Thesis (Ph.D.). / Written for the Dept. of Pharmacology and Therapeutics. Title from title page of PDF (viewed 2008/05/09). Includes bibliographical references.

Made available in DSpace on 2014-12-05T18:40:15Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) diego_rodrigues_ioc_mest_2009.pdf: 6237150 bytes, checksum: 6688132dca9a5eef5cf8d01c0a302000 (MD5) Previous issue date: 2014-11-18 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / O vírus da imunodeficiência humana tipo 1 (HIV-1), agente etiológico da síndrome da imunodeficiência adquirida (AIDS), representa um dos patógenos de maior interesse clínico das últimas décadas. Durante a infecção pelo HIV-1 muitas células CD4+ morrem; enquanto outras, como os macrófagos, sobrevivem e sustentam a replicação do HIV-1 por longos períodos, transformando-se em reservatórios virais. Desta forma, o reconhecimento de fatores que possam manter esses reservatórios celulares vivos e influenciar a replicação do HIV-1 é uma das maiores tarefas para o desenvolvimento de uma estratégia terapêutica mais eficaz. Uma vez demonstrado que a infecção pelo HIV-1 aumenta a secreção do fator de crescimento do nervo (NGF) e que esta molécula é crucial para a sobrevivência de macrófagos, nós analisamos se o NGF poderia modular a replicação do HIV-1 em PBMCs e macrófagos e os mecanismos relacionados a esse fenômeno. Células mononucleares de sangue periférico (PBMCs) e macrófagos infectados in vitro com HIV-1 foram tratadas com NGF. A replicação viral foi medida por ELISA para p24 em sobrenadantes de cultura. Diferentes inibidores farmacológicos foram utilizados para a análise de possíveis vias de sinalização intracelular envolvidas com a replicação de HIV-1 induzida por NGF. A participação de APOBEC3G foi avaliada em macrófagos expostos ao NGF, utilizando RT-PCR e \201Cimmunoblotting\201D. Infecções com HIV-1 sincronizadas foram utilizadas para estudar se o NGF poderia influenciar a ligação e entrada do vírus. A integração e a transcrição do HIV-1 foram avaliadas por PCR e real-time PCR, respectivamente. Nossos resultados demonstraram que o NGF estimulou a replicação do HIV-1 em macrófagos mas não em PBMCs atingindo um aumento de até 20 vezes em relação ao controle quando tratado com 10ng/mL. Esta neurotrofina não afetou a adsorção, a penetração, nem a integração do DNA proviral. Desta forma, o NGF deve atuar através da estimulação da transcrição das proteínas virais. A via disparada por NGF que estimula a transcrição do HIV-1 é dependente do receptor TrKA, que leva a mobilização de cálcio intracelular derivado do reticulo endoplasmático e na ativação de PKC. Uma vez disparado, PKC ativa ERK1/2, p38quinase e NFkB. A diminuição da produção de APOBEC3G em macrófagos tratados com NGF, também foi observada, inclusive quando tratado com interferon-y. Em conjunto, nossos resultados sugerem que o NGF estimula a produção de HIV-1 em macrófagos. Esse efeito envolve o receptor TrKA e está associado com a regulação negativa de APOBEC3G. Nosso estudo evidencia uma nova forma de interação entre o HIV-1 e a célula hospedeira, trazendo bases para uma melhor compreensão sobre esta complexa reação / The human immunodeficiency virus type 1 (HIV - 1), the e tiological agent of the acquired immunodeficiency syndrome (AIDS), represents one of the main pathogens of clinical interest of the last decades. During HIV - 1 infection many CD4 + cells die; while others, such as macrophages, survive and sustain HIV - 1 repli cation for long periods, becoming viral reservoirs. Therefore, the recognition of factors that can maintain these reservoir cells alive and influence HIV - 1 replication is one of the main tasks for the development of a more efficient therapeutic strategy. S ince it has been shown that HIV - 1 infection increases nerve growth factor (NGF) secretion , and that this molecule is crucial for macrophage survival, we further analyzed whether NGF could modulate HIV - 1 replication in PBMCs and macrophages and the mechanis ms underlying this phenomenon. Peripheral blood mononuclear cells (PBMC) and macrophages infected i n vitro by HIV - 1 were treated with recombinant human NGF. Viral replication was measured by p24 - ELISA in culture supernatants. Different pharmacological inhi bitors were employed to analyze the possible signaling pathway involved in NGF - induced HIV - 1 replication. Participation of APOBEC3G was evaluated in macrophages exposed to NGF, using RT - PCR and immunoblotting assays. Synchronized HIV - 1 infections were also used to study whether NGF would influence HIV - 1 biding/entry. HIV - 1 integration and transcription were evaluated by PCR and real - time PCR, respectively. Our results demonstrated that NGF stimulated HIV - 1 replication in macrophages, but not in PBMCs reach ing as much as a 20 - fold increase at 10 ng/mL. This neurotrophin did not affect viral adsorption and penetration, nor integration of proviral DNA. Therefore, NGF probably act trough the stimulation of viral transcription. The pathway triggered by NGF to st imulate HIV - 1 transcription was dependent on the engagement of high affinity receptor TrKA, which led to a mobilization of intracellular calcium, derived from endoplasmatic reticulum, and to PKC signaling. Once triggered, PKC activated ERK1/2, p38kinase, a nd NF - kB. We also observed a decrease of APOBEC3G production in NGF - treated macrophages, even when they were stimulated with interferon -  . All together, o ur results suggest that NGF stimulates HIV - 1 production in an important HIV - 1 reservoir, such as macro phages. This effect involves TrKA engagement and is associated with APOBEC3G down - regulation. Our study evidences a new feature of HIV - 1/cell host interaction, providing basis for a better comprehension of this complex interaction

To understand the functional interactions between the TrkA and p75 nerve growth factor (NGF) receptors, we employed several lines of investigation including biophysical, biochemical and cellular assays. A high-affinity nerve growth factor (NGF) receptor is thought to be a complex of two receptors, p75 and the receptor tyrosine kinase, TrkA. The existence of a gp75-TrkA complex was demonstrated by a copatching technique. p75 on the surface of intact cells is patched with an anti-p75 antibody and fluorescent secondary antibody, the cells are then fixed to prevent further antibody-induced redistributions, and the distribution of TrkA is probed with an anti-TrkA antibody and fluorescent secondary antibody. We utilize a baculovirus-insect cell expression system, which allows high level expression of wild-type and mutated NGF receptors. TrkA and p75 copatch in both the absence and presence of NGF. This association is specific, since p75 does not copatch with other tyrosine kinase receptors, including TrkB, platelet-derived growth factor receptor-β and Torso (Tor). To determine which domains of TrkA are required for copatching, we used a series of TrkA-Tor chimeric receptors and show that the extracellular domain of TrkA is sufficient for copatching with p75. A chimeric receptor with TrkA transmembrane and intracellular domains shows partial copatching with p75. Deletion of the intracellular domain of p75 decreases but does not eliminate copatching. A point mutation that inactivates the TrkA kinase has no effect on copatching, indicating that this enzymatic activity is not required for association with p75. Hence, although interactions between the p75 and TrkA extracellular domains are sufficient for complex formation, interactions involving other receptor domains also play a role. To study what signal transduction mechanisms were activated by the two receptors to bring about differentiation and survival, we stably transfected LAN5 neuroblastoma cells with an expression vector for ET-R, a chimeric receptor with the extracellular domain of the epidermal growth factor receptor (EGFR) and the TrkA transmembrane and intracellular domains. EGF activated the ET-R kinase and induced partial differentiation. NGF, which can bind to endogenous p75, did not induce differentiation, but enhanced the EGF-induced response, leading to differentiation of almost all of the cells. A mutated NGF, 3T-NGF, that binds to TrkA but not to p75 did not synergize with EGF. Enhancement of EGF-induced differentiation required at least nanomolar concentrations of NGF, consistent with the low-affinity p75 binding site. EGF may induce a limited number of neuronal cells because it also enhances apoptosis. Both NGF and a caspase inhibitor reduced apoptosis and, thereby, enhanced differentiation. NGF appears to enhance survival through the phosphatidylinositol-3 kinase (PI3K) pathway. Consistent with this hypothesis, Akt, a downstream effector of the PI3K pathway, was hyperphosphorylated in the presence of EGF+NGF. These results demonstrate that TrkA kinase initiates differentiation, and p75 enhances differentiation by rescuing differentiating cells from apoptosis via the PI3K pathway. Even though both EGF and NGF are required for differentiation of LAN5/ET-R cells, only NGF is required for survival of the differentiated cells. In the absence of NGF, the cells die by an apoptotic mechanism, involving caspase-3. An anti-p75 antibody blocked the survival effect of NGF. Brain-derived neurotrophic factor also enhanced cell survival, indicating that in differentiated cells, NGF acts through the p75 receptor to prevent apoptosis.