Em estudos anteriores desenvolvidos por este grupo de pesquisa uma expressão mais elevada de alvos transcricionais e componentes da via NF-kB, bem como altos níveis de NOTCH1, foi identificada em células-tronco hematopoéticas (CTH) CD34+ de sangue de cordão umbilical (SCU) quando comparadas às CTH CD34+ de medula óssea (MO). Este grupo verificou ainda, por comparação das células CD34+ com as CD133+ (mais primitivas) que diversos fatores de transcrição (FT) envolvidos com o potencial de hemangioblasto, com a autorenovação das CTH, e com a diferenciação linfóide; como: RUNX1/AML1, GATA3, USF1, TAL1/SCL, HOXA9 e HOXB4 apresentaram-se mais expressos em células mais primitivas. A potencial participação das vias Notch e NF-kB na regulação destes FT tem importância conceitual e prática no entendimento da biologia das CTH, e dos processos envolvidos na diferenciação destas células. Com isto em vista, este projeto teve como objetivo, estudar o papel da via NF-kB e da via Notch na regulação destes FT. Para isso, um modelo experimental in vitro, de diferenciação de CTH CD34+ em linfócitos T, foi utilizado e a influência de fatores agonistas e inibidores farmacológicos destas vias, foram avaliados por citometria de fluxo e PCR em tempo real. Nossos resultados evidenciam o papel da via Notch na regulação transcricional de HOXB4 e GATA3 em células-tronco hematopoéticas CD34+ humanas, o que foi confirmado com base na expressão dos alvos diretos de Notch (HEY1 e HES1). Notamos ainda, que a expressão dos transcritos HES1, GATA3 e HOXB4 é prejudicada pela síntese protéica das CTH, uma vez que quando empregamos o prétratamento com a droga CHX há aumento da transcrição dos mesmos. Também podemos inferir que a ação do TNF- é positiva sobre esses transcritos, já que quando o utilizamos há elevação do nível de expressão desses transcritos, com exceção a HES1. Em relação ao cocultivo das CTH com as células estromais de camundongos, verificamos que apenas a linhagem OP9-DL1 detém a capacidade de promover a diferenciação celular T, e isso foi comprovado pelo surgimento de células comprometidas com a linhagem linfocítica T, através da presença dos marcadores de superfície específico CD7+ e CD1a+. Esses resultados auxiliarão na compreensão dos mecanismos moleculares de regulação transcricional envolvidos não apenas na diferenciação de linfócitos T, mas também na manutenção de um estado mais primitivo das CTH. Este conhecimento pode vir a contribuir com o desenvolvimento ou otimização de protocolos laboratoriais visando à expansão de CTH ou geração de células T para usos terapêuticos. / In previous studies by this research group a higher expression of transcriptional targets and components via NF-kB, as well as high levels of NOTCH1, was identified in hematopoietic stem cells (HSC) CD34 + cells from umbilical cord blood (UCB) compared to CD34 + hematopoietic stem cells from bone marrow (BM). This group also found, by comparing the CD34 + cells with CD133 + (more primitive) that several transcription factors (TF) involved in the potential of hemangioblast, with self-renewal of hematopoietic stem cells and to differentiated lymphocytic; as Runx1 / AML1, GATA3, USF1, TAL1/SCL, HOXB4 and HOXA9 were more expressed in more primitive cells. The potential involvement of Notch signaling pathways and NF-kB in the regulation of FT has conceptual and practical importance in understanding the biology of HSC, and the processes involved in differentiation of these cells. With this in mind, this project aimed to study the role of NF-kB pathway and Notch signaling in the regulation of FT. For this, an experimental model in vitro differentiation of CD34 + hematopoietic stem cells into T lymphocytes, was used and the influence of pharmacological agonists and inhibitors of these pathways were evaluated by flow cytometry and real-time PCR. Our results highlight the role of Notch signaling in the transcriptional regulation of GATA3 and HOXB4 in hematopoietic stem cells CD34 + human, which was confirmed based on the expression of direct targets of Notch (HES1 and HEY1). We also note that the expression of transcripts HES1, GATA3 and HOXB4 protein synthesis is hampered by the HSC, since when we use the pre-treatment with the drug there CHX increased transcription thereof. We can also infer that the action of TNF- is positive about these transcripts, since when we use it for raising the level of expression of these transcripts, except the HES1. In relation to the HSC coculture with stromal cells of mice, we found that only the line-DL1 Op9 has the ability to promote T cell differentiation, and this was evidenced by the appearance of cells committed to the T lymphocyte lineage, through the presence of specific surface markers CD7 + and CD1a +. These results will help understand the molecular mechanisms of transcriptional regulation involved not only in the differentiation of T lymphocytes, but also in maintaining a more primitive state of HSC. This knowledge may contribute to the development or optimization of laboratory protocols aimed at the expansion of HSC or generation of T cells for therapeutic use.

Diversos estudos epidemiológicos mostram que nutrientes e outros compostos bioativos presentes nos alimentos (CBA) apresentam atividade quimiopreventiva contra o câncer. Assim, destaca-se o estudo dos isoprenóides devido a sua ação promissora tanto na prevenção quanto na terapia do câncer. Todavia, apesar dessas evidências, pouco se sabe a respeito da ação dessas substâncias nos processos de proliferação celular e apoptose in vivo. Assim, 141 ratos Wistar foram tratados durante duas semanas consecutivas com farnesol (grupo FR, 25 mg/100 g de peso corporal) ou óleo de milho (grupo OM; controle, 0,25 mL/100 g de peso corporal) e sacrificados em diferentes momentos após a hepatectomia parcial (HP; 0 h, 30 min, 2 h, 4 h, 8 h, 12 h, 18 h e 24 h). Os parâmetros hepáticos analisados foram a proliferação celular (núcleos marcados para PCNA/mm2), apoptose (corpúsculos apoptóticos [CA\'s] por mm2) e expressão de p65, ciclina D1 (\"western blot\") e HMG-CoA redutase (\"dot-blot\"). Os animais tratados com o isoprenóide, assim como o grupo controle, apresentaram reduzida taxa de proliferação celular até 8h após a cirurgia. No entanto, a partir desse momento, o grupo FR passou a apresentar taxa de proliferação celular inferior ao grupo OM, diferença esta que atingiu significância estatística (p<0,05) 24h após a HP. Com relação a apoptose, animais tratados com FR apresentaram maior número de CA\'s (p<0,05) do que o grupo OM 30 min após a HP. Já em relação à ação do FR em âmbito molecular, houve uma redução de 40% e 50% na expressão de p65 e ciclina D1 30min e 24h após a HP, respectivamente, embora essas diferenças não tenham atingido significância estatística (p>0,05). Além disso, animais tratados com o isoprenóide apresentaram maior (p<0,05) expressão do gene que codifica para HMG-CoA redutase 2 h e 12 h após a cirurgia. Assim, tanto a inibição da proliferação celular quanto a indução de apoptose podem ser reflexo das alterações da expressão hepática dos genes para HMG-CoA redutase, p65 e ciclina D1 por parte do isoprenóide. / Epidemiological data have shown that nutrients and others bioactive compounds in food have chemopreventive activities against cancer. Among these compounds, isoprenoids are suggested either as a chemopreventive or chemotherapy agents. However, despite these evidences, studies focused on the isoprenoids activities on cell proliferation and apoptosis in vivo are rare. Thus, the effect of the 15-carbon isoprenoid farnesol on liver regeneration after partial hepatectomy was evaluated. Wistar rats were treated for two consecutive weeks with farnesol (FR group, 25 mg/100 g body weight) or corn oil (OM group, control, 0,25 mL/100 g body weight) and killed at different time points after partial hepatectomy (HP; 0 h, 30 min, 2 h, 4 h, 8 h, 12 h, 18 h and 24 h). Still, hepatic cell proliferation (PCNA lebeled nuclei), apoptosis (quantification of apoptotic bodies), p65 and cyclin D1 protein expression (western blot) and HMG-CoA reductase mRNA expression (dot blot) were also evaluated. Comparing to OM group, farnesol treatment significantly inhibited (p<0,05) hepatic cell proliferation 24 h after HP. Regarding apoptosis, also compared to controls, farnesol treated rats presented more (p<0,05) apoptotic bodies at 30 min. Besides, there were a suggestion of a higher number of apoptotic bodies 2 and 12 hours after HP in FR group comparing to OM group. According to western blot analysis, comparing to controls, this 15-carbon isoprenoid reduced 40% and 50% p65 and cyclin D1 hepatic protein expression, 30 min and 24 h after partial hepatectomy, respectively, although the differences did not also reach the statistical significance. Furthermore, farnesol treated rats had higher (p<0,05) HMG-CoA reductase mRNA levels than controls 2 h and 12 h after the surgery. Theses data suggest that the alterations on p65, cyclin D1 and HMG¬-CoA reductase gene expression observed in FR group might be associated with the inhibition of cell proliferation and the induction of apoptosis by farnesol.

Das 1817 erstmals schriftlich erwähnte Selen galt lange Zeit nur als toxisch und sogar als procancerogen, bis es 1957 von Schwarz und Foltz als essentielles Spurenelement erkannt wurde, dessen biologische Funktionen in Säugern durch Selenoproteine vermittelt werden. Die Familie der Glutathionperoxidasen nimmt hierbei eine wichtige Stellung ein. Für diese sind konkrete Funktionen und die dazugehörigen molekularen Mechanismen, welche über die von ihnen katalysierte Hydroperoxidreduktion und damit verbundene antioxidative Kapazität hinausgehen, bislang nur unzureichend beschrieben worden. <br><br> Die Funktion der gastrointestinalen Glutathionperoxidase (GI-GPx) wird als Barriere gegen eine Hydroperoxidabsorption im Gastrointestinaltrakt definiert. Neuen Erkenntnissen zufolge wird die GI-GPx aber auch in verschiedenen Tumoren verstärkt exprimiert, was weitere, bis dato unbekannte, Funktionen dieses Enzymes wahrscheinlich macht.<br> Um mögliche neue Funktionen der GI-GPx, vor allem während der Cancerogenese, abzuleiten, wurde hier die transkriptionale Regulation der GI-GPx detaillierter untersucht. Die Sequenzanalyse des humanen GI-GPx-Promotors ergab das Vorhandensein von zwei möglichen "antioxidant response elements" (ARE), bei welchen es sich um Erkennungssequenzen des Transkriptionsfaktors Nrf2 handelt. Die meisten der bekannten Nrf2-Zielgene gehören in die Gruppe der Phase-II-Enzyme und verfügen über antioxidative und/oder detoxifizierende Eigenschaften. Sowohl auf Promotorebene als auch auf mRNA- und Proteinebene konnte die Expression der GI-GPx durch typische, in der Nahrung enthaltene, Nrf2-Aktivatoren wie z.B. Sulforaphan oder Curcumin induziert werden. Eine direkte Beteiligung von Nrf2 wurde durch Cotransfektion von Nrf2 selbst bzw. von Keap1, das Nrf2 im Cytoplasma festhält, demonstriert. Somit konnte die GI-GPx eindeutig als Nrf2-Zielgen identifiziert werden. Ob sich die GI-GPx in die Gruppe der antiinflammatorischen und anticancerogenen Phase-II-Enzyme einordnen lässt, bleibt noch zu untersuchen. <br><br> Die Phospholipidhydroperoxid Glutathionperoxidase (PHGPx) nimmt aufgrund ihres breiten Substratspektrums, ihrer hohen Lipophilie und ihrer Fähigkeit, Thiole zu modifizieren, eine Sonderstellung innerhalb der Familie der Glutathionperoxidasen ein. Mit Hilfe eines PHGPx-überexprimierenden Zellmodells wurden deshalb Beeinflussungen des zellulären Redoxstatus und daraus resultierende Veränderungen in der Aktivität redoxsensitiver Transkriptionsfaktorsysteme und in der Expression atheroskleroserelevanter Adhäsionsmoleküle untersucht. Als Transkriptionsfaktoren wurden NF-kB und Nrf2 ausgewählt. Die Bindung von NF-kB an sein entsprechendes responsives Element in der DNA erfordert das Vorhandensein freier Thiole, wohingegen Nrf2 durch Thiolmodifikation von Keap1 freigesetzt wird und in den Kern transloziert. Eine erhöhte Aktivität der PHGPx resultierte in einer Erhöhung des Verhältnisses von GSH zu GSSG, andererseits aber in einer verminderten Markierbarkeit freier Proteinthiole. PHGPx-Überexpression reduzierte die IL-1-induzierte NF-kB-Aktivität, die sich in einer verminderten NF-kB-DNA-Bindefähigkeit und Transaktivierungsaktivität ausdrückte. Auch war die Proliferationsrate der Zellen vermindert. Die Expression des NF-kB-regulierten vaskulären Zelladhäsionsmoleküls, VCAM-1, war ebenfalls deutlich verringert. Umgekehrt war in PHGPx-überexprimierenden Zellen eine erhöhte Nrf2-Aktivität und Expression der Nrf2-abhängigen Hämoxygenase-1 zu verzeichnen. Letzte kann für die meisten der beobachteten Effekte verantwortlich gemacht werden.<br><br> Die hier dargestellten Ergebnisse verdeutlichen, dass eine Modifizierung von Proteinthiolen als wichtige Determinante für die Regulation der Expression und Funktion von Glutathionperoxidasen angesehen werden kann. Entgegen früheren Vermutungen, welche oxidative Vorgänge generell mit pathologischen Veränderungen assoziierten, scheint ein moderater oxidativer Stress, bedingt durch eine transiente Thiolmodifikation, durchaus günstige Auswirkungen zu haben, da, wie hier dargelegt, verschiedene, miteinander interagierende, cytoprotektive Mechanismen ausgelöst werden. Hieran wird deutlich, dass sich "antioxidative Wirkung" oder "oxidativer Stress" keineswegs nur auf "gute" oder "schlechte" Vorgänge beschränken lassen, sondern im Zusammenhang mit den beeinflussten (patho)physiologischen Prozessen und dem Ausmaß der "Störung" des physiologischen Redoxgleichgewichtes betrachtet werden müssen. / Selenium was discovered in 1817 by the Swedish chemist Berzelius and was for a long time considered as being toxic and even procarcinogenic. In 1957, however, Schwarz and Foltz realized that selenium is an essential trace element which elicits its biological functions in mammals as a structural component of selenoproteins among which the family of glutathione peroxidases plays a dominant role. Glutathione peroxidases reduce hydroperoxides to the corresponding alcohols and contribute to the antioxidative capacity of a cell. However, other functions of glutathione peroxidases and the according molecular mechanisms have hardly been described.>br><br> The gastrointestinal glutathione peroxidase (GI-GPx) is believed to build a barrier against the absorption of foodborne hydroperoxides. In addition, GI-GPx expression is increased in different tumors. This indicates further, still unknown, functions of this enzyme.<br> In order to elucidate new possible functions of GI-GPx, especially during carcinogenesis, the transcriptional regulation of GI-GPx was analyzed in more detail. An analysis of the GI-GPx promoter sequence revealed the presence of two putative "antioxidant response elements" (ARE) which are recognition sites for the transcription factor Nrf2. Most of the known Nrf2 target genes either belong to the group of phase-II detoxification enzymes or possess antioxidative and/or detoxifying properties. On promoter level as well as on mRNA- and protein level the expression of GI-GPx was induced by typical Nrf2-activating compounds such as sulforaphane or curcumin that are contained in the diet. A direct involvement of Nrf2 was demonstrated by cotransfection of Nrf2 itself or by cotransfection of Keap1 which retains Nrf2 in the cytosol. Thus, the GI-GPx gene was unequivocally identified as a new target for Nrf2. Whether GI-GPx also belongs in the category of antiinflammatory and anticarcinogenic enzymes remains to be elucidated.<br><br> The phospholipid hydroperoxide glutathione peroxidase (PHGPx) is exceptional among the glutathione peroxidases because of its broad range of substrates, its high lipophilicity, and its ability to modify protein thiols. With PHGPx-overexpressing cells, the influence of PHGPx on the cellular redox state and on resulting changes in the activity of redox-sensitive transcription factors and on the expression of proatherogenic adhesion molecules was analyzed. For this, the redox-sensitive transcription factors NF-kB and Nrf2 were chosen. NF-kB requires free thiols for being able to bind to its responsive element within the DNA, whereas Nrf2 is released from Keap1 and translocates to the nucleus upon a modification of protein thiols. PHGPx-overexpression resulted in an increase in the ratio of GSH to GSSG, in a reduced amount of intracellular protein thiols, and in a diminished proliferation rate. Furthermore, PHGPx-overexpressing cells displayed a reduced IL-1-dependent NF-kB activity as was assessed by a reduced NF-kB DNA-binding ability and activity of a NF-kB-driven reporter gene. In addition, the expression of the NF-kB-dependent vascular cell adhesion molecule (VCAM-1) was also inhibited by overexpression of PHGPx. On the other hand, PHGPx-overexpressing cells displayed an increased activity of Nrf2 that was accompanied by an increased expression of the Nrf2-dependent heme oxygenase-1. Heme oxygenase-1 most likely is responsible for most of the aforementioned effects.<br><br> The data presented here show that a modification of protein thiols can be regarded as an important determinant for the regulation and for the functions of glutathione peroxidases. In contrast to the previous assumption that oxidative processes are always linked to pathologic changes, a moderate oxidative stress seems to have beneficial effects, because it triggers different cytoprotective mechanisms. It can be concluded that the terms "antioxidative effect" or "oxidative stress" cannot simply be restricted to "good" or "bad" processes, but need to be seen in context with the modulated (patho)physiological processes and the degree of "disturbance" of the physiologic redox balance.

Diversos estudos epidemiológicos mostram que nutrientes e outros compostos bioativos presentes nos alimentos (CBA) apresentam atividade quimiopreventiva contra o câncer. Assim, destaca-se o estudo dos isoprenóides devido a sua ação promissora tanto na prevenção quanto na terapia do câncer. Todavia, apesar dessas evidências, pouco se sabe a respeito da ação dessas substâncias nos processos de proliferação celular e apoptose in vivo. Assim, 141 ratos Wistar foram tratados durante duas semanas consecutivas com farnesol (grupo FR, 25 mg/100 g de peso corporal) ou óleo de milho (grupo OM; controle, 0,25 mL/100 g de peso corporal) e sacrificados em diferentes momentos após a hepatectomia parcial (HP; 0 h, 30 min, 2 h, 4 h, 8 h, 12 h, 18 h e 24 h). Os parâmetros hepáticos analisados foram a proliferação celular (núcleos marcados para PCNA/mm2), apoptose (corpúsculos apoptóticos [CA\'s] por mm2) e expressão de p65, ciclina D1 (\"western blot\") e HMG-CoA redutase (\"dot-blot\"). Os animais tratados com o isoprenóide, assim como o grupo controle, apresentaram reduzida taxa de proliferação celular até 8h após a cirurgia. No entanto, a partir desse momento, o grupo FR passou a apresentar taxa de proliferação celular inferior ao grupo OM, diferença esta que atingiu significância estatística (p<0,05) 24h após a HP. Com relação a apoptose, animais tratados com FR apresentaram maior número de CA\'s (p<0,05) do que o grupo OM 30 min após a HP. Já em relação à ação do FR em âmbito molecular, houve uma redução de 40% e 50% na expressão de p65 e ciclina D1 30min e 24h após a HP, respectivamente, embora essas diferenças não tenham atingido significância estatística (p>0,05). Além disso, animais tratados com o isoprenóide apresentaram maior (p<0,05) expressão do gene que codifica para HMG-CoA redutase 2 h e 12 h após a cirurgia. Assim, tanto a inibição da proliferação celular quanto a indução de apoptose podem ser reflexo das alterações da expressão hepática dos genes para HMG-CoA redutase, p65 e ciclina D1 por parte do isoprenóide. / Epidemiological data have shown that nutrients and others bioactive compounds in food have chemopreventive activities against cancer. Among these compounds, isoprenoids are suggested either as a chemopreventive or chemotherapy agents. However, despite these evidences, studies focused on the isoprenoids activities on cell proliferation and apoptosis in vivo are rare. Thus, the effect of the 15-carbon isoprenoid farnesol on liver regeneration after partial hepatectomy was evaluated. Wistar rats were treated for two consecutive weeks with farnesol (FR group, 25 mg/100 g body weight) or corn oil (OM group, control, 0,25 mL/100 g body weight) and killed at different time points after partial hepatectomy (HP; 0 h, 30 min, 2 h, 4 h, 8 h, 12 h, 18 h and 24 h). Still, hepatic cell proliferation (PCNA lebeled nuclei), apoptosis (quantification of apoptotic bodies), p65 and cyclin D1 protein expression (western blot) and HMG-CoA reductase mRNA expression (dot blot) were also evaluated. Comparing to OM group, farnesol treatment significantly inhibited (p<0,05) hepatic cell proliferation 24 h after HP. Regarding apoptosis, also compared to controls, farnesol treated rats presented more (p<0,05) apoptotic bodies at 30 min. Besides, there were a suggestion of a higher number of apoptotic bodies 2 and 12 hours after HP in FR group comparing to OM group. According to western blot analysis, comparing to controls, this 15-carbon isoprenoid reduced 40% and 50% p65 and cyclin D1 hepatic protein expression, 30 min and 24 h after partial hepatectomy, respectively, although the differences did not also reach the statistical significance. Furthermore, farnesol treated rats had higher (p<0,05) HMG-CoA reductase mRNA levels than controls 2 h and 12 h after the surgery. Theses data suggest that the alterations on p65, cyclin D1 and HMG¬-CoA reductase gene expression observed in FR group might be associated with the inhibition of cell proliferation and the induction of apoptosis by farnesol.

Em estudos anteriores desenvolvidos por este grupo de pesquisa uma expressão mais elevada de alvos transcricionais e componentes da via NF-kB, bem como altos níveis de NOTCH1, foi identificada em células-tronco hematopoéticas (CTH) CD34+ de sangue de cordão umbilical (SCU) quando comparadas às CTH CD34+ de medula óssea (MO). Este grupo verificou ainda, por comparação das células CD34+ com as CD133+ (mais primitivas) que diversos fatores de transcrição (FT) envolvidos com o potencial de hemangioblasto, com a autorenovação das CTH, e com a diferenciação linfóide; como: RUNX1/AML1, GATA3, USF1, TAL1/SCL, HOXA9 e HOXB4 apresentaram-se mais expressos em células mais primitivas. A potencial participação das vias Notch e NF-kB na regulação destes FT tem importância conceitual e prática no entendimento da biologia das CTH, e dos processos envolvidos na diferenciação destas células. Com isto em vista, este projeto teve como objetivo, estudar o papel da via NF-kB e da via Notch na regulação destes FT. Para isso, um modelo experimental in vitro, de diferenciação de CTH CD34+ em linfócitos T, foi utilizado e a influência de fatores agonistas e inibidores farmacológicos destas vias, foram avaliados por citometria de fluxo e PCR em tempo real. Nossos resultados evidenciam o papel da via Notch na regulação transcricional de HOXB4 e GATA3 em células-tronco hematopoéticas CD34+ humanas, o que foi confirmado com base na expressão dos alvos diretos de Notch (HEY1 e HES1). Notamos ainda, que a expressão dos transcritos HES1, GATA3 e HOXB4 é prejudicada pela síntese protéica das CTH, uma vez que quando empregamos o prétratamento com a droga CHX há aumento da transcrição dos mesmos. Também podemos inferir que a ação do TNF- é positiva sobre esses transcritos, já que quando o utilizamos há elevação do nível de expressão desses transcritos, com exceção a HES1. Em relação ao cocultivo das CTH com as células estromais de camundongos, verificamos que apenas a linhagem OP9-DL1 detém a capacidade de promover a diferenciação celular T, e isso foi comprovado pelo surgimento de células comprometidas com a linhagem linfocítica T, através da presença dos marcadores de superfície específico CD7+ e CD1a+. Esses resultados auxiliarão na compreensão dos mecanismos moleculares de regulação transcricional envolvidos não apenas na diferenciação de linfócitos T, mas também na manutenção de um estado mais primitivo das CTH. Este conhecimento pode vir a contribuir com o desenvolvimento ou otimização de protocolos laboratoriais visando à expansão de CTH ou geração de células T para usos terapêuticos. / In previous studies by this research group a higher expression of transcriptional targets and components via NF-kB, as well as high levels of NOTCH1, was identified in hematopoietic stem cells (HSC) CD34 + cells from umbilical cord blood (UCB) compared to CD34 + hematopoietic stem cells from bone marrow (BM). This group also found, by comparing the CD34 + cells with CD133 + (more primitive) that several transcription factors (TF) involved in the potential of hemangioblast, with self-renewal of hematopoietic stem cells and to differentiated lymphocytic; as Runx1 / AML1, GATA3, USF1, TAL1/SCL, HOXB4 and HOXA9 were more expressed in more primitive cells. The potential involvement of Notch signaling pathways and NF-kB in the regulation of FT has conceptual and practical importance in understanding the biology of HSC, and the processes involved in differentiation of these cells. With this in mind, this project aimed to study the role of NF-kB pathway and Notch signaling in the regulation of FT. For this, an experimental model in vitro differentiation of CD34 + hematopoietic stem cells into T lymphocytes, was used and the influence of pharmacological agonists and inhibitors of these pathways were evaluated by flow cytometry and real-time PCR. Our results highlight the role of Notch signaling in the transcriptional regulation of GATA3 and HOXB4 in hematopoietic stem cells CD34 + human, which was confirmed based on the expression of direct targets of Notch (HES1 and HEY1). We also note that the expression of transcripts HES1, GATA3 and HOXB4 protein synthesis is hampered by the HSC, since when we use the pre-treatment with the drug there CHX increased transcription thereof. We can also infer that the action of TNF- is positive about these transcripts, since when we use it for raising the level of expression of these transcripts, except the HES1. In relation to the HSC coculture with stromal cells of mice, we found that only the line-DL1 Op9 has the ability to promote T cell differentiation, and this was evidenced by the appearance of cells committed to the T lymphocyte lineage, through the presence of specific surface markers CD7 + and CD1a +. These results will help understand the molecular mechanisms of transcriptional regulation involved not only in the differentiation of T lymphocytes, but also in maintaining a more primitive state of HSC. This knowledge may contribute to the development or optimization of laboratory protocols aimed at the expansion of HSC or generation of T cells for therapeutic use.

Eines der prävalentesten humanen Pathogene ist das Magenbakterium Helicobacter pylori, welches ca. die Hälfte der Weltbevölkerung infiziert. Die Persistenz geht mit einer chronischen Gastritis einher, welche bis zu Magenkrebs fortschreiten kann. H.pylori bedient sich diverser Mechanismen um sich der Erkennung des Immunsystems zu entziehen und somit eine chronische Infektion zu ermöglichen. Erhöhte Expression des Immunzellinhibitors PD-L1 wurde in Magenepithelzellen gefunden, welche mit diesem Gram-negativen Erreger infiziert wurden. In dieser Arbeit wurde die Regulation auf in vitro Ebene untersucht, wobei zwei unterschiedliche Mechanismen identifiziert wurden. Ursächlich für die frühe PD-L1-Induktion ist die ADP-heptose/ALPK1 Signalkaskade. Der bakterielle Metabolit ADP-heptose, welcher für die Bildung von LPS benötigt wird, wurde als PAMP identifiziert, welcher durch das Sekretionssystems cagT4SS in die infizierte transportiert und anschließend von der Host Kinase ALPK1 erkannt wird. Gegensätzlich hierzu, wurde festgestellt, dass die zweite PD-L1-Hochregulation auf der metabolischen Reprogrammierung des Wirts beruht. Ein Merkmal von H. pylori ist dessen Bedarf an Cholesterin, welches es aus dem Medium oder aus membranösen Lipidregionen des Wirts extrahiert wird. Es konnte bewiesen werden, dass dieser Sterol-Abbauprozess zu einer erhöhten Stoffwechselaktivität führt, die spezifisch mit einer Zunahme der Glykolyse verbunden ist und mit einer Expressionsverschiebung des ersten Glykolyseenzyms Hexokinase von der Isoform 1 zu 2 einhergeht. Knockdown und Knockout- Experimente wiesen auf einen Zusammenhang mit der Regulation des Immunzellinhibitoren PD-L1 hin. / One of the most prevalent bacteria is the gastric bacterium Helicobacter pylori, which infects half of the world’s population. Persistence is accompanied with chronic gastritis which can progress towards gastric cancer. Several strategies are used by H.pylori to evade the immune system, enabling chronic infection. Heightened expression of the immune cell inhibitor PD-L1 was found in gastric epithelial cells, infected with this Gram-negative pathogen. Within this thesis, upregulation was studied in in vitro models, revealing two distinct mechanism. Causative for early PD-L1 induction is the ADP heptose/ALPK1 signaling axis. The bacterial metabolite ADP heptose, which is needed for LPS synthesis, was identified as PAMP, which is transported through the secretion system cagT4SS into the infected cell and is recognized by the host kinase ALPK1. In contrast, late upregulation of PD-L1 was found to be linked to metabolic reprogramming upon infection. Characteristic to H.pylori is its need of cholesterol, which it has to extract from the surrounding medium or lipid-rich regions within the host membrane. It could be shown that this sterol extraction process is accompanied with an increased metabolic activity which is linked with enhanced glycolysis and an expression shift of the glycolytic enzyme hexokinase isoform 1 to 2. Knockdown and knockout experiments showed a link between HK2 and regulation of the immune checkpoint PD-L1.

No description available.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Mast cells play a critical role in both acute and chronic inflammation and mature in peripheral tissues from bone marrow-derived progenitors that circulate in the blood as immature precursors. Mast cell progenitors are likely to encounter the serum-borne bioactive sphingolipid metabolite, sphingosine-1-phosphate (S1P), during migration to target tissues. Mast cells developed from human cord blood-derived progenitors cultured with stem cell factor (SCF) alone express intragranular tryptase (MCT), the phenotype predominant in the lung. S1P accelerated the development of cord blood-derived mast cells (CB-MCs) and strikingly increased the numbers of mast cells expressing chymase. These mast cells have functional FcepsilonRI, and similar to skin mast cells that express both tryptase and chymase (MCTC), also express CD88, the receptor for C5a, and are activated by anaphylatoxin C5a and the secretagogue compound 48/80. S1P induced release of IL-6, a cytokine known to promote development of functionally mature MCTC, from cord blood cultures containing adherent macrophages, and from highly purified macrophages, but not from macrophage-depleted CB-MCs. In contrast, S1P stimulated secretion of the chemokine, monocyte chemoattractant protein 1 (MCP-1/CCL2), from these macrophage-depleted and purified CB-MCs.

Les stratégies de traitement biologiques ciblant le TNF-α se sont avérées efficaces pour réduire l'inflammation et les symptômes cliniques dans plusieurs maladies inflammatoires chroniques et sont maintenant couramment utilisées pour les patients qui ne répondent pas aux AINS au cours de la spondyloarthrite (SpA). Cependant, 30 à 40% des patients ne répondent pas aux anti-TNF, et il est actuellement impossible de prédire la réponse des patients à ces biomédicaments. Pour améliorer les résultats cliniques, nous avons besoin d’une part d’une meilleure compréhension des mécanismes d’action des anti-TNF sur le système immunitaire, et d’autre part de biomarqueurs permettant de prédire la réponse à ces biomédicaments afin de guider la décision thérapeutique. Mon projet de doctorat a porté sur deux objectifs complémentaires: (i) l'objectif principal était de progresser dans notre compréhension des mécanismes pathogéniques impliqués dans la SpA axiale et de définir de quelle façon les anti-TNF-α affectent les réponses immunitaires des patients, (ii) de développer des biomarqueurs pour prédire la réponse thérapeutique aux inhibiteurs du TNF. En collaboration avec l'équipe du Pr. Dougados à l'Hôpital Cochin, nous avons recruté deux cohortes indépendantes de patients SpA ayant une maladie active et pour lesquels nous avons collecté des échantillons de sang avant l'initiation du traitement par anti-TNF puis 1 semaine et 3 mois après le début du traitement. Les réponses immunitaires de ces patients ont été analysées à l'aide de tests hautement standardisés réalisés ex-vivo sur sang circulant. Ces tests "TruCulture" se présentent sous forme de seringues, dans lesquelles 1 ml de sang total est mis à incuber avec un stimulus spécifique ; 20 stimuli différents ont été testé et validé avant et après traitement dans les deux cohortes de patients. Nous avons observé une réduction très significative de la sécrétion de IL-1ra, IL-1β, IL-8, and MIP-1β en réponse à des stimuli microbiens et à des agonistes des TLR dans les échantillons de sang prélevés 7 jours et/ou 3 mois après le début du traitement. Pour identifier les bases moléculaires de l’action des inhibiteurs du TNF nous avons analysé l'expression des gènes dans ces différentes conditions de stimulation. L'analyse bioinformatique quantitative de l'expression des gènes (QuSAGE) a révélé que les gènes les plus modulés par le traitement anti-TNF étaient NF-KB et les gènes cibles de NF-kB, y compris le TNF lui-même et l’IL1B. Nos données suggèrent que les inhibiteurs du TNF agissent principalement en perturbant une boucle autorégulatrice pilotée par NF-kB. Afin d'identifier les signatures immunologiques de réponse aux anti-TNF avant le début du traitement, nous avons corrélé les réponses immunitaires chez les patients analysés au temps 0 à la réponse thérapeutique aux anti-TNF mesurée à 3 mois. Nos résultats suggèrent que les patients atteints de SpA et exprimant des niveaux inférieurs de PAX5 et des niveaux supérieurs de SPP1 en réponse à la stimulation avec SEB avant l'initiation de la thérapie anti-TNF ont les meilleures réponses thérapeutiques. Notre recherche montre que les tests TruCulture sont un outil efficace pour étudier les fonctions immunitaires chez les patients atteints de SpA et que les effets du traitement anti-TNF peuvent être mesurés lorsque les cellules immunitaires sont stimulées. En terme de recherche translationnelle, nous avons identifié des molécules qui pourront être utilisés comme biomarqueurs pour aider les cliniciens à prédire les réponses thérapeutiques aux traitements anti TNF / The introduction of anti-TNF therapy has proven effective to reduce inflammation and clinical symptoms in several chronic inflammatory diseases. However, 30-40% of patients do not respond to TNF blockers and it is currently not possible to predict responsiveness of patients to anti-TNF therapy. Furthermore, their impact on the immune system is incompletely understood. The goals of my PhD project were (i) to define the impact of anti-TNF therapy on immune responses to microbial challenges and stimuli targeting specific immune pathways in spondyloarthritis (SpA) patients, and (ii) to identify immunological correlates associated with therapeutic responses to TNF-blockers.Using a set of whole-blood, syringe-based assays to perform ex vivo stimulation while preserving physiological cellular interactions (TruCulture assays), we have performed a pilot study in SpA patients and investigated immune responses to 20 different stimuli before and 3 months after initiation of anti-TNF therapy. These findings were validated in a replication cohort, also assessing the effects of anti-TNF agents after only one week of treatment. We observed a highly significant reduction of the secretion of IL-1ra, IL-1β, IL-8 and MIP-1β in response to selected stimuli after 3 months of treatment compared to the baseline. Interestingly, these changes were already detectable after a single injection of an anti-TNF agent. To gain insight into the molecular mechanism of TNF blockers, we profiled gene expression in the stimulation cultures from all patients. Quantitative set analysis for gene expression (QuSAGE) revealed that the gene modules most affected by anti-TNF therapy are NF-kB transcription factors and inhibitors and NF-kB target genes, including TNF itself and IL1B. Our data suggest that TNF-blockers primarily act by disrupting an autoregulatory loop driven by NF-kB. We also tested whether there is a correlation between the responses of immune cells to specific stimuli and the clinical response to TNF-blockers. The decision tree model that we trained and validated suggests that SpA patients who expressed lower levels of PAX5 and higher levels of SPP1 in response to SEB stimulation before initiation of anti-TNF therapy had the best therapeutic responses. Our study shows that TruCulture assays are an efficient and robust tool to monitor immune functions in SpA patients and that the effects of anti-TNF therapy can be measured when immune cells are challenged, but not at steady state. Our data also indicate that analyzing immune responses in patients before therapy is a promising strategy to develop biomarkers for prediction of therapeutic responses to TNF-blockers