and SDS-PAGE analysis showed similar DNA and proteic profiles between classical vaccinal strains and the new isolate. Comparative analysis about the efficacy of vaccines in protecting mice against the intracerebral challenge showed that the 21A1 vaccine was able to induce the highest mouse protection when compared with other B. pertussis vaccines. The results presented here indicate that the inclusion of selected new strain isolates in the composition of the B. pertussis vaccines can be an alternative to maintain or increase vaccine potency.
The low cost of sperm sexing methods combined with in vitro embryo production in genetic improvem... more The low cost of sperm sexing methods combined with in vitro embryo production in genetic improvement programs can increase the profitability of cattle production, in particular when it does not decrease reproductive efficiency. The aim of this work was to evaluate the sex ratio deviation of thawed bovine semen processed by density gradient centrifugation and swim-up. Semen doses were collected from ten bulls of different breeds, and each experimental group was replicated ten times.A Percoll™ gradient was prepared by mixing Dulbecco’s Modified Eagle Medium (DMEM; Sigma Aldrich, St. Louis, MO, USA) isotonic solutions with Percoll™ (GE Healthcare Bio-Science AB, Uppsala, Sweden) stock solution with 0.3% BSA, resulting in densities ranging from 1.110 to 1.123 g mL-1. The layers of discontinuous density gradient were disposed from the larger density (bottom of the tube) to the smaller into 15-mL conical centrifuge tubes. About 40 million sperm were overlaid on Percoll™ gradient and were ...
Ischemia/reperfusion injury (IRI) is a leading cause of acute renal failure. The definition of th... more Ischemia/reperfusion injury (IRI) is a leading cause of acute renal failure. The definition of the molecular mechanisms involved in renal IRI and counter protection promoted by ischemic pre-conditioning (IPC) or Hemin treatment is an important milestone that needs to be accomplished in this research area. We examined, through an oligonucleotide microarray protocol, the renal differential transcriptome profiles of mice submitted to IRI, IPC and Hemin treatment. After identifying the profiles of differentially expressed genes observed for each comparison, we carried out functional enrichment analysis to reveal transcripts putatively involved in potential relevant biological processes and signaling pathways. The most relevant processes found in these comparisons were stress, apoptosis, cell differentiation, angiogenesis, focal adhesion, ECM-receptor interaction, ion transport, angiogenesis, mitosis and cell cycle, inflammatory response, olfactory transduction and regulation of actin cytoskeleton. In addition, the most important overrepresented pathways were MAPK, ErbB, JAK/STAT, Toll and Nod like receptors, Angiotensin II, Arachidonic acid metabolism, Wnt and coagulation cascade. Also, new insights were gained about the underlying protection mechanisms against renal IRI promoted by IPC and Hemin treatment. Venn diagram analysis allowed us to uncover common and exclusively differentially expressed genes between these two protective maneuvers, underscoring potential common and exclusive biological functions regulated in each case. In summary, IPC exclusively regulated the expression of genes belonging to stress, protein modification and apoptosis, highlighting the role of IPC in controlling exacerbated stress response. Treatment with the Hmox1 inducer Hemin, in turn, exclusively regulated the expression of genes associated with cell differentiation, metabolic pathways, cell cycle, mitosis, development, regulation of actin cytoskeleton and arachidonic acid metabolism, suggesting a pleiotropic effect for Hemin. These findings improve the biological understanding of how the kidney behaves after IRI. They also illustrate some possible underlying molecular mechanisms involved in kidney protection observed with IPC or Hemin treatment maneuvers.
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Papers by Carlos Moreira