Thesis (doctoral)--Rijksuniversiteit te Utrecht.

Rheumatoid arthritis (RA) is a debilitating chronic inflammatory disease causing bone and cartilage degradation. Macrophages are known to play a role in RA pathology by producing pro-inflammatory cytokines and chemokines, which activates immune cells, drives inflammation and facilitates the degradation of bone and cartilage. Alterations in epigenetic mechanisms, processes that regulate gene expression, have been implicated in the regulation of pro-inflammatory cytokines in RA. Therefore, the aim of this thesis was to determine specific epigenetic variation between RA patient blood and synovial fluid (SF)-derived macrophages (SF MLS). Granulocyte and macrophages colony stimulating factor (GM-CSF) was used to differentiate healthy donor and RA patient blood monocytes into macrophages. Lipopolysaccharide (LPS) was used to stimulate blood and SF-derived macrophages to initiate inflammatory cytokine production. A library of small molecule inhibitors was used to identify key epigenetic regulators of pro-inflammatory cytokine production. Bromodomain and extra-terminal (BET) protein inhibitors (JQ1, I-BET151, PFI-1) were the only class of inhibitor to show consistent down regulation of pro-inflammatory cytokines in both healthy and RA patient-derived macrophages. However, only JQ1 was shown to reduce TNFα production significantly in SF MLS. Transcriptional profiling of RA patient SF MLS indicated a preference for a pro-inflammatory phenotype, and a resistance to steroids (a trait found in 30% of RA patients); SF MLS production of chemokines and cytokines were not downregulated by glucocorticoids in comparison to corresponding blood-derived macrophages. However, JQ1 treatment successfully suppressed these genes. In addition, silencing of BRD4 in blood-derived macrophages from healthy donors reduced pro-inflammatory cytokine production. Chromatin immunoprecipitation studies showed BRD4 was localised to pro-inflammatory promoter regions upon LPS stimulation and displaced in the presence of JQ1. These studies identified BET proteins BRD2, 3 and 4, as essential epigenetic regulators of pro-inflammatory cytokine and chemokine production in both healthy donors and RA patient macrophages. Furthermore, the observation that BET inhibitors can regulate genes that are steroid resistant in RA patient SF MLS, highlights their therapeutic potential in RA.

The Streptococcus Anginosus/Milleri Group (SMG) is made up of three closely related but distinct bacterial species: Streptococcus intermedius, Streptococcus constellatus, and Streptococcus anginosus. The SMG are recovered from about one-third of healthy, asymptomatic individuals. Despite this, the SMG cause more incidences of invasive streptococcal disease than Group A and Group B Streptococcus combined. Members of this group are somehow able to live a dual lifestyle. Little work has been conducted on the molecular pathogenicity of the SMG and host factors that contribute to host susceptibility to this group have been under-investigated. My research works towards discovering how the host recognizes the SMG as well as what enables the SMG to evade clearance by the immune system. I hypothesize that: 1) recognition of the SMG by toll-like receptor 2 (TLR2) plays a key role in triggering a cytokine response by the innate immune branch (which coordinates the immune response to the SMG), 2) the expression of cytolysins and extracellular polysaccharides by members of the SMG enables evasion of innate immune recognition and cytokine responses. hTLR2 reporter and monocyte-like cell lines as well as human blood samples from healthy donors were used to investigate the host factors that contribute to SMG infection. Five clinical reference SMG strains and a transposon mutant library were used to probe the contributing bacterial factors. It was found that TLR2 activation plays an important role in the cytokine response to the SMG, but there is heterogeneity between strains in their ability to activate TLR2. It was also found that intermedilysin expression by S. intermedius strains enables evasion of recognition; however, different hosts display varying susceptibility to this cytolysin. This study reveals that investigation of both host and microbial factors is essential to build an understanding of the mechanisms of SMG transition from commensalism to pathogenicity. / Thesis / Master of Science (MSc) / The Streptococcus Miller/Anginosus Group (SMG) is a group of bacteria comprised of three species. Members of this group are recovered from roughly one-third of healthy individuals. However, the SMG are also found in samples collected from patients with invasive disease. It is not well understood why some human-SMG relationships are pathogenic and others are not. However, it is likely that the combination of both human and SMG factors determine the nature of the relationship formed between the two. In this study, the human and SMG factors that contribute to infection were investigated. The ways by which human cells recognize members of the SMG and defend themselves from damage was explored. Additionally, SMG factors that potentially contribute to infection were probed to discover their effect on human cells. By investigating both the bacterial and host factors that lead to infection, disease treatments and preventative strategies can be tailored to individual cases.

O fator IX (FIX) da coagulação sanguínea é uma proteína dependente de vitamina K de grande valor farmacêutico no tratamento da Hemofilia B, o qual é baseado na administração do fator de coagulação derivado de plasma humano ou da proteína recombinante produzida em células murinas. A terapia baseada nestas abordagens apresenta alto custo e está associada às contaminações com vírus e príons, além do desenvolvimento de inibidores de FIX. Esses efeitos aumentam o risco de morbidade e mortalidade relacionadas às hemorragias. Neste trabalho, clonamos o cDNA do FIX em um vetor lentiviral e avaliamos a expressão da proteína recombinante em duas linhagens celulares humanas. A clonagem do cDNA do FIXh no vetor de expressão lentiviral 1054 foi confirmada através da análise com enzimas de restrição específicas obtendo-se as bandas esperadas de 1407 pb e 10054 pb visualizadas em gel de agarose. As linhagens celulares 293T e SK-Hep-1 foram transduzidas com o vetor lentiviral 1054-FIX gerado em nosso laboratório e as células que apresentaram maior expressão de EGFP foram selecionadas e separadas por citometria de fluxo. A quantificação da expressão de FIXrh foi realizada por ensaios de ELISA e cromogênico. A quantificação de FIXrh total foi de 500 ng/106 células para a linhagem 293T e 803 ng/106 células para a linhagem SK-Hep-1. A atividade biológica específica de FIXh nas células 293T e SK-Hep-1 foi 0,047 UI/106 células e 0,186 UI/106 células, respectivamente. Com o intuito de avaliar o perfil de produção de FIXrh ativo ao longo do tempo, foi realizado um acompanhamento de 180 dias, no qual foi observado que a linhagem SK-Hep-1 cessou a expressão de FIX, enquanto as células 293T mantiveram a expressão durante o período. O FIXrh foi caracterizado por western blot confirmando a presença de uma banda imunoreativa esperada de 57 kDa. As linhagens 293T e SK-Hep-1 apresentaram 7,67 e 17 cópias do vetor inserido/célula, respectivamente. Considerando a importância do processo de ?-carboxilação, foi realizada uma análise da expressão gênica dos genes envolvidos neste processo, tais como o VKORC1, ?-carboxilase e o inibidor calumenina, nas linhagens celulares. Os resultados demonstraram razões elevadas entre os genes VKORC1 e calumenina e VKORC1 e ?-carboxilase nas duas linhagens. A cinética de crescimento das células foi realizada por um período de 7 dias apresentando diferenças significativas entre as células SK-Hep-1 transduzidas e não transduzidas, enquanto que as células 293T não presentaram diferenças estatísticas no crescimento celular. A suplementação do meio de cultura com íons Ca+2 e Mg+2 foi testada para avaliar sua influência na expressão de FIXrh ativo. As células 293T apresentaram melhor desempenho nas concentrações de 0,5 mmol/L de Ca+2 e 1,0 mmol/L de Mg+2 e as células SK-Hep-1 no meio de cultura não suplementado. Nossos dados indicam que a linhagem hepática SK-Hep-1 é a melhor produtora de FIXrh funcional e as comparações realizadas entre os dois tipos celulares são importantes na caracterização do comportamento de linhagens geneticamente modificadas voltadas para a expressão de proteínas recombinantes heterólogas e abre novos caminhos para futuros estudos que visam o melhoramento da produção desse tipo de proteína. / Blood coagulation factor IX is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the treatment of Hemophilia B which is based on the plasma-derived coagulation factors or recombinant protein produced in murine cells. Coagulation therapy based on these approaches has high costs and is closely associated with prion and virus contamination besides the FIX inhibitors development. These effects increase the risk for bleeding-related morbidity and mortality. The purpose of this study was to clone hFIX into a lentiviral vector and evaluate the expression of the recombinant protein in two human cell lines. The cloning of the hFIX cDNA into 1054 lentiviral expression vector was confirmed by enzymatic restriction obtaining the expected 1407 bp and 10054 bp bands in agarose gel. The 293T and SK-Hep-1 cell lines have been stable transduced with 1054-FIX lentiviral vector generated in our laboratory and the cells with higher expression of EGFP were selected and separated by flow cytometry. The quantification of the expression of rhFIX was performed by ELISA and chromogenic assays. The concentration of total rhFIX was 500 ng/106 cells in 293T cell line and 803 ng/106 cells in SK-Hep-1 cell line. The biological activity of FIX secreted by 293T and SK-Hep-1 was 0,047 UI/106 cells and 0,186 UI/106 cells, respectively. In order to evaluate the active rhFIX production profile over time, we conducted a monitoring of 180 days, which was noted that the SK-Hep-1 cell line ceased FIX expression, while 293T cells maintained the expression during this period. rhFIX was characterized by western blot analysis confirming the presence of a expected 57 kDa immunereactive band. The 293T and SK-Hep-1 cell lines showed 7.67 and 17 integrated vector copies/cell, respectively. Considering the importance of the ?-carboxylation process, we performed a gene expression analysis of genes involved in this process, such as VKORC1, ?-carboxylase and calumenin, in cell lines. The results showed high ratios among the genes VKORC1 and calumenin and among VKORC1 and ?-carboxylase in both cell lines. The cell growth kinetics was performed by a 7-day period, showed significant differences between SK-Hep-1 transduced cells and non-transduced cells, whereas 293T cells showed no difference in cell growth. Enrichment of culture medium with Ca +2 and Mg +2 ions was tested to evaluate its influence on the expression of active FIX. 293T cells showed better performance in 0.5 mmol/L Ca+2 and 1.0 mmol/L Mg +2 concentrations and SK-Hep-1 cells in culture medium control. Our data indicate that transduced SK-Hep-1 cells are the best producer of functional rhFIX, and comparisons between these two cell lines are important in characterizing the behavior of genetically modified cell lines focused on the heterologous expression of recombinant proteins and opens new avenues for future studies aimed at improving the production of this type of protein.

Thesis (Ph.D.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Experimental Surgery, Department of Surgery. Title from pdf file main screen (viewed on April 24, 2010). Includes bibliographical references.

O fator IX (FIX) da coagulação sanguínea é uma proteína dependente de vitamina K de grande valor farmacêutico no tratamento da Hemofilia B, o qual é baseado na administração do fator de coagulação derivado de plasma humano ou da proteína recombinante produzida em células murinas. A terapia baseada nestas abordagens apresenta alto custo e está associada às contaminações com vírus e príons, além do desenvolvimento de inibidores de FIX. Esses efeitos aumentam o risco de morbidade e mortalidade relacionadas às hemorragias. Neste trabalho, clonamos o cDNA do FIX em um vetor lentiviral e avaliamos a expressão da proteína recombinante em duas linhagens celulares humanas. A clonagem do cDNA do FIXh no vetor de expressão lentiviral 1054 foi confirmada através da análise com enzimas de restrição específicas obtendo-se as bandas esperadas de 1407 pb e 10054 pb visualizadas em gel de agarose. As linhagens celulares 293T e SK-Hep-1 foram transduzidas com o vetor lentiviral 1054-FIX gerado em nosso laboratório e as células que apresentaram maior expressão de EGFP foram selecionadas e separadas por citometria de fluxo. A quantificação da expressão de FIXrh foi realizada por ensaios de ELISA e cromogênico. A quantificação de FIXrh total foi de 500 ng/106 células para a linhagem 293T e 803 ng/106 células para a linhagem SK-Hep-1. A atividade biológica específica de FIXh nas células 293T e SK-Hep-1 foi 0,047 UI/106 células e 0,186 UI/106 células, respectivamente. Com o intuito de avaliar o perfil de produção de FIXrh ativo ao longo do tempo, foi realizado um acompanhamento de 180 dias, no qual foi observado que a linhagem SK-Hep-1 cessou a expressão de FIX, enquanto as células 293T mantiveram a expressão durante o período. O FIXrh foi caracterizado por western blot confirmando a presença de uma banda imunoreativa esperada de 57 kDa. As linhagens 293T e SK-Hep-1 apresentaram 7,67 e 17 cópias do vetor inserido/célula, respectivamente. Considerando a importância do processo de ?-carboxilação, foi realizada uma análise da expressão gênica dos genes envolvidos neste processo, tais como o VKORC1, ?-carboxilase e o inibidor calumenina, nas linhagens celulares. Os resultados demonstraram razões elevadas entre os genes VKORC1 e calumenina e VKORC1 e ?-carboxilase nas duas linhagens. A cinética de crescimento das células foi realizada por um período de 7 dias apresentando diferenças significativas entre as células SK-Hep-1 transduzidas e não transduzidas, enquanto que as células 293T não presentaram diferenças estatísticas no crescimento celular. A suplementação do meio de cultura com íons Ca+2 e Mg+2 foi testada para avaliar sua influência na expressão de FIXrh ativo. As células 293T apresentaram melhor desempenho nas concentrações de 0,5 mmol/L de Ca+2 e 1,0 mmol/L de Mg+2 e as células SK-Hep-1 no meio de cultura não suplementado. Nossos dados indicam que a linhagem hepática SK-Hep-1 é a melhor produtora de FIXrh funcional e as comparações realizadas entre os dois tipos celulares são importantes na caracterização do comportamento de linhagens geneticamente modificadas voltadas para a expressão de proteínas recombinantes heterólogas e abre novos caminhos para futuros estudos que visam o melhoramento da produção desse tipo de proteína. / Blood coagulation factor IX is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the treatment of Hemophilia B which is based on the plasma-derived coagulation factors or recombinant protein produced in murine cells. Coagulation therapy based on these approaches has high costs and is closely associated with prion and virus contamination besides the FIX inhibitors development. These effects increase the risk for bleeding-related morbidity and mortality. The purpose of this study was to clone hFIX into a lentiviral vector and evaluate the expression of the recombinant protein in two human cell lines. The cloning of the hFIX cDNA into 1054 lentiviral expression vector was confirmed by enzymatic restriction obtaining the expected 1407 bp and 10054 bp bands in agarose gel. The 293T and SK-Hep-1 cell lines have been stable transduced with 1054-FIX lentiviral vector generated in our laboratory and the cells with higher expression of EGFP were selected and separated by flow cytometry. The quantification of the expression of rhFIX was performed by ELISA and chromogenic assays. The concentration of total rhFIX was 500 ng/106 cells in 293T cell line and 803 ng/106 cells in SK-Hep-1 cell line. The biological activity of FIX secreted by 293T and SK-Hep-1 was 0,047 UI/106 cells and 0,186 UI/106 cells, respectively. In order to evaluate the active rhFIX production profile over time, we conducted a monitoring of 180 days, which was noted that the SK-Hep-1 cell line ceased FIX expression, while 293T cells maintained the expression during this period. rhFIX was characterized by western blot analysis confirming the presence of a expected 57 kDa immunereactive band. The 293T and SK-Hep-1 cell lines showed 7.67 and 17 integrated vector copies/cell, respectively. Considering the importance of the ?-carboxylation process, we performed a gene expression analysis of genes involved in this process, such as VKORC1, ?-carboxylase and calumenin, in cell lines. The results showed high ratios among the genes VKORC1 and calumenin and among VKORC1 and ?-carboxylase in both cell lines. The cell growth kinetics was performed by a 7-day period, showed significant differences between SK-Hep-1 transduced cells and non-transduced cells, whereas 293T cells showed no difference in cell growth. Enrichment of culture medium with Ca +2 and Mg +2 ions was tested to evaluate its influence on the expression of active FIX. 293T cells showed better performance in 0.5 mmol/L Ca+2 and 1.0 mmol/L Mg +2 concentrations and SK-Hep-1 cells in culture medium control. Our data indicate that transduced SK-Hep-1 cells are the best producer of functional rhFIX, and comparisons between these two cell lines are important in characterizing the behavior of genetically modified cell lines focused on the heterologous expression of recombinant proteins and opens new avenues for future studies aimed at improving the production of this type of protein.

Orientador: João Ernesto de Carvalho / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T17:18:09Z (GMT). No. of bitstreams: 1 Zafred_RafaelRosolenTeixeira_M.pdf: 2402931 bytes, checksum: 1695b8ad500015e973737d3b719cde53 (MD5) Previous issue date: 2014 / Resumo: Astronium fraxinifolium Schott., conhecido popularmente como "Gonçalo Alves", é uma espécie vegetal característica de regiões tropicais e tem sua distribuição no Cerrado Brasileiro, possui o caule e as folhas ricos em substâncias tânicas. Relatados populares indicam a utilização de A. fraxinifolium no tratamento de diarreias, disenterias, anti-séptico, anti-microbiano, antihemorrágico, cicatrizante, anti-inflamatório e antiulcerogênico. Devido o uso popular e a insuficiência de dados na literatura, este trabalho teve por objetivo avaliar as potenciais atividades farmacológicas desta espécie em modelos experimentais in vitro (atividade antiproliferativa em cultura de células) e in vivo (toxicidade aguda, modelos de nocicepção, inflamação e tumor sólido murino) O material vegetal (cascas do caule e folhas) obtido das coletas foram secos e triturados, o pó obtido foi utilizado para obtenção dos extratos por maceração mecânica e sistema Soxhlet. Estes foram avaliados conforme seu rendimento e demonstraram a presença de taninos hidrolisáveis, como compostos majoritários, e flavonoides. No teste realizado em cultura de células tumorais e não tumorais de diferentes origens os extratos apresentaram, em sua maioria, uma ação citostática na maior concentração testada (250µg/mL). Por outro lado, nos testes relacionados com atividade antinociceptiva extrato bruto das folhas demonstrou atividade nas doses de 100 e 200mg/Kg no modelo da formalina em ambas as fases. Nos modelos inflamatórios (úlcera induzida por indometacina, edema de pata induzido por carragenina e edema de orelha induzido por óleo de cróton) as doses de 100 e 200mg/Kg, no modelo de edema de pata induzido por carragenina, e as doses de 150 e 300mg/Kg, no modelo de edema de orelha induzido por óleo de cróton, demonstraram atividade anti-inflamatória. Considerando que aproximadamente 25% dos tumores estão relacionados com inflamações crônicas, o extrato das folhas de A. fraxinifolium foi avaliado no modelo experimental do tumor sólido de Ehrlich no flanco, demonstrando inibição do crescimento tumoral na dose de 100mg/Kg. Sendo assim, os resultados experimentais sugerem que a espécie A. fraxinifolium possui atividade antiinflamatória dos componentes do extrato bruto, corroborando com sua utilização popular. Além de promover inibição do crescimento tumoral indicando que os componentes do extrato podem atuar em outros processos além do inflamatório. Porém, outros estudos necessitam ser realizados para identificar a classe de compostos que promoveram as atividades farmacológicas da espécie / Abstract: Astronium fraxinifolium Schott., popularly known as "Gonçalo Alves", is a species characteristic of tropical regions with their distribution in the Brazilian Cerrado, has stems and leaves rich in tannic substances. Reported the use of folk medicinal indicate A. fraxinifolium in the treatment of diarrhea, dysentery, antiseptic, anti-microbial, antihaemorrhagic, wound healing, anti-inflammatory and antiulcerogenic. Due to popular use and insufficient data in the literature, this study aimed to evaluate potential pharmacological activities of this species in vitro experimental models (antiproliferative activity in cell culture) and in vivo (acute toxicity models of nociception, inflammation and tumor solid murine) The plant material (stems and leaves) samples were obtained from dried and crushed, the powder obtained was used to obtain the extracts, mechanical maceration and soxhlet system. These were evaluated according to their yield and showed the presence of hydrolyzable tannins, as main compounds, and flavonoids. Testing conducted on cultured tumor and non-tumor cells of different origins, the extracts showed mostly a cytostatic action at the highest concentration tested (250?g/mL). Moreover, in tests related antinociceptive crude extract of the leaves showed activity at doses of 100 and 200mg/Kg in the formalin model in both phases. In inflammatory models (indomethacin-induced ulcer, paw edema induced by carrageenan and ear edema induced by croton oil) doses of 100 and 200mg/Kg in paw edema induced by carrageenan model, and the doses of 150 and 300mg/kg, in the ear edema induced by croton oil model, demonstrated anti-inflammatory activity. Considering that approximately 25% of tumors are associated with chronic inflammation, the extract of A. fraxinifolium was assessed in the experimental model of Ehrlich solid tumor in the flank, demonstrating inhibition of tumor growth at a dose of 100mg/kg. Thus, experimental results suggest that the species A. fraxinifolium has anti-inflammatory component of the crude extract activity, confirming its popular use. In addition to promoting tumor growth inhibition indicating that extract components can act in addition to other inflammatory processes. However, other studies should be performed to identify the class of compounds that promoted the pharmacological activity of the species / Mestrado / Fármacos, Medicamentos e Insumos para Saúde / Mestre em Biociências e Tecnologia de Produtos Bioativos

Eukaryotic initiation factor 4E (eIF4E) is a key part of initiation and regulation of translation in human cells. Three members of human eIF4E proteins have been characterized: eIF4E1, eIF4E2 and eIF4E3. Cellular stress causes translation initiation inhibition followed by disassembly of the polysomes, those processes are accompanied by the assembly of cytoplasmic RNA granules, called stress granules (SG). Stress granules are dynamic structures whose composition may vary depending on the cell type and the stress stimulus. In this study, human cells were subjected to the following stress conditions: high temperature (HS), sodium arsenite (AS) or hypoxia. Using fluorescence microscopy, pairs of human translational initiation factors from the 4E protein family were visualized and their localization to SG was assessed with one GFP- 4E incorporated in the stable cell line and the other one detected endogenously. Here we show eIF4E1 being a part of all the SGs, both in HS and AS conditions. Next, the eIF4E1 and eIF4E3 proteins together form more SGs than proteins eIF4E1, respectively eIF4E3, with eIF4E2. And last, that the presence of the particular 4E protein has no effect on the composition of SGs. Furthermore, selected groups of proteins were assessed for their potential to localize to the SGs under HS...

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