Gene expression analyses using native populations can provide information on the genetic and molecular mechanisms that determine intraspecific variation and contribute to the understanding of plant development and adaptation in multiple ways. Using quantitative real time – polymerase chain reaction (qRT-PCR), we analyzed the expression of 111 genes with probable roles in wood development in 400 loblolly pine individuals belonging to a population covering much of the natural range. Association mapping techniques are increasingly being used in plants to dissect complex genetic traits and identify genes responsible for the quantitative variation of these traits. We used candidate-gene based association studies to associate single nucleotide polymorphisms (SNPs) in candidate genes with the variation in gene expression. The specific objectives established for this study were to study natural variation in expression of xylem development genes in loblolly pine (Pinus taeda L.) using qRT-PCR, to associate SNPs in candidate genes with the variation in gene expression using candidate-gene based association analyses and to detect loblolly pine promoter polymorphisms and study their effect on gene expression. Out of the 111 genes analyzed using qRT-PCR, there were significant differences in expression among clones for 106 genes. Candidate-gene based association studies were performed between 3937 single nucleotide polymorphisms (SNPs) and gene expression to associate SNPs in candidate genes with the variation in gene expression. To the best of our knowledge, this is the first association genetic study where expression of a large number of genes, analyzed in a natural population, has been the phenotypic trait of interest. We cloned and sequenced promoters of 19 genes, 16 of which are transcription factors involved in wood development and drought response. SNP discovery was done in 13 of these promoters using a panel of 24 loblolly pine clones (unique genotypes). SNP genotyping is underway in the entire association population and association analyses will be done to study the effects of promoter SNPs on gene expression. The results from this project are promising and once these associations have been tested and proved, we believe that they will help in our understanding of the genetics of complex traits.

O Lúpus Eritematoso Sistêmico (LES) é uma doença autoimune inflamatória crônica que possui uma etiopatogênese complexa. Diversos fatores participam da patogênese da doença, dentre eles alterações no balanço de citocinas e quimiocinas. As quimiocinas e seus receptores são fundamentais na regulação da migração de leucócitos durante a inflamação e acredita-se que elas possam ter um papel importante na patogênese de doenças autoimunes, inclusive no LES. Diversos estudos abordaram o papel de quimiocinas e seus receptores no LES, porém, principalmente se tratando dos receptores de quimiocinas CCR5 e CCR2, não existe um consenso. Devido à falta de consenso em relação ao papel dos receptores de quimiocinas na patogênese do LES e considerando a necessidade de mais estudos nesta área, o presente trabalho tem por objetivo investigar o possível papel de polimorfismos na região promotora do CCR5 no desenvolvimento do LES, comparando as frequências dos genótipos e haplótipos entre pacientes e controles, e analisar o possível envolvimento destes polimorfismos nas manifestações clínicas/laboratoriais da doença. O estudo incluiu 382 pacientes com LES (289 Euro-descendentes e 93 Afro-descendentes) e 375 controles (243 Euro-descendentes e 132 Afro-descendentes) genotipados para os polimorfismos CCR2-64I G>A (rs1799864), CCR5-59353 C>T (rs1799988), CCR5-59356 C>T (rs41469351), CCR5-59402 A>G (rs1800023) e CCR5-59653 C>T (rs1800024) através de PCR-RFLP e sequenciamento, respectivamente. Dados prévios de nosso grupo em relação ao CCR5delta32 foram incluídos no estudo para a inferência dos haplótipos e como um possível fator de confusão na regressão binária logística. Os resultados obtidos indicam que, em pacientes Euro-descendentes, as frequências reduzidas o polimorfismo CCR5delta32 e o haplótipo HHG*2 observadas em pacientes quando comparados com controles foram associadas com a doença (p=0,001; OR 3,5; 95%CI 1,6-7,5 e 2,0% vs. 7,2%; presidual=2,9E-5; respectivamente). Em pacientes Afrodescendentes, as frequências dos haplótipos HHA/HHB, HHC e HHG*2 foram diferentes em pacientes e controles (10% vs. 20,5%, presidual = 0,003; 29,4% vs. 17,4%; presidual=0,003 e 3,9% vs. 0,8%; presidual=0,023; respectivamente). Em relação às manifestações clínicas da doença, a presença do CCR5delta32 foi confirmada como um fator de susceptibilidade para nefrite classe IV em pacientes Afro-descendentes e no grupo de pacientes como um todo (pcorrigido=0,012; OR 3,0; 95%CI 3,0-333,3 e pcorrigido=0,0006; OR 6,8; 95%CI 1,9-2,48; respectivamente). Em conclusão, o presente estudo indica que polimorfismos na região promotora do CCR5 podem atuar como modificadores no LES. Os resultados observados reforçam o papel do polimorfismo CCR5delta32 como um fator de proteção para o desenvolvimento do LES em Euro-descendentes e como um fator de susceptibilidade à nefrite classe IV em pacientes Afro-descendentes. Além disto, também foram descritos a redução da frequência dos haplótipos HHA/HHB e o aumento da frequência dos haplótipos HHC e HHG*2 em pacientes Afro-descendentes, que possivelmente podem estar associados com uma maior expressão do CCR5 em subtipos específicos celulares e com uma menor expressão deste receptor de maneira geral. / Systemic Lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease, characterized by a complex etiopathogenesis. Many factors are known to participate in the pathogenesis of SLE, including alterations in the cytokines or chemokines balance. Chemokines and their receptors are central players in the regulation of leucocytes chemotaxis in inflammation and they are thought to have an important role in the pathogenesis of autoimmune diseases, including SLE. Several studies have addressed the role of chemokines and their receptors in SLE, however there is no consensus regarding their involvement on the pathogenesis of the disease. Given the lack of consensus considering the role of chemokine receptors in SLE pathogenesis and the need for more studies in this area, the present work aims to investigate a possible role of the CCR5 promoter region polymorphisms in the development of SLE comparing the frequencies of the genotypes and haplotypes with ethnically matched controls and analyze if there is a possible involvement of the polymorphisms in the clinical outcome of the disease. This study included 388 SLE patients (289 classified as Europeanderived and 93 as African-derived) and 375 controls (243 European-derived and 132 African-derived) genotyped for the CCR2-64I G>A (rs1799864), CCR5-59353 C>T (rs1799988), CCR5-59356 C>T (rs41469351), CCR5-59402 A>G (rs1800023) and CCR5-59653 C>T (rs1800024) polymorphisms though PCRRFLP and direct sequencing, respectively. Previous data from CCR5delta32 were included in the study to infer the haplotypes and also as a possible confounding factor in the binary logistic regression. Our results indicated that, in Europeanderived patients, CCR5delta32 and the HHG*2 haplotype reduced frequencies in patients when compared to controls were associated with the disease (p=0.001; OR 3.5; 95%CI 1.6-7.5 and 2.0%, vs. 7.2% residual p= 2.9E-5, respectively). In African-derived patients, the HHA/HHB, HHC and HHG*2 haplotype frequencies differed between patients and controls (10% vs. 20.5%, residual p= 0.003; 29.4% vs. 17.4%, residual p=0.003 and 3.9% vs. 0.8%, residual p=0.023; respectively). Considering the clinical manifestations of the disease, CCR5delta32 presence was confirmed as a susceptibility factor to class IV nephritis in the African-derived group and when patients were considered together (pcorrected=0.012; OR 3.0; 95%CI 3.0-333.3 and pcorrected= 0.0006; OR 6.8; 95%CI 1.9-2.48, respectively). In conclusion, this study indicates that CCR5 promoter polymorphisms are important disease modifiers in SLE. Present data reinforces CCR5delta32 polymorphism as a protective factor for the development of the disease in European-derived patients and as a susceptibility factor for class IV nephritis in African-derived patients. Furthermore, we also describe a reduced frequency of HHA/HHB and an enhanced frequency of HHC and HHG*2 haplotypes in our African-derived patients, which potentially could reflect in a higher expression of CCR5 in specific cell subsets and in a lower expression of CCR5 overall.

O Lúpus Eritematoso Sistêmico (LES) é uma doença autoimune inflamatória crônica que possui uma etiopatogênese complexa. Diversos fatores participam da patogênese da doença, dentre eles alterações no balanço de citocinas e quimiocinas. As quimiocinas e seus receptores são fundamentais na regulação da migração de leucócitos durante a inflamação e acredita-se que elas possam ter um papel importante na patogênese de doenças autoimunes, inclusive no LES. Diversos estudos abordaram o papel de quimiocinas e seus receptores no LES, porém, principalmente se tratando dos receptores de quimiocinas CCR5 e CCR2, não existe um consenso. Devido à falta de consenso em relação ao papel dos receptores de quimiocinas na patogênese do LES e considerando a necessidade de mais estudos nesta área, o presente trabalho tem por objetivo investigar o possível papel de polimorfismos na região promotora do CCR5 no desenvolvimento do LES, comparando as frequências dos genótipos e haplótipos entre pacientes e controles, e analisar o possível envolvimento destes polimorfismos nas manifestações clínicas/laboratoriais da doença. O estudo incluiu 382 pacientes com LES (289 Euro-descendentes e 93 Afro-descendentes) e 375 controles (243 Euro-descendentes e 132 Afro-descendentes) genotipados para os polimorfismos CCR2-64I G>A (rs1799864), CCR5-59353 C>T (rs1799988), CCR5-59356 C>T (rs41469351), CCR5-59402 A>G (rs1800023) e CCR5-59653 C>T (rs1800024) através de PCR-RFLP e sequenciamento, respectivamente. Dados prévios de nosso grupo em relação ao CCR5delta32 foram incluídos no estudo para a inferência dos haplótipos e como um possível fator de confusão na regressão binária logística. Os resultados obtidos indicam que, em pacientes Euro-descendentes, as frequências reduzidas o polimorfismo CCR5delta32 e o haplótipo HHG*2 observadas em pacientes quando comparados com controles foram associadas com a doença (p=0,001; OR 3,5; 95%CI 1,6-7,5 e 2,0% vs. 7,2%; presidual=2,9E-5; respectivamente). Em pacientes Afrodescendentes, as frequências dos haplótipos HHA/HHB, HHC e HHG*2 foram diferentes em pacientes e controles (10% vs. 20,5%, presidual = 0,003; 29,4% vs. 17,4%; presidual=0,003 e 3,9% vs. 0,8%; presidual=0,023; respectivamente). Em relação às manifestações clínicas da doença, a presença do CCR5delta32 foi confirmada como um fator de susceptibilidade para nefrite classe IV em pacientes Afro-descendentes e no grupo de pacientes como um todo (pcorrigido=0,012; OR 3,0; 95%CI 3,0-333,3 e pcorrigido=0,0006; OR 6,8; 95%CI 1,9-2,48; respectivamente). Em conclusão, o presente estudo indica que polimorfismos na região promotora do CCR5 podem atuar como modificadores no LES. Os resultados observados reforçam o papel do polimorfismo CCR5delta32 como um fator de proteção para o desenvolvimento do LES em Euro-descendentes e como um fator de susceptibilidade à nefrite classe IV em pacientes Afro-descendentes. Além disto, também foram descritos a redução da frequência dos haplótipos HHA/HHB e o aumento da frequência dos haplótipos HHC e HHG*2 em pacientes Afro-descendentes, que possivelmente podem estar associados com uma maior expressão do CCR5 em subtipos específicos celulares e com uma menor expressão deste receptor de maneira geral. / Systemic Lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease, characterized by a complex etiopathogenesis. Many factors are known to participate in the pathogenesis of SLE, including alterations in the cytokines or chemokines balance. Chemokines and their receptors are central players in the regulation of leucocytes chemotaxis in inflammation and they are thought to have an important role in the pathogenesis of autoimmune diseases, including SLE. Several studies have addressed the role of chemokines and their receptors in SLE, however there is no consensus regarding their involvement on the pathogenesis of the disease. Given the lack of consensus considering the role of chemokine receptors in SLE pathogenesis and the need for more studies in this area, the present work aims to investigate a possible role of the CCR5 promoter region polymorphisms in the development of SLE comparing the frequencies of the genotypes and haplotypes with ethnically matched controls and analyze if there is a possible involvement of the polymorphisms in the clinical outcome of the disease. This study included 388 SLE patients (289 classified as Europeanderived and 93 as African-derived) and 375 controls (243 European-derived and 132 African-derived) genotyped for the CCR2-64I G>A (rs1799864), CCR5-59353 C>T (rs1799988), CCR5-59356 C>T (rs41469351), CCR5-59402 A>G (rs1800023) and CCR5-59653 C>T (rs1800024) polymorphisms though PCRRFLP and direct sequencing, respectively. Previous data from CCR5delta32 were included in the study to infer the haplotypes and also as a possible confounding factor in the binary logistic regression. Our results indicated that, in Europeanderived patients, CCR5delta32 and the HHG*2 haplotype reduced frequencies in patients when compared to controls were associated with the disease (p=0.001; OR 3.5; 95%CI 1.6-7.5 and 2.0%, vs. 7.2% residual p= 2.9E-5, respectively). In African-derived patients, the HHA/HHB, HHC and HHG*2 haplotype frequencies differed between patients and controls (10% vs. 20.5%, residual p= 0.003; 29.4% vs. 17.4%, residual p=0.003 and 3.9% vs. 0.8%, residual p=0.023; respectively). Considering the clinical manifestations of the disease, CCR5delta32 presence was confirmed as a susceptibility factor to class IV nephritis in the African-derived group and when patients were considered together (pcorrected=0.012; OR 3.0; 95%CI 3.0-333.3 and pcorrected= 0.0006; OR 6.8; 95%CI 1.9-2.48, respectively). In conclusion, this study indicates that CCR5 promoter polymorphisms are important disease modifiers in SLE. Present data reinforces CCR5delta32 polymorphism as a protective factor for the development of the disease in European-derived patients and as a susceptibility factor for class IV nephritis in African-derived patients. Furthermore, we also describe a reduced frequency of HHA/HHB and an enhanced frequency of HHC and HHG*2 haplotypes in our African-derived patients, which potentially could reflect in a higher expression of CCR5 in specific cell subsets and in a lower expression of CCR5 overall.

O Lúpus Eritematoso Sistêmico (LES) é uma doença autoimune inflamatória crônica que possui uma etiopatogênese complexa. Diversos fatores participam da patogênese da doença, dentre eles alterações no balanço de citocinas e quimiocinas. As quimiocinas e seus receptores são fundamentais na regulação da migração de leucócitos durante a inflamação e acredita-se que elas possam ter um papel importante na patogênese de doenças autoimunes, inclusive no LES. Diversos estudos abordaram o papel de quimiocinas e seus receptores no LES, porém, principalmente se tratando dos receptores de quimiocinas CCR5 e CCR2, não existe um consenso. Devido à falta de consenso em relação ao papel dos receptores de quimiocinas na patogênese do LES e considerando a necessidade de mais estudos nesta área, o presente trabalho tem por objetivo investigar o possível papel de polimorfismos na região promotora do CCR5 no desenvolvimento do LES, comparando as frequências dos genótipos e haplótipos entre pacientes e controles, e analisar o possível envolvimento destes polimorfismos nas manifestações clínicas/laboratoriais da doença. O estudo incluiu 382 pacientes com LES (289 Euro-descendentes e 93 Afro-descendentes) e 375 controles (243 Euro-descendentes e 132 Afro-descendentes) genotipados para os polimorfismos CCR2-64I G>A (rs1799864), CCR5-59353 C>T (rs1799988), CCR5-59356 C>T (rs41469351), CCR5-59402 A>G (rs1800023) e CCR5-59653 C>T (rs1800024) através de PCR-RFLP e sequenciamento, respectivamente. Dados prévios de nosso grupo em relação ao CCR5delta32 foram incluídos no estudo para a inferência dos haplótipos e como um possível fator de confusão na regressão binária logística. Os resultados obtidos indicam que, em pacientes Euro-descendentes, as frequências reduzidas o polimorfismo CCR5delta32 e o haplótipo HHG*2 observadas em pacientes quando comparados com controles foram associadas com a doença (p=0,001; OR 3,5; 95%CI 1,6-7,5 e 2,0% vs. 7,2%; presidual=2,9E-5; respectivamente). Em pacientes Afrodescendentes, as frequências dos haplótipos HHA/HHB, HHC e HHG*2 foram diferentes em pacientes e controles (10% vs. 20,5%, presidual = 0,003; 29,4% vs. 17,4%; presidual=0,003 e 3,9% vs. 0,8%; presidual=0,023; respectivamente). Em relação às manifestações clínicas da doença, a presença do CCR5delta32 foi confirmada como um fator de susceptibilidade para nefrite classe IV em pacientes Afro-descendentes e no grupo de pacientes como um todo (pcorrigido=0,012; OR 3,0; 95%CI 3,0-333,3 e pcorrigido=0,0006; OR 6,8; 95%CI 1,9-2,48; respectivamente). Em conclusão, o presente estudo indica que polimorfismos na região promotora do CCR5 podem atuar como modificadores no LES. Os resultados observados reforçam o papel do polimorfismo CCR5delta32 como um fator de proteção para o desenvolvimento do LES em Euro-descendentes e como um fator de susceptibilidade à nefrite classe IV em pacientes Afro-descendentes. Além disto, também foram descritos a redução da frequência dos haplótipos HHA/HHB e o aumento da frequência dos haplótipos HHC e HHG*2 em pacientes Afro-descendentes, que possivelmente podem estar associados com uma maior expressão do CCR5 em subtipos específicos celulares e com uma menor expressão deste receptor de maneira geral. / Systemic Lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease, characterized by a complex etiopathogenesis. Many factors are known to participate in the pathogenesis of SLE, including alterations in the cytokines or chemokines balance. Chemokines and their receptors are central players in the regulation of leucocytes chemotaxis in inflammation and they are thought to have an important role in the pathogenesis of autoimmune diseases, including SLE. Several studies have addressed the role of chemokines and their receptors in SLE, however there is no consensus regarding their involvement on the pathogenesis of the disease. Given the lack of consensus considering the role of chemokine receptors in SLE pathogenesis and the need for more studies in this area, the present work aims to investigate a possible role of the CCR5 promoter region polymorphisms in the development of SLE comparing the frequencies of the genotypes and haplotypes with ethnically matched controls and analyze if there is a possible involvement of the polymorphisms in the clinical outcome of the disease. This study included 388 SLE patients (289 classified as Europeanderived and 93 as African-derived) and 375 controls (243 European-derived and 132 African-derived) genotyped for the CCR2-64I G>A (rs1799864), CCR5-59353 C>T (rs1799988), CCR5-59356 C>T (rs41469351), CCR5-59402 A>G (rs1800023) and CCR5-59653 C>T (rs1800024) polymorphisms though PCRRFLP and direct sequencing, respectively. Previous data from CCR5delta32 were included in the study to infer the haplotypes and also as a possible confounding factor in the binary logistic regression. Our results indicated that, in Europeanderived patients, CCR5delta32 and the HHG*2 haplotype reduced frequencies in patients when compared to controls were associated with the disease (p=0.001; OR 3.5; 95%CI 1.6-7.5 and 2.0%, vs. 7.2% residual p= 2.9E-5, respectively). In African-derived patients, the HHA/HHB, HHC and HHG*2 haplotype frequencies differed between patients and controls (10% vs. 20.5%, residual p= 0.003; 29.4% vs. 17.4%, residual p=0.003 and 3.9% vs. 0.8%, residual p=0.023; respectively). Considering the clinical manifestations of the disease, CCR5delta32 presence was confirmed as a susceptibility factor to class IV nephritis in the African-derived group and when patients were considered together (pcorrected=0.012; OR 3.0; 95%CI 3.0-333.3 and pcorrected= 0.0006; OR 6.8; 95%CI 1.9-2.48, respectively). In conclusion, this study indicates that CCR5 promoter polymorphisms are important disease modifiers in SLE. Present data reinforces CCR5delta32 polymorphism as a protective factor for the development of the disease in European-derived patients and as a susceptibility factor for class IV nephritis in African-derived patients. Furthermore, we also describe a reduced frequency of HHA/HHB and an enhanced frequency of HHC and HHG*2 haplotypes in our African-derived patients, which potentially could reflect in a higher expression of CCR5 in specific cell subsets and in a lower expression of CCR5 overall.

Two polymorphisms in the promoter region of macrophage migration inhibitory factor (MIF)—rs755622 and rs5844572—exhibit prognostic relevance in inflammatory diseases. The aim of this study was to investigate a correlation between these MIF promoter polymorphisms and the severity of hepatitis C virus (HCV)-induced liver fibrosis. Our analysis included two independent patient cohorts with HCV-induced liver fibrosis (504 and 443 patients, respectively). The genotype of the single nucleotide polymorphism (SNP) -173 G/C and the repeat number of the microsatellite polymorphism -794 CATT5–8 were determined in DNA samples and correlated with fibrosis severity. In the first cohort, homozygous carriers of the C allele in the rs755622 had lower fibrosis stages compared to heterozygous carriers or wild types (1.25 vs. 2.0 vs. 2.0; p = 0.03). Additionally, 7 microsatellite repeats were associated with lower fibrosis stages (<F2) (p = 0.04). Comparable tendencies were observed in the second independent cohort, where fibrosis was assessed using transient elastography. However, once cirrhosis had been established, the C/C genotype and higher microsatellite repeats correlated with impaired liver function and a higher prevalence of hepatocellular carcinoma. Our study demonstrates that specific MIF polymorphisms are associated with disease severity and complications of HCV-induced fibrosis in a stage- and context-dependent manner.