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.

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.

Dortmund, Universiẗat, Diss., 2001.

The phenomenon of nuclear magnetic resonance is closely related to the molecular beam experiments and to microwave spectroscopy. Its significant feature is that the magnetic resonance principle, first applied to the molecular beam technique, has been extended to solids, liquids and gases in their normal physical states. In addition to providing yet another important method of measuring nuclear magnetic properties, this newer technique gives a means of investigating the establishment of the thermal equilibrium which is essential to the methods of obtaining very low temperatures; further, the resonance absorption spectrum yields information of crystal structures, phase transitions in solids and information about hindered rotation of molecules in solids. Thus the phenomenon can be used to study certain problems of the solid state. This thesis gives an account of such an application.

Indiana University-Purdue University Indianapolis (IUPUI) / Papillomaviruses (PVs) are non-enveloped DNA viruses that are the primary etiological agents of cervical and oropharyngeal cancers. Vaccines for H(human)PV have proven to be effective prophylactic treatments; however, there is no treatment available for those currently infected. To develop new therapies, we require a clear understanding of viral pathogenesis and regulation. The Papillomavirus E2 protein is a sequence specific DNA binding protein that recruits cellular factors to its genome in infected epithelial cells. E2 also binds to and loads the viral E1 DNA helicase at the origin of replication. Post-translational modifications of PV E2 have been identified as potential regulators of E2 functions. We recently reported lysine (K) 111 as a target of p300 acetylation in B(bovine)PV that is involved in the regulation of viral transcription. K111 is conserved in most papillomaviruses, so we pursued a mutational approach to query the functional significance of lysine in HPV E2. Amino acid substitutions that prevent acetylation, including arginine, were unable to stimulate transcription and E1 mediated DNA replication. The arginine K111 mutant retained E2 transcriptional repression, nuclear localization, DNA and chromatin binding, and association with E2 binding partners involved in PV transcription and replication. When directly investigating origin unwinding, the replication defective E2 K111R mutant recruited E1 to the viral replication origin, but surprisingly, unwinding of the duplex DNA did not occur. In contrast, the glutamine K111 mutant increased origin melting and stimulated replication compared to wild type E2. We have identified Topoisomerase I as a key host factor involved in viral replication whose recruitment is dependent on K111 acetylation, and propose a new model for viral origin dynamics during replication initiation. This work reveals a novel activity of E2 necessary for denaturing the viral origin that likely depends on acetylation of highly conserved lysine 111.

A fagocitose de células apoptóticas, também denominada eferocitose, é um processo dinâmico e de fundamental importância para homeostase dos tecidos após uma injúria. Estudos demonstraram previamente que a fagocitose de células apoptóticas promove a síntese de mediadores anti-inflamatórios como PGE2, TGF-? e IL-10, podendo resultar num microambiente supressor e aumento da susceptibilidade do hospedeiro contra agentes infecciosos. Entretanto, a fagocitose de células apoptóticas infectadas por células dendríticas promove a geração não apenas de citocinas anti-inflamatórias como TGF-?, mas também de IL-6 e IL-23, levando a um efeito imunoestimulador, a diferenciação de células Th17. A atuação da PGE2 na imunidade adaptativa vem sendo investigada quanto à diferenciação e ativação de linfócitos Th1, Treg e Th17. Nossos resultados demonstram que a fagocitose de células apoptóticas infectadas com E. coli promove a ativação e migração de células dendríticas, assim como a produção de citocinas pró- e anti-inflamatórias e altos níveis de PGE2. No entanto, diferente da hipótese inicial, a presença de altas concentrações de PGE2 inibe drasticamente a diferenciação de células Th17 no contexto de fagocitose de células apoptóticas infectadas com E. coli por células dendríticas, in vitro. O tratamento de linfócitos T CD4+naive com antagonistas e agonistas de EP2/EP4 demonstram que o efeito supressor de PGE2 é mediado primordialmente pelo receptor EP4. Por fim, nossos resultados in vivo comprovam os resultados obtidos in vitro, demonstrando o papel supressor de PGE2 na diferenciação de células Th17 no contexto de fagocitose de células apoptóticas infectadas em modelo de infecção pulmonar. / The phagocytosis of apoptotic cells, also called efferocytosis, is a dynamic process critical for tissue homeostasis after injury. We and other groups previously have shown that phagocytosis of apoptotic cells promotes the synthesis of anti-inflammatory mediators such as PGE2, TGF-? and IL-10, that may result in the suppression of host defense against microorganisms. However, an elegant study using infected apoptotic cells showed that phagocytosis of these cells promote not only the generation of anti-inflammatory cytokines such as TGF-? but also IL-6 and IL-23, resulting in an immunostimulatory effect, the differentiation of Th17 cells. The role of PGE2 in adaptive immunity has been investigated regarding differentiation and activation of Th1, Th17 and Treg. Our results demonstrate that engulfment of E.coli infected apoptotic cells promotes the activation and migration of dendritic cells as well as production of pro and anti-inflammatory cytokines together with high levels of PGE2. However, differing from our hypothesis, high levels of PGE2 inhibits drastically the differentiation of Th17 cells on the context of engulfment of E.coli infected apoptotic cells by dendritic cells in vitro. The treatment of T CD4+naive cells with antagonist or agonists of EP2/EP4 receptors demonstrates the suppressor effect is mainly mediated by EP4 receptor. Finally, the instillation of E.coli infected apoptotic cells in E.coli infected animals resulted on modest Th17 increase but treatment with cox inhibitor increased Th17 cell differentiation. Therefore, our in vivo results prove the in vitro results.

A fagocitose de células apoptóticas, também denominada eferocitose, é um processo dinâmico e de fundamental importância para homeostase dos tecidos após uma injúria. Estudos demonstraram previamente que a fagocitose de células apoptóticas promove a síntese de mediadores anti-inflamatórios como PGE2, TGF-? e IL-10, podendo resultar num microambiente supressor e aumento da susceptibilidade do hospedeiro contra agentes infecciosos. Entretanto, a fagocitose de células apoptóticas infectadas por células dendríticas promove a geração não apenas de citocinas anti-inflamatórias como TGF-?, mas também de IL-6 e IL-23, levando a um efeito imunoestimulador, a diferenciação de células Th17. A atuação da PGE2 na imunidade adaptativa vem sendo investigada quanto à diferenciação e ativação de linfócitos Th1, Treg e Th17. Nossos resultados demonstram que a fagocitose de células apoptóticas infectadas com E. coli promove a ativação e migração de células dendríticas, assim como a produção de citocinas pró- e anti-inflamatórias e altos níveis de PGE2. No entanto, diferente da hipótese inicial, a presença de altas concentrações de PGE2 inibe drasticamente a diferenciação de células Th17 no contexto de fagocitose de células apoptóticas infectadas com E. coli por células dendríticas, in vitro. O tratamento de linfócitos T CD4+naive com antagonistas e agonistas de EP2/EP4 demonstram que o efeito supressor de PGE2 é mediado primordialmente pelo receptor EP4. Por fim, nossos resultados in vivo comprovam os resultados obtidos in vitro, demonstrando o papel supressor de PGE2 na diferenciação de células Th17 no contexto de fagocitose de células apoptóticas infectadas em modelo de infecção pulmonar. / The phagocytosis of apoptotic cells, also called efferocytosis, is a dynamic process critical for tissue homeostasis after injury. We and other groups previously have shown that phagocytosis of apoptotic cells promotes the synthesis of anti-inflammatory mediators such as PGE2, TGF-? and IL-10, that may result in the suppression of host defense against microorganisms. However, an elegant study using infected apoptotic cells showed that phagocytosis of these cells promote not only the generation of anti-inflammatory cytokines such as TGF-? but also IL-6 and IL-23, resulting in an immunostimulatory effect, the differentiation of Th17 cells. The role of PGE2 in adaptive immunity has been investigated regarding differentiation and activation of Th1, Th17 and Treg. Our results demonstrate that engulfment of E.coli infected apoptotic cells promotes the activation and migration of dendritic cells as well as production of pro and anti-inflammatory cytokines together with high levels of PGE2. However, differing from our hypothesis, high levels of PGE2 inhibits drastically the differentiation of Th17 cells on the context of engulfment of E.coli infected apoptotic cells by dendritic cells in vitro. The treatment of T CD4+naive cells with antagonist or agonists of EP2/EP4 receptors demonstrates the suppressor effect is mainly mediated by EP4 receptor. Finally, the instillation of E.coli infected apoptotic cells in E.coli infected animals resulted on modest Th17 increase but treatment with cox inhibitor increased Th17 cell differentiation. Therefore, our in vivo results prove the in vitro results.

GB virus C (GBV-C) is a nonpathogenic lymphotropic virus that replicates in B and T lymphocytes. Infection with GBV-C is documented worldwide and is common: between 1% and 5% of healthy blood donors are viremic at the time of donation. Antibodies to GBV-C proteins are not usually detected during viremia, and antibodies to the GBV-C envelope glycoprotein E2 develop following the clearance of viremia. Although GBV-C viremia may persist for decades, viremia usually clears within 2 years following infection in the majority of individuals infected by blood transfusion. A chimpanzee variant of GBV-C, designated GBV-Ccpz, is found in captive and noncaptive chimpanzees and its prevalence and natural history are uncharacterized. GBV-C research was initially performed by viral hepatitis research groups because it was predicted to cause hepatitis. The realization that GBV-C did not cause hepatitis resulted in a marked reduction in research activity. Because Hepatitis C virus co-infection worsens the clinical course of HIV-infected patients, researchers hypothesized that the related virus, GBV-C, may impact HIV disease. In 1998, researchers found that HIV-infected individuals who were co-infected with GBV-C survived longer than those without GBV-C. These findings provide the rationale for examining the relationship of GBV-C and HIV and the development of GBV-C as a novel therapeutic for HIV. GBV-C infection of PBMCs inhibits the replication of HIV isolates and one of the mechanisms for this is the induction of the release of soluble ligands for HIV entry receptors (RANTES, macrophage inflammatory proteins (MIP)-1α and MIP-1β and SDF-1) by GBV-C. The GBV-C envelope glycoprotein E2 contributes directly to the inhibition of HIV infection. Incubation of recombinant E2 with PBMCs at 4°C prior to HIV infection results in a decrease in HIV replication, and only HIV gp160 enveloped pseudoparticle transduction, not VSV-G enveloped pseudoparticle transduction, is inhibited by GBV-C E2. This suggests that GBV-C E2 inhibits HIV infection at an entry step when the HIV gp160 envelope protein interacts with cellular receptors and membranes. How GBV-C E2 interacts with cellular surfaces and which cellular proteins are utilized for GBV-C binding and entry are unknown. Here, we characterize GBV-C E2 binding to human PBMCs, murine cells, and multiple transformed cell lines to identify the PBMC subset which E2 binds and to identify candidate cellular receptors involved in GBV-C binding and entry. Understanding how GBV-C E2 interacts with cellular surfaces is critical to determining how it inhibits HIV entry. Anti-GBV-C E2 antibodies are also associated with improved survival in HIV-infected individuals. Recent studies demonstrated that anti-E2 antibodies neutralize HIV infection in vitro and immunoprecipitate HIV virions. In these studies, we describe how anti-E2 antibodies immunoprecipitate retroviral particles regardless of the specific viral envelope protein on the surface, but only neutralize particles bearing the HIV gp160 envelope protein. We also found that the cellular antigen recognized by anti-E2 antibodies is accessible only in permeabilized cells and not on the cell surface. These studies provide insight into the HIV-inhibitory mechanisms of anti-E2 antibodies, which should aid in the development of GBV-C E2 as an immunogen in an HIV vaccine. Finally, no animal models exist for studying GBV-C infection or GBV-C vaccines as HIV therapeutics in vivo. We examined the natural history GBV-Ccpz in a captive chimpanzee population, and found that the prevalence of GBV-Ccpz viremia and anti-E2 antibodies, as well as the length of persistent infection, were similar to those found in healthy human blood donors. The GBV-Ccpz 5#8217;ntr and RdRp sequences from chimpanzee subspecies troglodytes and verus shared a high level of sequence identity and indicate that the chimpanzee variant should be designated GBV-Ccpz rather than the currently used GBV-Ctrog. These findings demonstrate that GBV-Ccpz viremia and E2 antibody status should be tested in animals involved in clinical research trials because affected animals may have altered responses to GBV-C infection or HIV vaccines, and that the chimpanzee would be a good animal model in which to study GBV-C infection.

No description available.

Thesis advisor: Junona Moroianu / Human papillomaviruses (HPVs) are the most common sexually transmitted infection in the United States. High risk HPV types, including HPV 16, can cause cervical carcinomas upon infecting squamous basal epithelial cells. The HPV E2 protein is a multifunctional protein that regulates viral DNA replication and expression of a large number of cellular and viral genes, including the E6 and E7 viral oncogenes. Previous research in the Moroianu lab has identified a novel alpha-helical nuclear localization signal (NLS) in the C-terminal domain of HPV 16 E2 protein (75). Here, we focused on continuing the dissection of the HPV 16 E2 NLS and on identification of the nuclear import mechanism used by this protein. We identified several residues in the C-terminal domain of HPV 16 E2 (327KHK329) and within the NLS (K299, C300) that enhance the function of the NLS. Additionally, we determined that dimerization of the C-terminal domain plays an important role in the nuclear import of HPV 16 E2 as a mutation that disrupted it led to a significant decrease in the nuclear localization of the protein. We discovered that importin 11 karyopherin is a nuclear import receptor for HPV 16 E2. Our data suggest a nuclear import mechanism for HPV 16 E2 whereby UbcM2/UBE2E3 E2-type ubiquitin-conjugating enzyme acts as an adapter to bind HPV 16 E2 to importin 11 karyopherin for its nuclear import. This is a previously undescribed nuclear import mechanism which may have implications for the control of HPV 16 E2 functions. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.