<p>Glutathione transferases (GSTs) constitute a family of multifarious enzymes that conjugate glutathione (GSH) with a wide range of electrophiles. GSTs are grouped into different classes based on protein sequence similarities. Despite high sequence identities between GSTs of the same class they often display different substrate specificites. Human GST M1-1 is efficiently catalyzing the conjugation of GSH and various epoxide substrates, whereas the 84% sequence-identical GST M2-2 has low activities with the same substrates.</p><p>Evolutionary rate analysis was used to identify hypervariable amino acid positions among GST Mu class sequences. A Thr to Ser conversion of the variable residue 210 in GST M2-2 elicited a drastic increase in catalytic activity with epoxides, which is the characteristic activity of GST M1-1. This provides support for the usefulness of evolutionary analysis in identifying functionally important residues, although the additional mutations of two other variable residues did not confer any noteworthy changes in activity.</p><p>To further investigate the functional importance of residue T210 in GST M2-2 it was replaced by all other commonly occurring amino acids. The replacements caused marked changes in substrate specificity, stability, and expressivity, indicating how functionalities of a duplicated Mu class GST may easily be altered by point mutations. </p><p>The stereo- and regioselectivity in epoxide-conjugation catalyzed by GSTs M1-1 and M2-2 was investigated. The results show that a serine in position 210 is beneficial for high enantioselectivity with trans-stilbene oxide. However, an alanine in position 210 is more favorable for stereo- and regioselectivity with the smaller epoxide substrate styrene-7,8-oxide. </p><p>The low enantioselectivity of GST M1-1 was improved 10- and 9- fold with styrene-7,8-oxide and 1-phenylpropylene oxide, respectively, through different combination of site-specific mutations and posttranslational chemical modifications. The approach can be employed in more extensive screening experiments where a large variety of modifications easily can be tested.</p>

Glutathione transferases (GSTs) constitute a family of multifarious enzymes that conjugate glutathione (GSH) with a wide range of electrophiles. GSTs are grouped into different classes based on protein sequence similarities. Despite high sequence identities between GSTs of the same class they often display different substrate specificites. Human GST M1-1 is efficiently catalyzing the conjugation of GSH and various epoxide substrates, whereas the 84% sequence-identical GST M2-2 has low activities with the same substrates. Evolutionary rate analysis was used to identify hypervariable amino acid positions among GST Mu class sequences. A Thr to Ser conversion of the variable residue 210 in GST M2-2 elicited a drastic increase in catalytic activity with epoxides, which is the characteristic activity of GST M1-1. This provides support for the usefulness of evolutionary analysis in identifying functionally important residues, although the additional mutations of two other variable residues did not confer any noteworthy changes in activity. To further investigate the functional importance of residue T210 in GST M2-2 it was replaced by all other commonly occurring amino acids. The replacements caused marked changes in substrate specificity, stability, and expressivity, indicating how functionalities of a duplicated Mu class GST may easily be altered by point mutations. The stereo- and regioselectivity in epoxide-conjugation catalyzed by GSTs M1-1 and M2-2 was investigated. The results show that a serine in position 210 is beneficial for high enantioselectivity with trans-stilbene oxide. However, an alanine in position 210 is more favorable for stereo- and regioselectivity with the smaller epoxide substrate styrene-7,8-oxide. The low enantioselectivity of GST M1-1 was improved 10- and 9- fold with styrene-7,8-oxide and 1-phenylpropylene oxide, respectively, through different combination of site-specific mutations and posttranslational chemical modifications. The approach can be employed in more extensive screening experiments where a large variety of modifications easily can be tested.

The mutability of transcription factors (TF) is thought to be of high importance for the evolutionary change of living organisms. Transcription factors, coactivators, coregulators, kinases, chromatin remodelers conditional factors and other proteins together govern the timing and level of gene expression. About 10% of the genes in the human genome are predicted to be TFs and mutational changes in these genes or in the target regulatory sequences they bind will potentially give rise to evolutionary advantages or malfunctions for the organism. Recent research has suggested that the parts of the transcription factors that are not structurally defined in solution, so called intrinsically disordered regions (IDRs), have a higher potential for evolutionary diversification than more structurally rigid regions. This suggests that these domains that earlier have been considered mostly unimportant may have an important potential for evolutionary diversification. This project aimed to further evaluate evidence supporting the hypothesis that variable-structured domains in transcription factors are of significant importance for functional diversification. This was be done by comparing the rate of synonymous and non-synonymous genetic variation in the coding regions of 12 selected TFs within a highly conserved clade of European wine yeasts and by comparing this variation to divergent phenotypic patterns within the strains. The frequency of non-synonymous mutations was much greater than for synonymous mutations indicating an important role of positive selection acting on these TFs during diversification of the different strains. No significant connections were discovered between the distribution of DNA variation and phenotypic patterns.

HIV-1 genetic diversity is a major obstacle for developing an effective vaccine. My hypothesis is that HIV-1 genetic diversity can be characterized and that cross-clade immunogens can be predicted at the population level. I systematically investigated positive selection (PS) pressures on HIV-1 Env and Gag proteins based on the analysis of the sequences collected from the Los Alamos Sequence Database. I identified PS sites, investigated PS patterns, correlated PS with the known functional sites of the two proteins, calculated frequencies of HLA alleles targeting CTL epitopes, and compared PS patterns among major subtypes. The results showed that PS pressure was widely dispersed across the entire regions of both HIV-1 Env and Gag proteins, suggesting the conserved regions are under host immune response pressure. The neutralizing antibody, non-neutralizing antibody, and CTL responses were found to be the major forces driving genetic diversity of HIV-1 env and gag genes at population level. However, PS pressures on both Env and Gag proteins remain stable over time, suggesting genetic diversity of HIV-1 driven by host immune responses changed very little over the last 29 years. Furthermore, the results also demonstrated that up to 70% PS sites were shared among the major HIV-1 clades, implying the existence of cross-clade immunogenicity. A number of potential cross-clades immunogens were predicted to elicit CTL or neutralizing antibody responses from Env and Gag proteins. I also detected a significant correlation between HLA allele frequencies and host CTL responses elicited by Accessory/Regulator’s proteins at population level. Moreover, I detected an association between the frequency of HLA-B7 supertype and the number of identified optimal CTL epitopes. The results suggest HLA class I allele frequencies in a population influence the evolution of HIV-1. I also systematically evaluated the utility of ultra-deep pyrosequencing to characterize genetic diversity of HIV-1 gag genes within quasispecies. The results showed that ultra-deep pyrosequencing of amplified HIV genes is a better method than the traditional Sanger-clone-based method in the comprehensive characterization of genetic diversity of HIV-1 quasispecies, especially in detecting low frequency variations. In conclusion, my thesis provides important information for rational design of an effective HIV-1 vaccine.

HIV-1 genetic diversity is a major obstacle for developing an effective vaccine. My hypothesis is that HIV-1 genetic diversity can be characterized and that cross-clade immunogens can be predicted at the population level. I systematically investigated positive selection (PS) pressures on HIV-1 Env and Gag proteins based on the analysis of the sequences collected from the Los Alamos Sequence Database. I identified PS sites, investigated PS patterns, correlated PS with the known functional sites of the two proteins, calculated frequencies of HLA alleles targeting CTL epitopes, and compared PS patterns among major subtypes. The results showed that PS pressure was widely dispersed across the entire regions of both HIV-1 Env and Gag proteins, suggesting the conserved regions are under host immune response pressure. The neutralizing antibody, non-neutralizing antibody, and CTL responses were found to be the major forces driving genetic diversity of HIV-1 env and gag genes at population level. However, PS pressures on both Env and Gag proteins remain stable over time, suggesting genetic diversity of HIV-1 driven by host immune responses changed very little over the last 29 years. Furthermore, the results also demonstrated that up to 70% PS sites were shared among the major HIV-1 clades, implying the existence of cross-clade immunogenicity. A number of potential cross-clades immunogens were predicted to elicit CTL or neutralizing antibody responses from Env and Gag proteins. I also detected a significant correlation between HLA allele frequencies and host CTL responses elicited by Accessory/Regulator’s proteins at population level. Moreover, I detected an association between the frequency of HLA-B7 supertype and the number of identified optimal CTL epitopes. The results suggest HLA class I allele frequencies in a population influence the evolution of HIV-1. I also systematically evaluated the utility of ultra-deep pyrosequencing to characterize genetic diversity of HIV-1 gag genes within quasispecies. The results showed that ultra-deep pyrosequencing of amplified HIV genes is a better method than the traditional Sanger-clone-based method in the comprehensive characterization of genetic diversity of HIV-1 quasispecies, especially in detecting low frequency variations. In conclusion, my thesis provides important information for rational design of an effective HIV-1 vaccine.

Monócitos são células hematopoiéticas com função na imunidade inata e adquirida. De acordo com o estímulo que recebem, podem se diferenciar em macrófagos e potencializar suas funções efetoras, modulando a resposta imune e participando de vários processos fisiológicos e patológicos. Os macrófagos são muito heterogêneos e capazes de assumir diferentes fenótipos em resposta aos estímulos que recebem do microambiente. Em um ambiente gorvernado por interferon gama (IFN-γ), se diferenciam em células com aumentada capacidade de apresentação de antígenos e síntese de citocinas pró-inflamatórias (macrófagos M1). Por outro lado, quando são estimulados por interleucina-4 (IL-4), eles se diferenciam em um fenótipo antagonista com atividade de reparo (macrófagos M2). Dada a importância destas células no sistema imune, é necessário desenvolver e otimizar técnicas que sirvam de ferramentas para estudar a diferenciação de macrófagos em seus perfis fenotípicos bem como seu papel em doenças humanas. Assim, o objetivo deste estudo foi padronizar e comparar dois diferentes protocolos de isolamento de monócitos descritos na literatura e analisar seu impacto sobre a diferenciação de macrófagos. Isolamos monócitos do sangue periférico de cinco indivíduos saudáveis pelas técnicas de aderência e seleção positiva. Os monócitos foram diferenciados a macrófagos com suplementação de M-CSF. Depois da indução aos perfis M1 e M2, avaliamos marcadores de superfície celular, expressão de mRNA de citocinas, secreção de quimiocinas e fagocitose. Observamos que os métodos utilizados para isolar monócitos possuem diferentes purezas, mas que monócitos isolados por ambos métodos foram capazes de ser diferenciados a macrófagos em seus perfis M1 e M2. Análises de citometria de fluxo mostraram que há uma diminuição da expressão de CD14, principalmente em macrófagos M2, e manutenção (M2) ou aumento (m1) da expressão de HLA-DR. Monócitos (CD80-CD86high) induzidos aos fenótipo M1 são caracterizados pela regulação positiva de CD80 e regulação negativa de CD86 (CD80++CD86+). O perfil M2 foi caracterizado pela expressão de CD206high e ausência de CD163- (CD206highCD163-). A expressão do mRNA revelou que IL-1β e TNF-α foram marcadores de M1 e TGF-β e CCL18 foram marcadores de M2. Além disso, quimiocinas inflamatórias como CXCL9, CXCL10 e CCL5 foram significativamente aumentadas em macrophagos M1. Macrófagos M1 e M2 são ativos e funcionais como demonstrado no ensaio de fagocitose. Embora ambos métodos utilizados para isolar monócitos tiveram purezas diferentes, ambas técnicas forneceram monócitos capazes de serem diferenciados a macrófagos. / Monocytes are hematopoietic cells with a major role in innate and adaptative immunity. According to the stimulus they receive, they can differentiate into macrophages and enhance effector functions by modulating the immune response and participating in various physiological and pathophysiological processes. Macrophages are very heterogeneous and are able to assume different phenotypes in response to the different stimuli they receive from the microenvironment. In a proinflammatory milieu ruled by interferon gamma (IFN-γ), they differentiate into cells with increased capacity to present antigens and synthesis of proinflammatory cytokines (M1 macrophages). On the other hand, when they are stimulated with interleukin 4 (IL-4), they differentiate into an antagonist phenotype with repair activity (M2 macrophages). Given the importance of these cells in the immune system, it is necessary to develop and optimize techniques that serve as useful tools for studying the differentiation of macrophages in their different phenotypic profiles as well as their roles in human diseases. In this regard, the aim of this study was to standardize and compare two different human monocyte isolation protocols described in the literature and analyze their impact on macrophage differentiation. We isolated peripheral blood monocytes from five healthy subjects by the adherence technique and positive selection. Monocytes were differentiated into macrophages with M-CSF supplementation. After M1 or M2 induction, we evaluated cell surface markers, mRNA cytokine expression, chemokine secretion and phagocytosis. We found that the methods used to isolate monocytes have different purities, but monocytes isolated from both methods were able to differentiate into the M1 and M2 profile. The monocyte and macrophage flow cytometry analysis demonstrated that CD14 decreased expression, mainly in M2 macrophages, and maintained (M2) or increased (M1) the HLA-DR expression. Monocytes (CD80-CD86high) induced to an M1 phenotype were characterized by upregulation of CD80 and down regulation of CD86. (CD80++CD86+) The M2 profile was characterized by the expression of CD206high and absence of CD163- (CD206highCD163-). The mRNA expression revealed that IL-1β and TNF-α were M1 markers and TGF-β and CCL18 were M2 markers. Further more, inflammatory chemokines as CXCL9, CXCL10 and CCL5 were significantly increased in M1 macrophages. M1 and M2 macrophages were active and functional as shown in the phagocytic assay. Although methods used for the isolation of monocytes yielded different purities, both techniques provided monocytes able to differentiate to macrophages.

Submitted by Izabel Franco (izabel-franco@ufscar.br) on 2016-10-10T13:11:28Z No. of bitstreams: 1 DissJLO.pdf: 1924288 bytes, checksum: 2b2ce6a9b82a3dfb41ddb5f343fde1b7 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-20T19:39:32Z (GMT) No. of bitstreams: 1 DissJLO.pdf: 1924288 bytes, checksum: 2b2ce6a9b82a3dfb41ddb5f343fde1b7 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-20T19:39:39Z (GMT) No. of bitstreams: 1 DissJLO.pdf: 1924288 bytes, checksum: 2b2ce6a9b82a3dfb41ddb5f343fde1b7 (MD5) / Made available in DSpace on 2016-10-20T19:39:46Z (GMT). No. of bitstreams: 1 DissJLO.pdf: 1924288 bytes, checksum: 2b2ce6a9b82a3dfb41ddb5f343fde1b7 (MD5) Previous issue date: 2014-02-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / The genus Anastrepha (Tephritidae) has several species of great economic importance due to their impact in fruticulture. We are particularly interested in the evolutionary history of Anastrepha fraterculus, the most relevant species in the closely related group of species fraterculus, which has undergone recent divergence and holds the majority of species of economic importance in the genus. To better understand the differentiation process and identification in the group, we need to find molecular and morphological markers that are involved with the differences between species. Reproductive genes have, in general, been very informative in this regard due to their rapid evolutionary rates, although few of them have been studied and characterized in this species so far. Therefore, the present study focuses on the exuperantia gene (exu), which participates in both oogenesis (localization of bicoid and oskar mRNAs) and spermatogenesis, since exu embryos from mutant mothers are unviable and males are sterile. Thus, the first step was to use Next generation sequencing strategies (RNA-seq) to structurally characterize and define expression patterns of this gene between sexes in the cephalic and reproductive tissues of A. fraterculus. A. fraterculus has similar structural and expression patterns to Drosophila melanogaster in reproductive tissues, in which the transcripts differ between sexes for the 5’ and 3’ untranslated regions (UTRs). We describe for the first time the expression of exu in cephalic tissues, involving a new isoform that is common to both sexes. All these alternative spliced transcripts share a common coding region, resulting in the same protein. We used the exu coding region, along with sequences from several Diptera to investigate the molecular evolution of exu in Cyclorrhapha. This group has experienced an enormous adaptive radiation, associated with molecular and morphological changes, and by comparing the dN/dS ratio between Cyclorrhapha and other Diptera we found that exu was subjected to positive selection in Cyclorrhapha for at least two sites in the coding region. One of the sites is present in the RNA exonuclease-like domain of exu. The second site is located in a not characterized region that is conserved and under purifying selection in Diptera, suggesting that it may be an important region of the protein. The adaptive changes found in exu may reflect an evolutionary gain that allowed its co-option for a new function the gene performs in Cyclorrhapha: the localization of bicoid in the anterior region of the oocyte, since bcd is a gene exclusive of Cyclorrhapha. / O gênero Anastrepha (Tephritidae) é de grande importância econômica devido ao seu impacto na fruticultura nacional. Estudamos aqui a história evolutiva de Anastrepha fraterculus, a espécie mais relevante do grupo fraterculus, o qual sofreu divergência recente e contém a maioria das espécies de importância econômica do gênero. Para entender melhor o processo de especiação nesse grupo, e auxiliar no processo de identificação taxonômica, precisamos estabelecer marcadores morfológicos e moleculares envolvidos com as diferenças entre as espécies. Genes envolvidos com a reprodução têm se mostrado bastante informativos para estes propósitos, devido às rápidas taxas evolutivas que apresentam, embora poucos tenham sido estudados e caracterizados nessas espécies até o momento. Nesse sentido, esse trabalho tem como foco o gene exuperantia (exu), que participa tanto da ovogênese (localização dos mRNAs bicoid e oskar) quanto da espermatogênese, uma vez que embriões formados a partir de fêmeas mutantes exu são inviáveis e machos mutantes exu são estéreis. Assim, o primeiro passo foi caracterizar estruturalmente e definir padrões de expressão desse gene entre os sexos nos tecidos cefálicos e reprodutivos em A. fraterculus. Utilizando sequenciamento de próxima geração (RNA-seq), identificamos um padrão estrutural e de expressão semelhante àquele de Drosophila melanogaster em tecidos reprodutivos: os transcritos diferem nas regiões não traduzidas (UTRs) 5’ e 3’ entre os sexos. Pela primeira vez identificamos a expressão de exu em tecidos cefálicos, envolvendo uma nova isoforma que é igual nos dois sexos. Em todos os casos, as regiões codificadoras são iguais, resultando na sequência proteica. Utilizamos essa região codificadora de exu junto com sequências de outras espécies de Diptera para investigar a evolução molecular de exu em Cyclorrhapha, que sofreu uma grande radiação adaptativa associada com alterações morfológicas e moleculares. Comparando a relação dN/dS entre Cyclorrhapha e outros Diptera, encontramos dois sítios sob seleção positiva em Cyclorrhapha, um deles presente no domínio semelhante à RNA exonuclease de exu. O segundo está presente em uma região não caracterizada para domínios proteicos, mas que é bem conservada e está sob seleção purificadora em Diptera, sugerindo ser uma região importante da proteína. As mudanças adaptativas encontradas em exu podem refletir um ganho evolutivo que permitiu sua co-optação para uma nova função em Cyclorrhapha: a de localização de bicoid na região anterior do ovócito, visto que bcd é um gene exclusivo de Cyclorrhapha.

Monócitos são células hematopoiéticas com função na imunidade inata e adquirida. De acordo com o estímulo que recebem, podem se diferenciar em macrófagos e potencializar suas funções efetoras, modulando a resposta imune e participando de vários processos fisiológicos e patológicos. Os macrófagos são muito heterogêneos e capazes de assumir diferentes fenótipos em resposta aos estímulos que recebem do microambiente. Em um ambiente gorvernado por interferon gama (IFN-γ), se diferenciam em células com aumentada capacidade de apresentação de antígenos e síntese de citocinas pró-inflamatórias (macrófagos M1). Por outro lado, quando são estimulados por interleucina-4 (IL-4), eles se diferenciam em um fenótipo antagonista com atividade de reparo (macrófagos M2). Dada a importância destas células no sistema imune, é necessário desenvolver e otimizar técnicas que sirvam de ferramentas para estudar a diferenciação de macrófagos em seus perfis fenotípicos bem como seu papel em doenças humanas. Assim, o objetivo deste estudo foi padronizar e comparar dois diferentes protocolos de isolamento de monócitos descritos na literatura e analisar seu impacto sobre a diferenciação de macrófagos. Isolamos monócitos do sangue periférico de cinco indivíduos saudáveis pelas técnicas de aderência e seleção positiva. Os monócitos foram diferenciados a macrófagos com suplementação de M-CSF. Depois da indução aos perfis M1 e M2, avaliamos marcadores de superfície celular, expressão de mRNA de citocinas, secreção de quimiocinas e fagocitose. Observamos que os métodos utilizados para isolar monócitos possuem diferentes purezas, mas que monócitos isolados por ambos métodos foram capazes de ser diferenciados a macrófagos em seus perfis M1 e M2. Análises de citometria de fluxo mostraram que há uma diminuição da expressão de CD14, principalmente em macrófagos M2, e manutenção (M2) ou aumento (m1) da expressão de HLA-DR. Monócitos (CD80-CD86high) induzidos aos fenótipo M1 são caracterizados pela regulação positiva de CD80 e regulação negativa de CD86 (CD80++CD86+). O perfil M2 foi caracterizado pela expressão de CD206high e ausência de CD163- (CD206highCD163-). A expressão do mRNA revelou que IL-1β e TNF-α foram marcadores de M1 e TGF-β e CCL18 foram marcadores de M2. Além disso, quimiocinas inflamatórias como CXCL9, CXCL10 e CCL5 foram significativamente aumentadas em macrophagos M1. Macrófagos M1 e M2 são ativos e funcionais como demonstrado no ensaio de fagocitose. Embora ambos métodos utilizados para isolar monócitos tiveram purezas diferentes, ambas técnicas forneceram monócitos capazes de serem diferenciados a macrófagos. / Monocytes are hematopoietic cells with a major role in innate and adaptative immunity. According to the stimulus they receive, they can differentiate into macrophages and enhance effector functions by modulating the immune response and participating in various physiological and pathophysiological processes. Macrophages are very heterogeneous and are able to assume different phenotypes in response to the different stimuli they receive from the microenvironment. In a proinflammatory milieu ruled by interferon gamma (IFN-γ), they differentiate into cells with increased capacity to present antigens and synthesis of proinflammatory cytokines (M1 macrophages). On the other hand, when they are stimulated with interleukin 4 (IL-4), they differentiate into an antagonist phenotype with repair activity (M2 macrophages). Given the importance of these cells in the immune system, it is necessary to develop and optimize techniques that serve as useful tools for studying the differentiation of macrophages in their different phenotypic profiles as well as their roles in human diseases. In this regard, the aim of this study was to standardize and compare two different human monocyte isolation protocols described in the literature and analyze their impact on macrophage differentiation. We isolated peripheral blood monocytes from five healthy subjects by the adherence technique and positive selection. Monocytes were differentiated into macrophages with M-CSF supplementation. After M1 or M2 induction, we evaluated cell surface markers, mRNA cytokine expression, chemokine secretion and phagocytosis. We found that the methods used to isolate monocytes have different purities, but monocytes isolated from both methods were able to differentiate into the M1 and M2 profile. The monocyte and macrophage flow cytometry analysis demonstrated that CD14 decreased expression, mainly in M2 macrophages, and maintained (M2) or increased (M1) the HLA-DR expression. Monocytes (CD80-CD86high) induced to an M1 phenotype were characterized by upregulation of CD80 and down regulation of CD86. (CD80++CD86+) The M2 profile was characterized by the expression of CD206high and absence of CD163- (CD206highCD163-). The mRNA expression revealed that IL-1β and TNF-α were M1 markers and TGF-β and CCL18 were M2 markers. Further more, inflammatory chemokines as CXCL9, CXCL10 and CCL5 were significantly increased in M1 macrophages. M1 and M2 macrophages were active and functional as shown in the phagocytic assay. Although methods used for the isolation of monocytes yielded different purities, both techniques provided monocytes able to differentiate to macrophages.

Monócitos são células hematopoiéticas com função na imunidade inata e adquirida. De acordo com o estímulo que recebem, podem se diferenciar em macrófagos e potencializar suas funções efetoras, modulando a resposta imune e participando de vários processos fisiológicos e patológicos. Os macrófagos são muito heterogêneos e capazes de assumir diferentes fenótipos em resposta aos estímulos que recebem do microambiente. Em um ambiente gorvernado por interferon gama (IFN-γ), se diferenciam em células com aumentada capacidade de apresentação de antígenos e síntese de citocinas pró-inflamatórias (macrófagos M1). Por outro lado, quando são estimulados por interleucina-4 (IL-4), eles se diferenciam em um fenótipo antagonista com atividade de reparo (macrófagos M2). Dada a importância destas células no sistema imune, é necessário desenvolver e otimizar técnicas que sirvam de ferramentas para estudar a diferenciação de macrófagos em seus perfis fenotípicos bem como seu papel em doenças humanas. Assim, o objetivo deste estudo foi padronizar e comparar dois diferentes protocolos de isolamento de monócitos descritos na literatura e analisar seu impacto sobre a diferenciação de macrófagos. Isolamos monócitos do sangue periférico de cinco indivíduos saudáveis pelas técnicas de aderência e seleção positiva. Os monócitos foram diferenciados a macrófagos com suplementação de M-CSF. Depois da indução aos perfis M1 e M2, avaliamos marcadores de superfície celular, expressão de mRNA de citocinas, secreção de quimiocinas e fagocitose. Observamos que os métodos utilizados para isolar monócitos possuem diferentes purezas, mas que monócitos isolados por ambos métodos foram capazes de ser diferenciados a macrófagos em seus perfis M1 e M2. Análises de citometria de fluxo mostraram que há uma diminuição da expressão de CD14, principalmente em macrófagos M2, e manutenção (M2) ou aumento (m1) da expressão de HLA-DR. Monócitos (CD80-CD86high) induzidos aos fenótipo M1 são caracterizados pela regulação positiva de CD80 e regulação negativa de CD86 (CD80++CD86+). O perfil M2 foi caracterizado pela expressão de CD206high e ausência de CD163- (CD206highCD163-). A expressão do mRNA revelou que IL-1β e TNF-α foram marcadores de M1 e TGF-β e CCL18 foram marcadores de M2. Além disso, quimiocinas inflamatórias como CXCL9, CXCL10 e CCL5 foram significativamente aumentadas em macrophagos M1. Macrófagos M1 e M2 são ativos e funcionais como demonstrado no ensaio de fagocitose. Embora ambos métodos utilizados para isolar monócitos tiveram purezas diferentes, ambas técnicas forneceram monócitos capazes de serem diferenciados a macrófagos. / Monocytes are hematopoietic cells with a major role in innate and adaptative immunity. According to the stimulus they receive, they can differentiate into macrophages and enhance effector functions by modulating the immune response and participating in various physiological and pathophysiological processes. Macrophages are very heterogeneous and are able to assume different phenotypes in response to the different stimuli they receive from the microenvironment. In a proinflammatory milieu ruled by interferon gamma (IFN-γ), they differentiate into cells with increased capacity to present antigens and synthesis of proinflammatory cytokines (M1 macrophages). On the other hand, when they are stimulated with interleukin 4 (IL-4), they differentiate into an antagonist phenotype with repair activity (M2 macrophages). Given the importance of these cells in the immune system, it is necessary to develop and optimize techniques that serve as useful tools for studying the differentiation of macrophages in their different phenotypic profiles as well as their roles in human diseases. In this regard, the aim of this study was to standardize and compare two different human monocyte isolation protocols described in the literature and analyze their impact on macrophage differentiation. We isolated peripheral blood monocytes from five healthy subjects by the adherence technique and positive selection. Monocytes were differentiated into macrophages with M-CSF supplementation. After M1 or M2 induction, we evaluated cell surface markers, mRNA cytokine expression, chemokine secretion and phagocytosis. We found that the methods used to isolate monocytes have different purities, but monocytes isolated from both methods were able to differentiate into the M1 and M2 profile. The monocyte and macrophage flow cytometry analysis demonstrated that CD14 decreased expression, mainly in M2 macrophages, and maintained (M2) or increased (M1) the HLA-DR expression. Monocytes (CD80-CD86high) induced to an M1 phenotype were characterized by upregulation of CD80 and down regulation of CD86. (CD80++CD86+) The M2 profile was characterized by the expression of CD206high and absence of CD163- (CD206highCD163-). The mRNA expression revealed that IL-1β and TNF-α were M1 markers and TGF-β and CCL18 were M2 markers. Further more, inflammatory chemokines as CXCL9, CXCL10 and CCL5 were significantly increased in M1 macrophages. M1 and M2 macrophages were active and functional as shown in the phagocytic assay. Although methods used for the isolation of monocytes yielded different purities, both techniques provided monocytes able to differentiate to macrophages.

Orientador: Wanderley Dias da Silveira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T23:45:44Z (GMT). No. of bitstreams: 1 Rojas_ThaisCabreraGalvao_D.pdf: 2609032 bytes, checksum: 23a0546c76c17eff8087d0160c69308d (MD5) Previous issue date: 2012 / Resumo: A bactéria Escherichia coli coloniza o trato intestinal de aves e humanos, de maneira comensal sem causar processos infecciosos. No entanto alguns clones adquiriram fatores de virulência específicos, permitindo o desenvolvimento de diferentes doenças como infecção do trato urinário, diarréia e meningite em humanos e colibacilose em aves. As linhagens que causam doença em aves são tipicamente denominadas APEC (Avian Pathogenic Escherichia coli). Neste trabalho foram sequenciados e anotados os genomas de quatro linhagens APECs (SCI-07, SEPT362, S17 e O8)que, juntamente com mais nove genomas referentes a linhagens de Escherichia coli patogênicas para aves e patogênicas para humanos foram utilizados para a busca de genes sob seleção positiva. Os genes homólogos foram agrupados,e posteriormente submetidos ao alinhamento de códons e das sequencias protéicas correspondentes. Uma árvore filogenética foi gerada para cada grupo de proteínas homólogas. Testes estatísticos determinaram qual entre os modelos de seleção neutra ou seleção positiva melhor explicou os dados existentes (alinhamentos de códons e árvores filogenéticas). Essas análises detectaram duzentas e cinquenta e quatro grupos de genes homólogos com evidência de seleção positiva. Para cada grupo foi realizado um teste de recombinação para verificar se o aumento na variação das sequencias não era devido à conversão gênica, resultando em cento e dezesseis grupos de genes homólogos sob seleção positiva. A proteína correspondente a um gene de cada grupo de genes homólogos foi identificada, por meio da ferramenta Blast. Diversos fatores de virulência, já conhecidos, e proteínas regulatórias puderem ser detectados. Os genes sob seleção positiva, também foram submetidos à anotação considerando o termo GO (Gene Ontology),apenas da categoria processo biológico. Dos cento e dezesseis genes apenas cinquenta e sete puderam ser identificados por meio dessa metodologia. O resultado da classificação dos genes dentro da classe GO, considerando o terceiro nível hierárquico,mostrou que a maioria dos genes anotados (31) tinha relação com o metabolismo primário.As proteínas cuja identificação, por meio do blast, não foi possível (proteínas hipotéticas)foram submetidas à análise de predição de localização subcelular e de peptídeo sinal. Essas análises revelaram que três proteínas desconhecidas (hypothetical proteinECIAI39_1028, hypothetical proteinZ0639e hypothetical proteinEC042_3791) são potenciais alvos para estudos que visam à busca de novos fatores de virulência de Escherichia coli patogênicas / Abstract: The bacterium Escherichia coli colonizesthe intestinal tract of birds and humans, in a commensal relationship without causing infection. However, some clones have acquired specific virulence factors allowing the development of various diseases such as urinary tract infection, diarrhea and meningitis in humans and colibacillosis in poultry. The strains that cause disease in birds are typically named APEC (Avian Pathogenic Escherichia coli). In this study we sequenced and annotated the genomes of four APECs strains (SCI-07, SEPT362, S17 and O8). These genomes and nine others avian pathogenic Escherichia coli and humans pathogenic strains genomes were used for studying genes under positive selection. The homologous genes were grouped and then subjected to codons and corresponding protein sequences alignment. A phylogenetic tree was generated for each group of homologous proteins. Statistical tests determined which among neutral or positive selection models best explains the existing data (codon alignments and phylogenetic trees). This analyzes detected two hundred fifty-four groups of homologous genes with positive selection evidence. For each group a recombination test was conducted to verify if the variation increase in the sequences was not due to gene conversion, resulting in one hundred and sixteen groups of homologous genes under positive selection. The protein corresponding to a gene of each group of homologous genes under positive selection was identified through Blast tool. Genes under positive selection were annotated considering the GO term (Gene Ontology), just for the biological process category. Only fifty-seven genes could be identified using this methodology. The gene classification within the GO classes, considering only the third hierarchical level showed that most of the annotated genes (31) were related with the primary metabolism. Proteins which blast identification was not possible (hypothetical proteins) were subjected to sub cellular localization and signal peptide prediction analyzes. These analyzes revealed that three unknown proteins (hypothetical protein ECIAI39_1028, hypothetical protein Z0639e hypothetical protein EC042_3791) are potential targets for studies, in order to search for new virulence factors of pathogenic Escherichia coli / Doutorado / Microbiologia / Doutora em Genética e Biologia Molecular