The serotonin-1A (5-HT1A) receptor plays an important role in the regulation of the serotonin (5-HT) system as an autoreceptor on 5-HT neurons. The transcription factor CC2D1A/Freud-1 is a potent repressor of the 5-HT1A promoter in neuronal but not in non-neuronal cells. The clinical relevance of Freud-1 is evident in a naturally occurring mutation, resulting in a truncated form of Freud-1 lacking its C-terminal half, that is associated with non syndromic mental retardation in humans. Thus, it is of interest to clarify the structure and function of Freud-1. As Freud-1 was shown to interact with the transcriptional regulator Brg1 at the 5-HT1A promoter, identification of the structural domains mediating the Brg1/Freud-1 interaction is required to assess the role of Brg1 in Freud-1 repression. In this study, I used pull-down assays with recombinant proteins, co-immunoprecipitation studies and immunofluorescent staining with confocal microscopy to show that Freud-1 interacts directly with the C-terminus of Brg1 and that the C-terminal domain of Freud-1 is required for this interaction.

The serotonin-1A (5-HT1A) receptor plays an important role in the regulation of the serotonin (5-HT) system as an autoreceptor on 5-HT neurons. The transcription factor CC2D1A/Freud-1 is a potent repressor of the 5-HT1A promoter in neuronal but not in non-neuronal cells. The clinical relevance of Freud-1 is evident in a naturally occurring mutation, resulting in a truncated form of Freud-1 lacking its C-terminal half, that is associated with non syndromic mental retardation in humans. Thus, it is of interest to clarify the structure and function of Freud-1. As Freud-1 was shown to interact with the transcriptional regulator Brg1 at the 5-HT1A promoter, identification of the structural domains mediating the Brg1/Freud-1 interaction is required to assess the role of Brg1 in Freud-1 repression. In this study, I used pull-down assays with recombinant proteins, co-immunoprecipitation studies and immunofluorescent staining with confocal microscopy to show that Freud-1 interacts directly with the C-terminus of Brg1 and that the C-terminal domain of Freud-1 is required for this interaction.

The serotonin-1A (5-HT1A) receptor plays an important role in the regulation of the serotonin (5-HT) system as an autoreceptor on 5-HT neurons. The transcription factor CC2D1A/Freud-1 is a potent repressor of the 5-HT1A promoter in neuronal but not in non-neuronal cells. The clinical relevance of Freud-1 is evident in a naturally occurring mutation, resulting in a truncated form of Freud-1 lacking its C-terminal half, that is associated with non syndromic mental retardation in humans. Thus, it is of interest to clarify the structure and function of Freud-1. As Freud-1 was shown to interact with the transcriptional regulator Brg1 at the 5-HT1A promoter, identification of the structural domains mediating the Brg1/Freud-1 interaction is required to assess the role of Brg1 in Freud-1 repression. In this study, I used pull-down assays with recombinant proteins, co-immunoprecipitation studies and immunofluorescent staining with confocal microscopy to show that Freud-1 interacts directly with the C-terminus of Brg1 and that the C-terminal domain of Freud-1 is required for this interaction.

CC2D1A (coiled-coil and C2 domain containing protein 1A), also known as Freud-1, has been identified as a transcriptional repressor of the serotonin receptor 5-HT1A, a regulator of endosomal budding and an activator of NF-KB signaling. It also acts as a scaffold that promotes activity of the PI3K/Akt pathway upon stimulation by the epidermal growth factor (EGF). Moreover, several studies highlight naturally occurring mutations of CC2D1A in humans that produce varying degrees of intellectual disorder and autism. Use of the Cre-LoxP system to conditionally knockout CC2D1A in mice has provided promising results regarding its effect on 5-HT1A expression and behaviour. This thesis aims to extend the use of this knockout model by studying cell signaling activity in mouse embryonic fibroblasts (MEFs), derived from the CC2D1Aflx/flx transgenic line, that have been treated with a commercially available Cre recombinase to completely knock out CC2D1A. I hypothesize that CC2D1A directly regulates EGF receptor activity and that its Cre-mediated knock down in vitro will entirely block cell signaling pathways activated by the EGF receptor. Western blot analysis demonstrated that, after Cre-mediated CC2D1A knockout, Akt and Erk1/2 phosphorylation were still maintained upon EGF treatment. In addition, overexpressing Freud-1 via transfection had no effect on cell signaling compared to the wild-type control. Analysis of recombinant Freud-1 constructs reveal that a C-terminal truncation enhances its ability to bind to PIP2 and PIP3 – phospholipids essential to the Akt pathway. In addition, immunocytochemistry analysis demonstrates a responsiveness of CC2D1A to EGF treatment. Altogether, these data highlight a unique and effective way in carrying out gene knockout in vitro while also emphasizing the need to further investigate CC2D1A’s importance in regulating cell signaling pathways and functional compensation by other homologous proteins

The mechanisms that govern the repression of 5-HT1A receptor gene expression mediated by a novel mental retardation gene, Freud-1, were examined in HEK293 and SKNSH cells. This study provides a possible mechanism of 5-HT1A receptor gene regulation by Freud-1, which, to mediate its action, recruits Swi/Snf and Sin3A/histone deacetylase (HDAC) complexes in non-neuronal HEK293 cells and Swi/Snf only in neuronal, 5-HT1A receptor-expressing SKNSH cells. Thus, Freud-1 has a dual mechanism of repression depending on cell type: HDAC dependent in HEK293 cells and HDAC independent in SKNSH cells. In addition, I present evidence that Freud-1 is not sumoylated at its consensus sumoylation sites and I present the lipid binding properties of Freud-1 and Freud-1 mutants.

The mechanisms that govern the repression of 5-HT1A receptor gene expression mediated by a novel mental retardation gene, Freud-1, were examined in HEK293 and SKNSH cells. This study provides a possible mechanism of 5-HT1A receptor gene regulation by Freud-1, which, to mediate its action, recruits Swi/Snf and Sin3A/histone deacetylase (HDAC) complexes in non-neuronal HEK293 cells and Swi/Snf only in neuronal, 5-HT1A receptor-expressing SKNSH cells. Thus, Freud-1 has a dual mechanism of repression depending on cell type: HDAC dependent in HEK293 cells and HDAC independent in SKNSH cells. In addition, I present evidence that Freud-1 is not sumoylated at its consensus sumoylation sites and I present the lipid binding properties of Freud-1 and Freud-1 mutants.

The mechanisms that govern the repression of 5-HT1A receptor gene expression mediated by a novel mental retardation gene, Freud-1, were examined in HEK293 and SKNSH cells. This study provides a possible mechanism of 5-HT1A receptor gene regulation by Freud-1, which, to mediate its action, recruits Swi/Snf and Sin3A/histone deacetylase (HDAC) complexes in non-neuronal HEK293 cells and Swi/Snf only in neuronal, 5-HT1A receptor-expressing SKNSH cells. Thus, Freud-1 has a dual mechanism of repression depending on cell type: HDAC dependent in HEK293 cells and HDAC independent in SKNSH cells. In addition, I present evidence that Freud-1 is not sumoylated at its consensus sumoylation sites and I present the lipid binding properties of Freud-1 and Freud-1 mutants.

The mechanisms that govern the repression of 5-HT1A receptor gene expression mediated by a novel mental retardation gene, Freud-1, were examined in HEK293 and SKNSH cells. This study provides a possible mechanism of 5-HT1A receptor gene regulation by Freud-1, which, to mediate its action, recruits Swi/Snf and Sin3A/histone deacetylase (HDAC) complexes in non-neuronal HEK293 cells and Swi/Snf only in neuronal, 5-HT1A receptor-expressing SKNSH cells. Thus, Freud-1 has a dual mechanism of repression depending on cell type: HDAC dependent in HEK293 cells and HDAC independent in SKNSH cells. In addition, I present evidence that Freud-1 is not sumoylated at its consensus sumoylation sites and I present the lipid binding properties of Freud-1 and Freud-1 mutants.

O presente trabalho procura compreender o conceito de trauma psíquico a partir das teorizações de S. Freud e S. Ferenczi. Discutem-se as ideias expostas pelos dois autores sobre os aspectos intra e interpsíquicos envolvidos na formação do trauma, com o intuito de articulá-las de um modo que contemple as diferentes experiências de traumatização. A dissertação está dividida em três partes. Na primeira, composta de cinco capítulos, o eixo são experiências que parecem ter um efeito disruptivo tão logo acontecem, imediatamente ou após um curto intervalo de tempo. Incluem-se, aqui, as neuroses traumáticas, em geral, e as neuroses de guerra, em particular, assim como eventos de menor intensidade, mas que demandam um trabalho característico do aparelho psíquico. A atenção de Freud e Ferenczi voltou-se para esse tipo de formação traumática, em um tempo, após a Primeira Guerra Mundial, devido à necessidade de compreender e tratar soldados com sintomas que remetiam às experiências de trauma. Devido ao despreparo, à ausência de contrainvestimento do sistema consciente, e à intensidade da estimulação, o psiquismo é obrigado a acionar medidas defensivas primitivas, na tentativa de ligar o afluxo de excitação. Na segunda parte deste estudo, dividida em quatro capítulos, discutem-se as teorizações dos autores-base que explicitam a formação do trauma em dois tempos distintos. O modelo deriva do observado nas experiências de sedução. Para Freud, algumas vivências não são traumáticas no momento em que ocorrem, mas ganham esse atributo posteriormente, ao serem reativadas por uma nova experiência que as ressignifica. Para Ferenczi, algumas formas de traumatização envolvem o que chama de duplo choque: uma experiência causa comoção psíquica e, quando o indivíduo busca no ambiente a validação e o reconhecimento de suas sensações e percepções, estas são negadas. Utilizando casos clínicos colhidos da literatura sobre o tema, e cotejando a relação pessoal entre os autores, procura-se articular os modelos de traumatização observados. No capítulo final, conclusivo, acompanha-se a tentativa de Michael Balint de decompor a formação do trauma em três fases, unindo as ideias de Freud e Ferenczi, e propõe-se, com base no que foi discutido, a hipótese de que alguns tipos de formação traumática envolvem três tempos: o momento do choque, a reação do ambiente após o evento, e a ressignificação a posteriori das experiências anteriores / In this work I advance an understanding of the concept of psychic trauma based on Freuds and Ferenczis theoretical deliberations. I discuss both authors ideas concerning intra- and inter-psychic aspects of trauma formation so as to articulate them in such a way that I may distinguish different traumatic experiences. This work is divided into three parts. In the first five chapters I show that the focal point of a particular type of trauma is a set of experiences that appear to have an immediate or shortly delayed disruptive effect. In this set one finds traumatic neuroses in general and wartime neuroses in particular, as well as less intense events that nonetheless require a characteristic effort on the part of the psychic apparatus. Owing to the need to understand and treat soldiers whose symptoms derived from their traumatic experiences, both Freud and Ferenczi directed their attention to this type of trauma formation after the World War I. Because of psychic unpreparedness, the conscious systems lack of counter-cathexis, and the stimulations intensity, the psyche is forced into activating primitive defenses in its attempts to bind the overwhelming excitation. In the second part (four chapters), I discuss these foundational authors theoretical deliberations concerning the formation of trauma in two different stages. The model here derives from what has been observed in experiences of seduction. For Freud some experiences are not traumatic when they occur, but they become traumatic later, when they are reactivated by another experience that redefines them. For Ferenczi, some forms of traumatization entail what he calls a double shock: an experience causes psychic commotion, and, when subjects search their environment for validation and for acknowledgement of their sensations and perceptions, these are denied. Using pertinent clinical cases found in the literature and availing myself of Freuds and Ferenczis personal relationship, I attempt to delineate these two models of traumatization. In my concluding chapter I examine Michael Balints attempt to break down trauma formation into three phases by uniting Freuds and Ferenczis points of view. I propose, based on what has come before, the hypothesis that some types of trauma formation entail three phases: the moment of shock, the environments reaction after the event, and the a posteriori reinterpretation of the prior experiences