Megakaryocytes (MKs) are a rare population of haematopoietic cells, which produce platelets. Platelet production is a complex process that is tightly regulated at the transcriptional level by lineage specific transcription factors such as p45 NF-E2. Understanding how transcriptional regulators operate is imperative to advance our knowledge of disease pathophysiology and to propose novel treatment options. Therefore, the aims of this study were to: i) study the effects of p45 NF-E2 overexpression on various stages of megakaryopoiesis; (ii) elucidate the nuclear transport mechanisms of p45 NF-E2; and iii) determine the impact of a p45 NF-E2 modification called SUMOylation on thrombopoiesis. Exogenous p45 NF-E2 was overexpressed in haematopoietic cells in culture and various aspects of megakaryopoiesis were examined. Overexpression of p45 NF-E2 enhanced multiple stages of MK differentiation such as colony forming unit (CFU)-MK formation and terminal MK maturation. Most importantly, p45 NF-E2 overexpression resulted in significant increases in proplatelet and functional platelet production in vitro. This latter result was confirmed in vivo using lethally irradiated mice transplanted with cells that overexpressed p45 NF-E2. Unexpectedly, the enhancement of MK differentiation was at the expense of myeloid development and, for the first time, identified p45 NF-E2 as a negative regulator of myeloid differentiation. Secondly, we determined the nuclear localisation signal of p45-NF-E2 and the pathway responsible for nuclear import. We also investigated the importance of p45 NF-E2 nuclear import in thrombopoiesis. Finally, we showed that p45 NF-E2 is modified mainly by SUMO-2/3 in bone marrow cells and this process is involved in the transcriptional activation of MK-specific genes and platelet release. Taken together, these results suggest that enforced expression of p45 NF-E2 selectively enhances many aspects of MK differentiation including early and terminal MK maturation, proplatelet formation and platelet release. Equally important, this thesis also indicates that white blood cell differentiation may be inhibited by p45 overexpression, while molecular processes such as the nuclear import and SUMOylation of p45 NF-E2 are vital for thrombopoiesis. These observations will facilitate subsequent studies into the feasibility of manipulating p45 NF-E2 protein levels for the treatment of conditions such as thrombocytopaenia and other platelet disorders.

Cellular response to inflammatory cytokines involves concerted changes in gene expression. Cytokines, such as IL-6 and TNF-α, regulate gene expression through multiple intracellular signaling pathways. The activation of transcription factors is one of the important mechanisms through which these cytokines regulate gene expression, and NF-κB is a key transcription factor that is activated by TNF-α during inflammation. In this study, we have utilized green fluorescent protein reporter cells along with fluorescence microscopy, image analysis and mechanistic modeling to determine the activation dynamics of NF-κB in H35 rat hepatoma cells upon TNF-α stimulation. NF-κB reporter cells were monitored for induction of GFP expression for 24 hours following continuous stimulation with 2.5ng/mL, 10ng/mL and 25ng/mL TNF-α. As expected, TNF-α addition resulted in a significant increase in fluorescence. Relative fluorescence profiles were generated from the fluorescence intensity data, and indicated that fluorescence increases up to 24 hours after an initial delay of approximately four hours. The fluorescence data was also used to develop a model describing significant events leading to NF-κB activation and GFP expression. In addition, a model describing regulatable expression of GFP upon stable integration into the genome was also developed. Comparing these two models led to the construction of a third model depicting NF-κB activation dynamics. Simulation of the model representing NF-κB activation dynamics yielded an NF- κB activation profile, which demonstrated that in the presence of constant TNF-α stimulation, there is an approximate 90 minute hour time delay followed by a rapid increase in nuclear NF-κB, that reaches a steady state value at approximately two hours. This study establishes a method to derive NF-κB activation from reporter cell fluorescence data, and can be used to infer dynamics of activation of other transcription factors using GFP reporter cell lines.

Le NF-κB est un facteur de transcription crucial dans la régulation de lexpression de gènes impliqués dans la réponse immune, la prolifération et la survie cellulaires. Ce facteur de transcription peut être activé par de nombreux inducteurs incluant notamment les espèces réactives de loxygène (ROS). En labsence de stimuli, NF-κB est séquestré dans le cytoplasme des cellules par la protéine IκBα. Lors dun stimulus, IκBα est phosphorylée et par la suite dégradée permettant ainsi la translocation du NF-κB dans le noyau. Au cours de ce travail, nous nous sommes intéressés au mécanisme dactivation du NF-κB par un stress oxydant via la phosphorylation dIκBα sur tyrosine (voie atypique). Nous avons eu recours à deux types dapproches : une première in vitro et une seconde in vivo, se rapprochant fortement de conditions physiopathologiques chez lhomme. Dans un premier temps, nous avons mis en évidence que la chaperonne Hsp90 jouait un rôle clé dans la voie dactivation du NF-κB par un inducteur de stress oxydant, le pervanadate de sodium (PV). Cette voie dactivation requiert la phosphorylation de la protéine IκBα sur la tyrosine 42 par la tyrosine kinase c-Src. Grâce à lutilisation dun inhibiteur dHsp90, la geldanamycine, nous avons observé que la phosphorylation dIκBα et lactivité de c-Src étaient inhibées par ce composé pharmacologique et par conséquent lactivation du NF-κB également. Dans la suite de ce travail, nous avons utilisé deux techniques expérimentales permettant dinduire un stress oxydant, in vivo, au niveau du cerveau murin. Celles-ci sont linjection intracérébrale de FeCl2 et lischémie/reperfusion cérébrale. En ce qui concerne linjection intracérébrale de FeCl2, nous avons confirmé que cette technique engendrait une production importante de ROS. Nous avons observé que linjection de FeCl2 induisait lactivation du NF-κB. De plus, nos résultats suggèrent que cet inducteur active NF-κB via la phosphorylation dIκBα sur la tyrosine 42. Les mêmes observations ont été effectuées lors dune ischémie/reperfusion cérébrale. En effet, celle-ci induit également une activation du NF-κB ainsi que la phosphorylation dIκBα sur la tyrosine 42. Lensemble de ces données suggère dune part quHsp90 joue un rôle clé dans la voie dactivation atypique du NF-κB et dautre part quun stress oxydant in vivo induit lactivation du NF-κB ainsi que la phosphorylation dIκBα sur la tyrosine 42.

Equine infectious anemia virus (EIAV) is a macrophage-tropic retrovirus that causes persistent disease in horses and ponies. In addition to its structural proteins, EIAV encodes four regulatory/accessory genes, tat, rev, ttm, and S2. It has been documented EIAV S2 gene expression is essential for disease expression of EIAV. Using a yeast two-hybrid assay, it was shown that S2 protein interacts with human NF-KB2. NF-KB2 plays a key role in the alternative or non-canonical NF-KB pathway. In order to determine if the interaction of S2 with NF-KB2 might be relevant to equine disease, a cDNA representing full length equine NF-KB2 was generated in our laboratory using PCR and rapid amplification of cDNA ends. To our knowledge this is the first time that equine NF-KB2 cDNAs have been recovered and characterized. The sequence of equine NF-KB2 was 95% homologous to human overall, however a major difference was found in the ankyrin repeat region where protein-protein interactions occur. Two splice variants of equine NF-KB2 were found that correspond to splice variants of human NF- KB2. We tested the interaction of EIAV S2 and equine NF-KB2 using the yeast two hybrid system (Y2H) and co-immunoprecipitation. Unfortunately we were not able to detect an interaction between EIAV S2 and equine NF-KB2 in either system. Despite this result, NF-KB2 is an important component in the immune response so we examined its expression in equine macrophages. Moreover we were interested to know if EIAV might affect expression levels of equine NF-KB2, as NF-KB2 is a target of other viruses. Hence, the expression level of equine NF-KB2 was measured in uninfected and infected primary equine monocyte- derived macrophage (eMDM). Using quantitative PCR we determined that equine NF-KB2 gene expression is decreased in eMDM after 3 days post plating, about the time that monocytes start to differentiate into mature macrophages. However EIAV infection of eMDM upregulated the expression level of NF-KB2.

Cellular response to inflammatory cytokines involves concerted changes in gene expression. Cytokines, such as IL-6 and TNF-α, regulate gene expression through multiple intracellular signaling pathways. The activation of transcription factors is one of the important mechanisms through which these cytokines regulate gene expression, and NF-κB is a key transcription factor that is activated by TNF-α during inflammation. In this study, we have utilized green fluorescent protein reporter cells along with fluorescence microscopy, image analysis and mechanistic modeling to determine the activation dynamics of NF-κB in H35 rat hepatoma cells upon TNF-α stimulation. NF-κB reporter cells were monitored for induction of GFP expression for 24 hours following continuous stimulation with 2.5ng/mL, 10ng/mL and 25ng/mL TNF-α. As expected, TNF-α addition resulted in a significant increase in fluorescence. Relative fluorescence profiles were generated from the fluorescence intensity data, and indicated that fluorescence increases up to 24 hours after an initial delay of approximately four hours. The fluorescence data was also used to develop a model describing significant events leading to NF-κB activation and GFP expression. In addition, a model describing regulatable expression of GFP upon stable integration into the genome was also developed. Comparing these two models led to the construction of a third model depicting NF-κB activation dynamics. Simulation of the model representing NF-κB activation dynamics yielded an NF- κB activation profile, which demonstrated that in the presence of constant TNF-α stimulation, there is an approximate 90 minute hour time delay followed by a rapid increase in nuclear NF-κB, that reaches a steady state value at approximately two hours. This study establishes a method to derive NF-κB activation from reporter cell fluorescence data, and can be used to infer dynamics of activation of other transcription factors using GFP reporter cell lines.

Megakaryocytes (MKs) are a rare population of haematopoietic cells, which produce platelets. Platelet production is a complex process that is tightly regulated at the transcriptional level by lineage specific transcription factors such as p45 NF-E2. Understanding how transcriptional regulators operate is imperative to advance our knowledge of disease pathophysiology and to propose novel treatment options. Therefore, the aims of this study were to: i) study the effects of p45 NF-E2 overexpression on various stages of megakaryopoiesis; (ii) elucidate the nuclear transport mechanisms of p45 NF-E2; and iii) determine the impact of a p45 NF-E2 modification called SUMOylation on thrombopoiesis. Exogenous p45 NF-E2 was overexpressed in haematopoietic cells in culture and various aspects of megakaryopoiesis were examined. Overexpression of p45 NF-E2 enhanced multiple stages of MK differentiation such as colony forming unit (CFU)-MK formation and terminal MK maturation. Most importantly, p45 NF-E2 overexpression resulted in significant increases in proplatelet and functional platelet production in vitro. This latter result was confirmed in vivo using lethally irradiated mice transplanted with cells that overexpressed p45 NF-E2. Unexpectedly, the enhancement of MK differentiation was at the expense of myeloid development and, for the first time, identified p45 NF-E2 as a negative regulator of myeloid differentiation. Secondly, we determined the nuclear localisation signal of p45-NF-E2 and the pathway responsible for nuclear import. We also investigated the importance of p45 NF-E2 nuclear import in thrombopoiesis. Finally, we showed that p45 NF-E2 is modified mainly by SUMO-2/3 in bone marrow cells and this process is involved in the transcriptional activation of MK-specific genes and platelet release. Taken together, these results suggest that enforced expression of p45 NF-E2 selectively enhances many aspects of MK differentiation including early and terminal MK maturation, proplatelet formation and platelet release. Equally important, this thesis also indicates that white blood cell differentiation may be inhibited by p45 overexpression, while molecular processes such as the nuclear import and SUMOylation of p45 NF-E2 are vital for thrombopoiesis. These observations will facilitate subsequent studies into the feasibility of manipulating p45 NF-E2 protein levels for the treatment of conditions such as thrombocytopaenia and other platelet disorders.

Understanding the biological mechanisms underlying the initiation and progression of breast cancer is an important step for its prevention and treatment. We used an in vitro and in vivo model to demonstrate that p100/p52 and RelB are strongly activated in BRCA1-deficient mouse mammary progenitor cells and human BRCA1-mutation carriers. We found that NF-κB activation induces stem and progenitor cell expansion and inhibits differentiation. Knockdown and pharmacological inhibition showed that the progesterone-independent growth of BRCA1-deficient progenitor cells requires the alternative NF-κB activation mediated by ATM. Remarkably, treatment of mice with the NF-κB inhibitor dimethylaminoparthenolide (DMAPT) resulted in prolonged repression of BRCA1-deficient progenitor cell proliferation, revealing a possible approach to cyclic chemoprevention.

Indiana University-Purdue University Indianapolis (IUPUI) / The nuclear factor κB (NF-κB) plays pivotal roles in inflammatory and immune responses and in cancer. Therefore, understanding its regulation holds great promise for disease therapy. Using validation-based insertional mutagenesis (VBIM), a powerful technique established by us, we discovered armadillo repeat containing protein 4 (ARMC4) as a novel negative regulator of NF-κB in colorectal cancer (CRC). ARMC4 is a rarely studied protein only known to date for its role in primary ciliary dyskinesia (PCD) and mouse spermatogenesis. Thus, my work reveals a completely new facet of ARMC4 function that has never been reported before. We showed that ARMC4 overexpression downregulated the expression of NF-κB-dependent genes, many of which are related to cancer. Additionally, compared to the vector control group, overexpression of ARMC4 in HEK293 cells or CRC HT29, DLD1, and HCT116 cells dramatically reduced NF-κB activity, cellular proliferation, anchorage-independent growth, and migratory ability in vitro, and unsurprisingly, significantly decreased xenograft tumor growth in vivo. In contrast, shARMC4 knockdown cells showed quite opposite effect. Furthermore, co-immunoprecipitation (Co-IP) experiment confirmed that ARMC4 may form a complex with the p65 subunit of NF-κB. Importantly, immunohistochemistry (IHC) data exhibited much lower ARMC4 expression level in CRC patient tumor tissues compared to normal tissues, indicating that ARMC4 may function as a tumor suppressor in CRC. To conclude, my important findings for the first time uncovered the negative regulatory function of ARMC4 in NF-κB signaling, and present ARMC4 as an innovative therapeutic target in CRC treatment. / 2022-05-06

The Inhibitor of Apoptosis (IAP) proteins are a highly conserved group of anti-apoptotic proteins. Cellular IAP 1 and 2 (cIAP1 and 2) are two members of the IAP protein family that regulate the activity of the Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor family. The role and mechanism of the IAPs in ubiquitination are not yet completely understood due to the complexity of this posttranslational modification process. Additionally, The IAPs are involved in a myriad of cellular processes, and many of the process-specific mechanisms by which the IAPs are involved is unknown. I aim to delve deeper into the signalling pathways that are controlled by cIAP1 and cIAP2 by discovering currently unknown protein-protein interactions. In doing so, I will determine which proteins interact with the cIAPs and what signalling pathways these proteins are involved in. Using a BioID approach, I sought to characterize the cIAP1 interactors involved in the canonical and non-canonical NF-κB pathways. I generated a stable cell line expressing TurboID-cIAP1 fusion protein in HEK 293T cells that expressTurboID-cIAP1 at levels comparable to endogenous cIAP1. I identified multiple potential cIAP1 interactors that have ties to the NF-κB pathway. These proteins regulate NF-κB signalling in multiple ways including influencing acetylation and nuclear retention of the NF-κB transcription factors, phosphorylation of NF-κB transcription factors, and RNA splicing of genes involved in the TNFR1 complex I. Further work needs to be done to confirm these interactions and to discover the mechanisms by which these interactions occur. NF-κB signalling is known to have widespread function within the cell and within diseases such as cancer; it will be beneficial to study these interactions to better understand how cancer develops and how to treat it best, especially in patients with a poor prognosis.

A imunidade inata é responsável pela resposta inicial aos microrganismos, uma vez que impede, controla ou elimina a infecção. Esse sistema consiste em barreiras epiteliais, proteínas plasmáticas e células circulantes e teciduais. Dentre esses componentes, os macrófagos possuem grande importância, sendo capazes de controlar e eliminar agentes patogênicos através da fagocitose e produção de espécies reativas de oxigênio e nitrogênio. A ativação de PRRs por constituintes oriundos dos patógenos em macrófagos desencadeia eventos da resposta imune inata, ativados por diversas vias de sinalização intracelular. A via das PI3Ks é conhecida por regular várias funções nas células, como a regulação do ciclo celular, migração e produção de espécies reativas de oxigênio e nitrogênio. O NO é um mediador central na imunidade inata que, após estímulos inflamatórios, é produzido em altas quantidades através da iNOS. Macrófagos deficientes em PI3K produzem menos NO e apresentam prejudicado controle da infecção quando infectados por T. cruzi. O objetivo do presente trabalho foi investigar o papel da via PI3K na produção de NO por macrófagos peritoneais estimulados com LPS. Os macrófagos empregados no estudo, WT e PI3K-/-, possuem o mesmo fenótipo. Observamos que macrófagos PI3K-/- possuem uma menor produção de NO e expressam menos iNOS. A reduzida expressão de iNOS, após estímulo com LPS, é também observada quando macrófagos WT são tratados com inibidores seletivos da PI3K e AKT. Além disso, demonstramos que, concomitantemente à menor expressão da iNOS, ocorre deficiência na fosforilação da AKT e diminuição da ativação do fator de transcrição NF-kB, sugerindo que a PI3K participa da ativação do NF-kB. Foi observado ainda que o tratamento com PTX também diminui a expressão da iNOS. No entanto, macrófagos PAFR-/- expostos ao LPS presentam maior expressão da iNOS, enquanto os macrófagos CCR2-/- apresentam menor expressão dessa enzima nessas condições. Para investigar a implicação da via PI3K in vivo foi administrado LPS i.v., como modelo de choque endotoxemico, no qual observamos maior sobrevida em animais PI3K-/- comparado aos animais WT e menores níveis de nitrito no soro. Nossos dados sugerem que a enzima PI3K é crítica para expressão de iNOS e produção de NO pelos macrófagos, possivelmente através da ativação do receptor CCR2, estando envolvida na fisiopatologia do choque induzido por LPS. / Innate immunity is the initial response to microorganisms, since it prevents, controls and eliminates infection. This system consists in epithelial barriers, plasma proteins and circulating and tissue cells. Among these components, macrophages have great importance, being capable of control and eliminate pathogen agents through phagocytosis and production of reactive oxygen and nitrogen species. Activation of PRRs by pathogens constituents in macrophages triggers events of the innate immune response, activated by various intracellular signaling pathways. PI3Ks pathway is known to regulate several functions in the cell, such as regulation of the cell cycle, migration and production of reactive oxygen and nitrogen species. NO is a central mediator in innate immunity, which after inflammatory stimuli, is produced in high levels by iNOS. PI3K-deficient macrophages produce less NO and exhibit impaired control of infection when infected by T. cruzi. The aim of the present study is to investigate the role of PI3K pathway in NO production by LPS-estimulated peritoneal macrophages. The macrophages used in this study, WT and PI3K- / -, have the same phenotype. We observed that PI3K- / - macrophages have a lower NO production and express less iNOS. The low expression of iNOS after stimulation with LPS was also observed in WT macrophages treated with selective inhibitors of PI3K and AKT. Furthermore, we demonstrate that, along to lower iNOS expression, there is deficiency in AKT phosphorylation and decreased activation of the transcription factor NF-kB, suggesting that PI3K participates of the NF-kB activation. It was also observed that PTX treatment has decreased iNOS expression. However, LPS-exposed PFAR-/- macrophages present greater expression of iNOS, while CCR2-/- macrophage exhibit lower expression of this enzyme under these conditions. To investigate involvement of the PI3K pathway has \"in vivo\",LPS was administered i.v., as an endotoxic model, in which we observed a higher survival in PI3K- / - animals compared to WT animals and lower nitrite levels in serum. Our data suggest that PI3K enzyme is critical to iNOS expression and NO production by macrophages, possibly through activation of the CCR2 receptor, being involved in the LPS-induced shock pathophysiology