Congenital insensitivity to pain (CIP) is an extremely rare inherited disorder characterised by the inability to perceive physical pain from birth, resulting in a number of injuries including self-mutilation, repeated burns and fractures. A number of different genes underlying CIP have been identified over the years and all act principally either to direct development or function of nociceptors. In this dissertation, a number of unrelated families with CIP were recruited and novel missense and splicing mutations in NTRK1, NGF and SCN9A were identified in the cohort. The findings presented in this dissertation demonstrate how mutations in the NTRK1 gene can cause the phenotype of CIP, and increase our knowledge of the functions and the role of key residues of TRKA within the cell. I have verified the importance of NGF in the development of nociceptors and demonstrate the overlapping roles of the precursor proNGF with mature NGF as well as providing insight into the role of proNGF as a neurotrophic molecule, in contrast to the wider consensus. Moreover, I provide further evidence that splicing mutations are also responsible for CIP and highlight that splicing mutations are potentially missed in diagnostic labs. Lastly, I have demonstrated that stem cells can be used to study and generate different types of sensory neurons, indicating a potential use as a suitable platform for investigating monogenic disorders. The identification of novel genes, in addition to the dissection of the residues and pathways of known genes, is essential for the development of new analgesics.

The goal of this dissertation research is to determine novel regulatory mechanisms of neurotrophin signaling mediated by protein phosphatase 2A (PP2A). PP2A is a ubiquitous Ser/Thr phosphatase that removes phosphates from proteins to switch their activity on or off. The substrate specificity and subcellular localization of PP2A is determined by almost 20 regulatory subunits that associate with a core dimer built of catalytic and scaffold subunits. Since there are more than 48 possible heterotrimers, studying the function of PP2A poses many challenges. Therefore we have devised a strategy, using scaffold subunit knockdown and mutant replacement, to discern the function of specific families of regulatory subunits. With this approach, I have identified specific PP2A holoenzymes that modulate nerve growth factor (NGF) signaling pathways by positively regulating TrkA receptor tyrosine kinase activity. Many studies have shown that NGF is required for the survival and differentiation of sensory and sympathetic neurons. Additionally, NGF is implicated in many neurodegenerative diseases including Alzheimer's disease, Parkinson's disease as well as neuropathic pain. NGF elicits its biological effect through sustained activity of the TrkA receptor and stimulated signaling cascades, including the MAP kinase pathway. Although PP2A has been shown to modulate the mitogen-activated protein (MAP) kinase pathway both positively and negatively at multiple levels, work described herein introduces yet another level of regulation. Specifically, I have shown that PP2A/B' holoenzymes complex with the TrkA neurotrophin receptor to potentiate receptor tyrosine kinase activity, downstream effector kinase activation, neurite outgrowth, and neuronal differentiation. On the other hand, extracellular signal regulated kinase (ERK), a terminal effector in the MAP kinase pathway was shown to phosphorylate a residue in the juxtamembrane region of TrkA and impose feedback inhibition of receptor activity. Collectively, these data suggest a model in which PP2A and ERK oppose each other in the regulation of TrkA receptor activity and downstream signaling cascades that govern neuronal differentiation and maintenance.

Medulloblastoma cells do not contain detectable amounts of the bZIP protein Zhangfei. However, previous work has shown that expression of this protein in cells of the ONS-76 line, derived from a human medulloblastoma, causes the cells to stop growing and develop processes that resemble neuritis (a characteristic of differentiated neurons). Zhangfei-expressing cells eventually die. My objective was to determine the molecular mechanisms by which Zhangfei influences ONS-76 cells. My strategy was to infect ONS-76 cells with adenovirus vectors expressing either Zhangfei or the control E. coli protein â-galactosidase (LacZ) and then to compare the following parameters in Zhangfei and LacZ-expressing cells: a) markers of apoptosis, autophagy and macropinocytosis (the three main pathways of cell death); b) transcripts for genes involved in neurogenesis and apoptosis; c) phosphorylation of peptide targets of selected cellular protein kinases; and d) active transcription factors. Zhangfei-expressing cells appeared to succumb to apoptosis as determined by the expression of phosphatidylserine on the cell surface and intensity of nuclear staining with the DNA dye Hoechst. Increased staining for autophagic vesicles and upregulated expression of autophagy response genes in these cells indicated that they were undergoing autophagy, possibly associated with apoptosis. My analysis of steady-state transcripts for genes involved in apoptosis and neurogenesis and functional protein kinases in Zhangfei-expressing cells indicated that the mitogen-activated protein kinase (MAPK) pathway was active in these cells. In addition, I found that the transcription factor Brn3a as well as factors implicated in differentiation were also active. These observations led me to hypothesize that Zhangfei enhances the expression of Brn3a, a known inducer of TrkA, the high-affinity receptor for nerve growth factor (NGF). TrkA then binds in an autocrine manner to NGF, triggering the MAPK pathway and leading to differentiation of ONS-76 cells into neuron and glia-like cells, eventually bringing about cell death by apoptosis and autophagy. I tested this hypothesis by showing that Zhangfei could enhance transcription from the isolated Brn3a promoter, that ONS-76 cells produce NGF as detected in a bioassay, and that antibodies against NGF and inhibitors of TrkA and selected components of the MAPK pathway could partially restore the growth of Zhangfei-expressing ONS-76 cells. My work supports previous work highlighting the importance of NGF-TrkA signaling in the outcome of medulloblastomas and shows how Zhangfei is able to trigger this pathway.

Medulloblastoma cells do not contain detectable amounts of the bZIP protein Zhangfei. However, previous work has shown that expression of this protein in cells of the ONS-76 line, derived from a human medulloblastoma, causes the cells to stop growing and develop processes that resemble neuritis (a characteristic of differentiated neurons). Zhangfei-expressing cells eventually die. My objective was to determine the molecular mechanisms by which Zhangfei influences ONS-76 cells. My strategy was to infect ONS-76 cells with adenovirus vectors expressing either Zhangfei or the control E. coli protein â-galactosidase (LacZ) and then to compare the following parameters in Zhangfei and LacZ-expressing cells: a) markers of apoptosis, autophagy and macropinocytosis (the three main pathways of cell death); b) transcripts for genes involved in neurogenesis and apoptosis; c) phosphorylation of peptide targets of selected cellular protein kinases; and d) active transcription factors. Zhangfei-expressing cells appeared to succumb to apoptosis as determined by the expression of phosphatidylserine on the cell surface and intensity of nuclear staining with the DNA dye Hoechst. Increased staining for autophagic vesicles and upregulated expression of autophagy response genes in these cells indicated that they were undergoing autophagy, possibly associated with apoptosis. My analysis of steady-state transcripts for genes involved in apoptosis and neurogenesis and functional protein kinases in Zhangfei-expressing cells indicated that the mitogen-activated protein kinase (MAPK) pathway was active in these cells. In addition, I found that the transcription factor Brn3a as well as factors implicated in differentiation were also active. These observations led me to hypothesize that Zhangfei enhances the expression of Brn3a, a known inducer of TrkA, the high-affinity receptor for nerve growth factor (NGF). TrkA then binds in an autocrine manner to NGF, triggering the MAPK pathway and leading to differentiation of ONS-76 cells into neuron and glia-like cells, eventually bringing about cell death by apoptosis and autophagy. I tested this hypothesis by showing that Zhangfei could enhance transcription from the isolated Brn3a promoter, that ONS-76 cells produce NGF as detected in a bioassay, and that antibodies against NGF and inhibitors of TrkA and selected components of the MAPK pathway could partially restore the growth of Zhangfei-expressing ONS-76 cells. My work supports previous work highlighting the importance of NGF-TrkA signaling in the outcome of medulloblastomas and shows how Zhangfei is able to trigger this pathway.

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

Le NGF est reconnu, et le signal qu’il véhicule est donc médié, par deux récepteurs membranaires : p75NTR et TrkA. Il a été démontré qu’au niveau de la membrane, p75NTR et TrkA sont localisées dans les radeaux membranaires, des microdomaines caractérisés par la présence de protéines cavéolines (Cav-1 et/ou Cav-2). Dans le présent travail, nous avons constaté que la surexpression de Cav-1 dans les neurones des ganglions de la racine dorsale diminue l’extension des neurites. De la même manière, la surexpression de Cav-1 dans les cellules PC12 inhibe les réponses cellulaires déclenchées par l’exposition au NGF. L’activation des effecteurs situés en aval de TrkA n’est pas inhibée. L’expression de Cav-1 provoque une inhibition de la sortie du récepteur des radeaux accompagné par la rétention au niveau de la surface cellulaire, des effecteurs situés en aval incluant Rsk2 phosphorylé. Dans le même temps, la présence de formes phosphorylées de CREB n’est plus détectable. En revanche, la surexpression de Cav-2 potentialise la différenciation des cellules induite par le NGF, ce qui est associé à une activation prolongée des effecteurs situés en aval et à une internalisation des récepteurs. Ces différents effets pourraient être dû à la localisation des cavéolines, qui résulte en une perturbation du microenvironnement des cellules et donc de la signalisation du NGF. En outre, l’expression d’une Cav-1 mutée sur la sérine 80 (S80V) dans des cellules PC12, ne gêne ni le trafic ni la signalisation de TrkA. Au contraire elles se comportent de façon semblable à des cellules Cav-2. Ces études soulignent également le rôle potentiel de Cav-1 et ses mutations dans des cancers NGF-dépendantes. / At the plasma membrane, both NGF receptors have been shown to localized to lipid rafts, specific subdomains that are enriched in cholesterol, sphingolipids and the presence of caveolin proteins (Cav1 and/or Cav2). The focus of this work is on this membrane microenvironment mediated modulation of NGF signaling which via two receptors: p75NTR and TrkA. In the present work we found that overexpression of Cav-1 in mouse dorsal root ganglia neurons significantly impacted neurite extension. Similarly, overexpression of Cav-1 in PC12 cells strongly inhibits their ability to grow neurites in response to NGF. It inhibits NGF signaling without, impairing transient MAPK pathway activation. Rather, it does so by sequestering NGF receptors in lipid rafts, which correlates with the cell surface localization of downstream effectors, and phosphorylated-Rsk2, resulting in the prevention of the phosphorylation of CREB. By contrast, overexpression of Cav-2 potentiates NGF induced differentiation, which is accompanied by sustained activation of downstream effectors, and standard internalization of the receptors. This differential effect could be due to the different localization of Caveolins, that modifies the microenvironment, thereby affecting NGF signaling. Furthermore, PC12 cells expressing the non-phosphorylatable Cav-1 mutant (S80V), neither TrkA trafficking or CREB phosphorylation are inhibited and the response resembles that observed in Cav-2 expressing PC12 cells. These studies underline the interplay between caveolins and NGF signalling, offering insight into the potential impact of Caveolin-1 and mutations thereof in certain cancers where NGF signaling is involved.

Herpes simplex viruses (HSV) establish latent infections in sensory neurons of their host and are maintained in this state by little understood mechanisms that, at least in part, are regulated by signalling through nerve growth factor (NGF) and its receptor tropomyosin related kinase, trkA. Previous studies have demonstrated that Zhangfei is a transcriptional factor that is expressed in differentiated neurons and is thought to influence HSV replication and latency. Zhangfei, like the HSV trans-activator VP16 and Luman, binds the ubiquitous nuclear protein host cell factor (HCF) inhibiting the ability of VP16 and Luman to initiate HSV replication. <p>Recently, Brn3a, another neuronal factor thought to influence HSV latency and reactivation was found to possess an HCF-binding domain and could potentially require HCF for activity. The neuronal POU IV domain protein, Brn3a, among its many regulatory functions has been described as an enhancer of the NGF receptor trkA, during development in mouse. I therefore investigated the possible link between Brn3a, TrkA, NGF signaling, HCF, Zhangfei and HSV-1 latency and reactivation. I hypothesized that Zhangfei would also suppress the ability of Brn3a to activate the expression of TrkA and that this would have an impact on NGF-TrkA signaling and, consequently on HSV-1 reactivation from latency.<p>My first study determined which Brn3a/trkA promoter interactions were important for trkA transcription. I constructed a plasmid that contains 1043 base pairs of genomic sequences that extend from 30 nucleotides upstream of trkA coding region. In contrast to previous data, a short 190 bp region that lies proximal to the trkA initiation codon was sufficient for Brn3a trans-activation in NGF-differentiated PC12, Vero and human medulloblastoma cells. At least two portions of the 190 bp fragment bind to Brn3a. In addition, Brn3a increased endogenous levels of trkA transcripts in PC12 cells and initiated trkA expression in medulloblastoma cells, which normally do not express trkA. <p>The second step was to determine the effects of HCF and Zhangfei association with Brn3a on trkA trans-activation. I found that Brn3a required HCF for activating the trkA promoter and that Zhangfei has a suppressive effect over Brn3a-trkA activation in non-neuronal cells. In sympathetic neuron-like NGF-treated PC12 cells, Zhangfei did not suppress the ability of Brn3a to activate the TrkA promoter, however, Zhangfei was able capable of inducing the expression of TrkA in the absence of Brn3a. Both Brn3a and Zhangfei induced the expression of endogenous trkA in PC12 cells.<p>Since Vero and PC12 cells are not from human origin I wanted to examine the ability of Zhangfei to induce trkA transcription in medulloblastoma cells, that because of its tumor nature do not express trkA. TrkA transfections in these cells have shown to drive them to cell arrest or apoptosis. Since Zhangfei is not express in medulloblastoma tumors I then used ONS-76 medulloblastoma cells as a model to determine Zhangfeis envolvement in the NGF-trkA signaling pathway.<p> I show herein that in ONS-76 medulloblastoma cells resveratrol, an inducer of apoptosis and differentiation, increased the expression of Zhangfei and trkA as well as Early Growth Response Gene 1 (Egr1), a gene normally activated by NGF-trkA signalling. ONS-76 cells stop growing soon after treatment with resveratrol and a portion of the cell undergo apoptosis. While the induction of Zhangfei in resveratrol-treated cells was modest albeit consistent, the infection of actively growing medulloblastoma cells with an adenovirus vector expressing Zhangfei mimicked the effects of resveratrol. Zhangfei activated the expression of trkA and Egr1 and caused these cells to display markers of apoptosis. The phosphorylation of Erk1, an intermediate kinase in the NGF-trkA signaling critical for differentiation, was observed in Zhangfei infected cells, supporting the hypothesis that Zhangfei is a mediator of trkA-NGF signaling in theses cells leading either to differentiation or apoptosis. Binding of HCF by Zhangfei did not appear to be required for this effect as a mutant of Zhangfei incapable of binding HCF was also able to induce the expression of trkA and Egr1. <p>In in vivo and in vitro models of HSV-1 latency, the virus reactivates when NGF supply to the neuron is interrupted. Based on the above evidence Zhangfei, in HSV-1 latently infected neurons, would have the ability to prolong a state of latency by inducing trkA expression allowing the activation of NGF-trkA signaling pathway. Since NGF is produced by many cell types it is possible that reactivation is triggered not by a decrease in NGF but by a down-regulation of TrkA expression.Therefore, if Zhangfei expression is suppress the trkA signaling could be interrupted or shifted towards apoptosis signaling, this would allow neuronal HCF-binding proteins like Luman, which can activate HSV IE expression, to initiate HSV IE expression and subsequently viral replication.

Herpes simplex viruses (HSV) establish latent infections in sensory neurons of their host and are maintained in this state by little understood mechanisms that, at least in part, are regulated by signalling through nerve growth factor (NGF) and its receptor tropomyosin related kinase, trkA. Previous studies have demonstrated that Zhangfei is a transcriptional factor that is expressed in differentiated neurons and is thought to influence HSV replication and latency. Zhangfei, like the HSV trans-activator VP16 and Luman, binds the ubiquitous nuclear protein host cell factor (HCF) inhibiting the ability of VP16 and Luman to initiate HSV replication. <p>Recently, Brn3a, another neuronal factor thought to influence HSV latency and reactivation was found to possess an HCF-binding domain and could potentially require HCF for activity. The neuronal POU IV domain protein, Brn3a, among its many regulatory functions has been described as an enhancer of the NGF receptor trkA, during development in mouse. I therefore investigated the possible link between Brn3a, TrkA, NGF signaling, HCF, Zhangfei and HSV-1 latency and reactivation. I hypothesized that Zhangfei would also suppress the ability of Brn3a to activate the expression of TrkA and that this would have an impact on NGF-TrkA signaling and, consequently on HSV-1 reactivation from latency.<p>My first study determined which Brn3a/trkA promoter interactions were important for trkA transcription. I constructed a plasmid that contains 1043 base pairs of genomic sequences that extend from 30 nucleotides upstream of trkA coding region. In contrast to previous data, a short 190 bp region that lies proximal to the trkA initiation codon was sufficient for Brn3a trans-activation in NGF-differentiated PC12, Vero and human medulloblastoma cells. At least two portions of the 190 bp fragment bind to Brn3a. In addition, Brn3a increased endogenous levels of trkA transcripts in PC12 cells and initiated trkA expression in medulloblastoma cells, which normally do not express trkA. <p>The second step was to determine the effects of HCF and Zhangfei association with Brn3a on trkA trans-activation. I found that Brn3a required HCF for activating the trkA promoter and that Zhangfei has a suppressive effect over Brn3a-trkA activation in non-neuronal cells. In sympathetic neuron-like NGF-treated PC12 cells, Zhangfei did not suppress the ability of Brn3a to activate the TrkA promoter, however, Zhangfei was able capable of inducing the expression of TrkA in the absence of Brn3a. Both Brn3a and Zhangfei induced the expression of endogenous trkA in PC12 cells.<p>Since Vero and PC12 cells are not from human origin I wanted to examine the ability of Zhangfei to induce trkA transcription in medulloblastoma cells, that because of its tumor nature do not express trkA. TrkA transfections in these cells have shown to drive them to cell arrest or apoptosis. Since Zhangfei is not express in medulloblastoma tumors I then used ONS-76 medulloblastoma cells as a model to determine Zhangfeis envolvement in the NGF-trkA signaling pathway.<p> I show herein that in ONS-76 medulloblastoma cells resveratrol, an inducer of apoptosis and differentiation, increased the expression of Zhangfei and trkA as well as Early Growth Response Gene 1 (Egr1), a gene normally activated by NGF-trkA signalling. ONS-76 cells stop growing soon after treatment with resveratrol and a portion of the cell undergo apoptosis. While the induction of Zhangfei in resveratrol-treated cells was modest albeit consistent, the infection of actively growing medulloblastoma cells with an adenovirus vector expressing Zhangfei mimicked the effects of resveratrol. Zhangfei activated the expression of trkA and Egr1 and caused these cells to display markers of apoptosis. The phosphorylation of Erk1, an intermediate kinase in the NGF-trkA signaling critical for differentiation, was observed in Zhangfei infected cells, supporting the hypothesis that Zhangfei is a mediator of trkA-NGF signaling in theses cells leading either to differentiation or apoptosis. Binding of HCF by Zhangfei did not appear to be required for this effect as a mutant of Zhangfei incapable of binding HCF was also able to induce the expression of trkA and Egr1. <p>In in vivo and in vitro models of HSV-1 latency, the virus reactivates when NGF supply to the neuron is interrupted. Based on the above evidence Zhangfei, in HSV-1 latently infected neurons, would have the ability to prolong a state of latency by inducing trkA expression allowing the activation of NGF-trkA signaling pathway. Since NGF is produced by many cell types it is possible that reactivation is triggered not by a decrease in NGF but by a down-regulation of TrkA expression.Therefore, if Zhangfei expression is suppress the trkA signaling could be interrupted or shifted towards apoptosis signaling, this would allow neuronal HCF-binding proteins like Luman, which can activate HSV IE expression, to initiate HSV IE expression and subsequently viral replication.

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

Receptor-tyrosine kinases (RTKs) are membrane bound receptors characterized by their intrinsic kinase activity. RTK activities play an essential role in several human diseases, including cancer, diabetes and neurodegenerative diseases. RTK activities have been regulated by the expression or silencing of several genes as well as by the utilization of small molecules. Ras Interference 1 (Rin1) is a multifunctional protein that becomes associated with activated RTKs upon ligand stimulation. Rin1 plays a key role in receptor internalization and in signal transduction via activation of Rab5 and association with active form of Ras. This study has two main objectives: (1) It determines the role of Rin1 in the regulation of several RTKs focusing on insulin receptor. This was accomplished by studying the Rin1-insulin receptor interaction using a variety of biochemical and morphological assays. This study shows a novel interaction between the insulin receptor and Rin1 through the Vps9 domain. Two more RTKs (epidermal growth factor receptor and nerve growth factor receptor) also interacted with the SH2 domain of Rin1. The effect of the Rin1-RTK interaction on the activation of both Rab5 and Ras was also studied during receptor internalization and intracellular signaling. Finally, the role of Rin1 was examined in two differentiation processes (adipogenesis and neurogenesis). Rin1 showed a strong inhibitory effect on 3T3-L1 preadipocyte differentiation but it seems to show a modest effect in PC12 neurite outgrowth. These data indicate a selective function and specific interaction of Rin1 toward RTKs. (2) It examines the role of the small molecule Dehydroleucodine (DhL) on several key signaling molecules during adipogenesis. This was accomplished by studying the differentiation of 3T3-L1 preadipocytes exposed to different concentrations of DhL in different days of the adipocyte formation process. The results indicate that DhL selectively blocked adipocyte formation, as well as the expression of PPARγ, and C/EBPα. However, DhL treatment did not affect Rin1 or Rab5 expression and their activities. Taken together, the data indicate a potential molecular mechanism by which proteins or small molecules regulate selective and specific RTK intracellular membrane trafficking and signaling during cell growth and differentiation in normal and pathological conditions.