The promoter sequence of the mouse high affinity neurotensin receptor, Ntr-1, gene was cloned and characterized, sequences required for positive regulation in N1E-115 cells were localized, and at least two different peptides from these cells were shown to make specific contacts within the most potent positive regulatory element. A mouse neuroblastoma cell line, N1E-115, treated with 1.5% DMSO for 72 hours induces gene expression of both endogenous Ntr-l, and reporter constructs driven by the NTR-1 promoter, by 3 - 4 fold. The sequence ofthe NTR-1 promoter has no canonical TATA box, but is GC rich and contains consensus SP1, CACCC, CRE, and initiator elements. These elements are located within a 193 base positive regulatory region required for DMSO responsive activity and contains the transcriptional start site. Detailed mutational analysis of this region revealed that a CACCC box and the central region of a large GC rich palindrome are crucial cis-regulatory elements for DMSO induction. The SP1 element, an NGFI-A-related element, and the 5' end of the positive regulatory region are required for maintaining basal expression in N1E-115 cells. Cell type differences in the cis-regulatory elements that mediate both DMSO induction and maintenance of basal expression are observed. Characterization of proteins in N1E-115 cells that make specific contacts within the CACCC element identified at least two peptides with predicted sizes of 57 kd and 97 kd. Two dimensional UV crosslinking indicates that these proteins might contribute to inducible gel shift complexes that require the CACCC element. Several previously characterized CACCC binding proteins, belonging to the Kruppel-like family of transcription factors, were tested by supershift analysis for their ability to contribute to NTR-1 CACCC complexes. In fact, a protein closely related to SP1 does bind the CACCC element in N1E-115 cells, but of the other Kruppel-like protein tested, only BKLF contributes to a minor complex in N1E-115 cells. These results provide evidence for the complex regulation of Ntr-1 gene expression mediated by the cooperation of several cis-regulatory elements including a CACCC Kruppel-like binding element.

O peptídeo liberador de gastrina (GRP) age como potente mitógeno em tecidos neoplásicos pela ativação do seu receptor (GRPR), que é super expresso em estágios avançados de neuroblastoma humano. GRPR knockdown ou antagonistas de GRPR mostraram inibir o crescimento de neuroblastoma. Investigou-se, em condições experimentais, que os antagonistas de GRPR podem promover, em vez de inibir o crescimento de células de neuroblastoma. O antagonista do GRPR, RC-3095 em 0,1 nM inibiu, enquanto a concentração de 100 nM estimulou a proliferação de células de neuroblastoma murino Neuro2a, in vitro. Os efeitos proliferativos de RC -3095 no crescimento celular foram impedidos pelo inibidor de deacetilase de histona (HDI), butirato sódico (NAB). Estes resultados fornecem a primeira evidência de que um antagonista do GRPR pode estimular o crescimento de células cancerosas, e sugerem que o GRPR pode interagir com os mecanismos epigenéticos na regulação do crescimento de células do neuroblastoma.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O objetivo do presente trabalho é comparar, do ponto de vista elétrico, a membrana do neurônio ganglionar com a da célula de neuroblastoma, analisando os efeitos das cargas fixas sobre o potencial elétrico nas superfícies da bicamada lipídica e também sobre o comportamento do perfil de potencial através da membrana, considerando as condiçõesfísico-químicas do estado de repouso e do estado de potencial de ação. As condições para a ocorrência dos referidos estados foram baseadas em valores numéricos de parâmetros elétricos e químicos, característicos dessas células, obtidos na literatura. O neurônio ganglionar exemplifica um neurônio sadio, e a célula de neuroblastoma, que é uma célula tumoral, exemplifica um neurônio patológico, alterado por esta condição. O neuroblastoma é um tumor que se origina das células da crista neural (neuroblastos), que é uma estrutura embrionária que dá origem a muitas partes do sistema nervoso, podendo surgir em diversos locais do organismo, desde a região do crânio até a área mais inferior da coluna. O modelo adotado para simular a membrana de neurônio inclui: (a) as distribuições espaciais de cargas elétricas fixas no glicocálix e na rede de proteínas citoplasmáticas; (b) as distribuições de cargas na solução eletrolítica dos meios externo e interno; e (c) as cargas superficiais da bicamada lipídica. Os resultados que obtivemos mostraram que, nos estados de repouso e de ação, os potenciais superficiais da bicamada interno (ÁSbc) e externo (ÁSgb) da célula de neuroblastoma não sofrem alteração mensurável, quando a densidade de carga na superfície interna (QSbc) torna-se 50 vezes mais negativa, tanto para uma densidade de carga na superfície externa da bicamada nula (QSgb = 0), como para um valor de QSgb 6= 0. Porém, no estado de repouso, uma leve queda em ÁSbc do neur^onio ganglionar pode ser observada com este nível de variação de carga, sendo que ÁSgb do neurônio ganglionar é mais negativo quando QSgb = 1=1100 e/A2. No estado de ação, para QSgb = 0, o aumento da negatividade de QSbc não provoca alteração detectável de ÁSbc e ÁSgb para os dois neurônios. Quando consideramos QSgb = 1=1100 e/A2, ÁSgb do neurônio ganglionar se torna mais negativo, não se observando variações detectáveis nos potenciais superficiais da célula de neuroblastoma. Tanto no repouso quanto no estado de ação, ÁSgb das duas células não sofre variação sensível com o aumento da negatividade da carga fixa distribuída espacialmente no citoplasma. Já a ÁSbc sofre uma queda gradativa nos dois tipos celulares; porém, no estado de ação, esta queda é mais rápida. Descobrimos diferenças importantes nos perfis de potencial das duas células, especialmente na região do glicocálix. / The aim of our work is to compare, from the electrical point of view, the ganglion neuron membrane with the neuroblastoma cell's membrane, analyzing the effects of fixed charges on the electric potential of the surfaces of the lipidic bilayer and on the behavior of the potential profile across the membrane, considering the physicochemical conditions of the resting state and of the action potential state. The conditions for the occurrence of these states were defined, based on numerical values of electrical and chemical parameters of these cells, obtained in the literature. The ganglion neuron portrays a healthy neuron,and the neuroblastoma cell, which is a tumor cell, represents a pathologic neuron, different from the ganglion cell, due to this condition. A neuroblastoma is a tumor, originated from neural crest cells (neuroblasts), which is an embryonic structure that gives rise to many parts of the nervous system and can arise in various body sites, from the region of the skull all the way to the lower spinal column area.The model used to simulate the neuron membrane includes: (a) the spatial distribution of the fixed electric charges on the glycocalyx and on the network of cytoplasmic proteins; (b) the distribution of the charges in the electrolytic solution of outer and inner resources; and (c) the surface charges of the lipidic bilayer. The results we obtained show that, in the resting and action states, the inner (ÁSbc) and outer (ÁSgb) surface potential of neuroblastoma cells do not change measurably, when the charge density on the inner surface (QSbc) becomes 50 times more negative, for both null charge density on the outer surface (QSgb = 0) and for QSgb 6= 0. However, a slight drop in ÁSbc of a ganglion neuron can be observed with this level of charge variation, but ÁSgb of ganglion neuron is more negative when QSgb = 1=1100 e/A2. At action potential state, for QSgb = 0, the negative increase of QSbc does not measurably change ÁSbc and ÁSgb , for both neurons. When we consider QSgb = 1=1100 e/A2, for the ganglion neuron ÁSgb becomes more negative, with no significant detectable changes in the neuroblastoma cell's surface potentials. At the resting and action states, ÁSgb of both cells does not undergo substantial changes with the negative increasing of fixed charges uniformly distributed in the cytoplasm. However,ÁSbc undergoes a gradual decrease in both cell types, although for the action state, this fall is faster. We discovered important dierences among the potential profile of the two cells, especially in the glicocalyx region.

O transtorno bipolar (TB) é caracterizado como um grave transtorno psiquiátrico, que apresenta curso crônico com notável prejuízo cognitivo e funcional nos pacientes. Nesta tese, através de duas revisões avaliamos as alterações de marcadores periféricos de estresse oxidativo, neurotrofinas e inflamação presentes em pacientes com TB e discutimos o envolvimento destes na toxicidade sistêmica, bem como uma possível associação destas alterações com as disfunções cognitivas e funcionais apresentadas pelos pacientes ao longo do transtorno. Neste sentido, muitos estudos têm sido realizados buscando compreender a fisiopatologia do TB, no entanto, para o estabelecimento de um modelo in vitro é necessário ter um modelo celular adequado e um desafio que possa mimetizar a fisiopatologia da doença. Neste contexto, não há na literatura, até o momento, um modelo adequado que compreenda toda a complexidade dos sintomas do TB, por isso o objetivo geral desta tese consistiu na caracterização gênica do modelo in vitro de diferenciação celular da linhagem de neuroblastoma humano SH-SY5Y, induzido por ácido retinóico, e a busca por um modelo experimental para a avaliação da toxicidade sistêmica apresentada pelos pacientes com TB. O modelo de diferenciação foi avaliado através da técnica de microarranjo, onde verificamos que nas células diferenciadas há maior expressão de processos biológicos envolvidos no desenvolvimento neuronal, enquanto nas células indiferenciadas observamos maior expressão de processos biológicos relacionados a proliferação e manutenção celular. Ainda, genes relacionados a função sináptica e a síntese dopaminérgica estavam mais expressos nas células diferenciadas. Estes achados contribuem para a validação de um modelo de origem humana, de fácil manuseio e que apresenta perfil neuronal; auxiliando assim no estudo de doenças que acometem o sistema nervoso central, dentre elas o TB. Para avaliar o perfil de toxicidade no soro de pacientes bipolares, tratamos as células SH-SY5Y diferenciadas com soro de pacientes com TB, tanto em estágio inicial quanto avançado do transtorno. Como resultado, verificamos que o soro dos pacientes em estágio avançado apresenta maior toxicidade quando comparado ao soro de indivíduos controles por causar uma diminuição da viabilidade celular e uma diminuição na densidade de neuritos. Analisados em conjunto, os achados desta tese apontam para um novo modelo in vitro para analisar a fisiopatologia do TB, bem como o efeito de medicações e vias metabólicas envolvidas. Além disso, corroboram com dados prévios da literatura de que perifericamente os pacientes apresentam um índice de toxicidade relevante quando comparado a indivíduos controles e que este índice estaria relacionado a progressão do transtorno. / Bipolar disorder (BD) is characterized as a serious psychiatric disorder that presents chronic course with remarkable cognitive and functional impairment. In this thesis, we analyzed in two reviews the changes in peripheral markers of oxidative stress, neurotrophins and inflammation, discussing their involvement in systemic toxicity, as well as a possible association of these changes with the cognitive and functional impairment presented by BD patients throughout the disorder. In this sense, many studies have been performed seeking to understand the pathophysiology of this illness. Moreover, research on BD and drug development is hampered by the lack of suitable in vitro models. To counteract this, many attempts to explore patient-derived samples have been undertaken, resulting in partial reproduction of disease aspects. However, still does not exist in the literature a suitable model to understand the complexity of the BD symptoms. Therefore, the aims of this thesis was to evaluate the differential gene expression of the cell differentiation model of human neuroblastoma cell line SH-SY5Y, induced by retinoic acid, and the search for an experimental model for the evaluation of systemic toxicity in patients with BD. The differentiation model was assessed by microarray analysis and we found that the differentiated cells had increased expression of biological processes involved in neuronal development, while undifferentiated cells showed higher expression of biological processes related to cellular proliferation and maintenance. Also, genes related to the synaptic function and dopaminergic synthesis were more expressed in differentiated cells. These findings contribute to the validation of a cellular model from human source, easy handling and featuring neuronal profile; thereby aiding in the study of diseases that affect the central nervous system, including BD. In order to evaluate the toxicity profile in serum of bipolar patients, differentiated SH-SY5Y cells were treated with sera of BD patients in early and late stages of the disorder. As a result, for the first time in the literature, it has been verified the potential neurotoxicity of bipolar patients serum directly in human cells with a neuronal profile and we found that the serum of patients at late stage would present higher toxicity when compared to the control sera, causing a decrease in cell viability and a reduced neurite outgrowth density. Taken together, the findings of this thesis point to a new in vitro model to analyze the pathophysiology of BD, as well as the effect of medications and metabolic pathways involved in this disorder. Moreover, corroborate previous peripherally data found in the literature where patients would have a toxicity index compared to control subjects and that this index would be related to the progression of the disorder.

Espécies reativas de oxigênio (ERO) são produzidas através da respiração aeróbica e durante processos inflamatórios. Além disso, agressões externas como radiações, poluição, estresse, alcoolismo e tabagismo aumentam a sua produção. Altos níveis de ERO podem ocasionar dano oxidativo à lipídios, proteínas e DNA, comprometendo a função normal da célula, podendo estar envolvidos na patogênese e agravamento de diversas doenças. Há evidências que sugerem que antioxidantes naturais presentes em alimentos conferem benefícios adicionais à saúde, atuando como anticarcinogênicos, antiinflamatórios ou agentes antimutagênicos. O orégano (Oreganum sp) é uma especiaria mediterrânea usada como condimento na alimentação e pela medicina popular para diversos tipos de moléstias. O óleo possui forte ação antimicrobiana, devido ao elevado conteúdo de monoterpenos, sendo os principais o carvacrol, o timol e o para-cimeno. O carvacrol (5-isopropil-2metilfenol) é um fenol monoterpênico, com sabor picante e odor característico e tem sido amplamente usado na indústria de alimentos como aditivo seguro para aumentar a vida útil dos alimentos, como aromatizante em produtos assados, doces, bebidas e gomas de mascar, e/ou agente antimicrobiano com atividades contra bactérias, fungos e leveduras. Estudos têm relatado efeito antidepressivo e ansiolítico do carvacrol em camundongos, assim como proteção contra a radiação UVB diminuindo a peroxidação lipídica, estresse oxidativo e danos no DNA em células linfocitárias humanas e atividade antioxidante em diferentes sistemas de lipídios. Nós avaliamos a viabilidade celular e parâmetros de citotoxicidade do carvacrol em células de neuroblastoma humano SH-SY5Y. Este parece modificar levemente a morfologia das células, sem modificar significativamente a biomassa celular, parecendo ser tóxico na concentração de 100 μg/mL. Nas demais concentrações (1 a 50 μg/mL) não houve citotoxicidade. No ensaio de DCFH-DA o carvacrol reduziu a produção de ERO intracelular e diminui significativamente a produção de radicais peroxil no ensaio TRAP. Esses dados reforçam a ideia do carvacrol ser um potencial antioxidante, sendo necessários mais estudos para avaliar o mecanismo de ação deste composto. / Reactive oxygen species (ROS) are produced through aerobic respiration and during inflammation. Besides, external aggressions such as radiation, pollution, stress, alcoholism and smoking increase their production. Elevated levels of ROS can cause oxidative damage to lipids, proteins and DNA, compromising the normal cell function, and may be involved in the pathogenesis and progression of several diseases. There is suggesting that natural antioxidants found in foods provide additional health benefits, acting as anticarcinogenic, anti-inflammatory agents or antimutagenic. Oregano (Oreganum sp) is a Mediterranean spice used in food as a condiment and in popular medicine to treat several types of diseases. The essential oil has strong antimicrobial activity, due to the high content of monoterpenes, the main ones being carvacrol, thymol and para-cymene. Carvacrol (5-isopropyl-2metilfenol) is a phenol monoterpene with spicy taste and odor and has been widely used in food industry as additive to preserve foods, as flavoring agent in baked goods, candy, drinks and chewing gums, and/or antimicrobial agent with activity against bacteria, fungi and yeasts. Studies have reported antidepressant and anxiolytic effects of carvacrol in mice, as well as protection against UVB radiation, decreased lipid peroxidation, oxidative stress and DNA damage in human lymphocyte cells, and antioxidant activity in different lipid systems. We evaluated cell viability and cytotoxicity parameters of carvacrol in human neuroblastoma cells SH-SY5Y. Carvacrol induced morphology changes in cells without significant modification of the cellular biomass content and it was toxic at the concentration of 100 μg/mL. In other concentrations (1-50 μg/mL) it showed no cytotoxicity. In DCFH-DA assay carvacrol reduced the intracellular ROS production and significantly decreased the production of peroxyl radicals in the TRAP assay. These data reinforce the idea of carvacrol as a potential antioxidant and more research is needed to evaluate the mechanism of action of this compound.

abstract: ABSTRACT Post Translational Modifications (PTMs) are a series of chemical modifications with the capacity to expand the structural and functional repertoire of proteins. PTMs can regulate protein-protein interaction, localization, protein turn-over, the active state of the protein, and much more. This can dramatically affect cell processes as relevant as gene expression, cell-cell recognition, and cell signaling. Along these lines, this Ph.D. thesis examines the role of two of the most important PTMs: glycosylation and phosphorylation. In chapters 2, 3 and 4, a 10,000 peptide microarray is used to analyze the glycan variations in a series lipopolysaccharides (LPS) from Gram negative bacteria. This research was the first to demonstrate that using a small subset of random sequence peptides, it was possible to identify a small subset with the capacity to bind to the LPS of bacteria. These peptides bound to LPS not only in the solid surface of the array but also in solution as demonstrated with surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and flow cytometry. Interestingly, some of the LPS binding peptides also exhibit antimicrobial activity, a property that is also analyzed in this work. In chapters 5 and 6, the role of protein phosphorylation, another PTM, is analyzed in the context of human cancer. High risk neuroblastoma, a very aggressive pediatric cancer, was studied with emphasis on the phosphorylations of two selected oncoproteins: the transcription factor NMYC and the adaptor protein ShcC. Both proteins were isolated from high risk neuroblastoma cells, and a targeted-directed tandem mass spectrometry (LC-MS/MS) methodology was used to identify the phosphorylation sites in each protein. Using this method dramatically improved the phosphorylation site detection and increased the number of sites detected up to 250% in comparison with previous studies. Several of the novel identified sites were located in functional domain of the proteins and that some of them are homologous to known active sites in other proteins of the same family. The chapter concludes with a computational prediction of the kinases that potentially phosphorylate those sites and a series of assays to show this phosphorylation occurred in vitro. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2014

La Multidrug resistance Protein 1 (MRP1) est impliquée dans le phénotype de résistance multiple aux médicaments (MDR) des cellules cancéreuses. Les substrats physiologiques de MRP1 comprennent notamment le glutathion (GSH). Certains médicaments anti-cancéreux tels que la doxorubicine sont co-transportés avec le glutathion. Pour contourner le phénotype MDR induit par MRP1, nous proposons une nouvelle stratégie thérapeutique basée sur la sensibilité collatérale (SC) des cellules résistantes surexprimant MRP1. Certains composés, comme le vérapamil, provoquent la mort sélective des cellules résistantes (les cellules témoins ne sont pas affectées) en stimulant l'efflux du glutathion médié par MRP1. La déplétion intracellulaire très rapide et très forte du glutathion induit probablement un stress oxydatif déclenchant la mort cellulaire. Nous avons identifié de nouveaux agents de sensibilité collatérale puissants in vitro et nous avons montré l'effet du meilleur composé sur la réduction de la croissance des tumeurs chimiorésistantes chez la souris. Nous avons étudié le mécanisme moléculaire d'action des agents de SC et identifié un résidu, localisé dans une région inattendue, impliqué dans la stimulation de l'efflux de glutathion induit par ces molécules / Multidrug resistance Protein 1 (MRP1) is involved in the multidrug resistance (MDR) phenotype of cancer cells. Physiological substrates of MRP1 include glutathione (GSH) and drugs such as doxorubicin are co-transported with glutathione. To circumvent the MDR phenotype induced by MRP1, we propose a new therapeutic strategy based on collateral sensitivity (CS) of resistant cell expressing MRP1, its overexpression becoming the Achilles heel of the cell. Some compounds, like verapamil, act as MRP1 modulators. They trigger selective death of resistant cells (control cells are not affected) by stimulating MRP1-mediated glutathione efflux. The fast and huge intracellular depletion of glutathione probably induces an oxidative stress triggering cell death. We identified new potent collateral sensitivity agents in vitro and we checked the effect of the strongest compound on reducing resistant tumor growth in nude mice. We studied the molecular mechanism of action of CS agents and identified an unexpected residue involved in the stimulation of glutathione efflux induced by these molecules

O peptídeo liberador de gastrina (GRP) age como potente mitógeno em tecidos neoplásicos pela ativação do seu receptor (GRPR), que é super expresso em estágios avançados de neuroblastoma humano. GRPR knockdown ou antagonistas de GRPR mostraram inibir o crescimento de neuroblastoma. Investigou-se, em condições experimentais, que os antagonistas de GRPR podem promover, em vez de inibir o crescimento de células de neuroblastoma. O antagonista do GRPR, RC-3095 em 0,1 nM inibiu, enquanto a concentração de 100 nM estimulou a proliferação de células de neuroblastoma murino Neuro2a, in vitro. Os efeitos proliferativos de RC -3095 no crescimento celular foram impedidos pelo inibidor de deacetilase de histona (HDI), butirato sódico (NAB). Estes resultados fornecem a primeira evidência de que um antagonista do GRPR pode estimular o crescimento de células cancerosas, e sugerem que o GRPR pode interagir com os mecanismos epigenéticos na regulação do crescimento de células do neuroblastoma.

Espécies reativas de oxigênio (ERO) são produzidas através da respiração aeróbica e durante processos inflamatórios. Além disso, agressões externas como radiações, poluição, estresse, alcoolismo e tabagismo aumentam a sua produção. Altos níveis de ERO podem ocasionar dano oxidativo à lipídios, proteínas e DNA, comprometendo a função normal da célula, podendo estar envolvidos na patogênese e agravamento de diversas doenças. Há evidências que sugerem que antioxidantes naturais presentes em alimentos conferem benefícios adicionais à saúde, atuando como anticarcinogênicos, antiinflamatórios ou agentes antimutagênicos. O orégano (Oreganum sp) é uma especiaria mediterrânea usada como condimento na alimentação e pela medicina popular para diversos tipos de moléstias. O óleo possui forte ação antimicrobiana, devido ao elevado conteúdo de monoterpenos, sendo os principais o carvacrol, o timol e o para-cimeno. O carvacrol (5-isopropil-2metilfenol) é um fenol monoterpênico, com sabor picante e odor característico e tem sido amplamente usado na indústria de alimentos como aditivo seguro para aumentar a vida útil dos alimentos, como aromatizante em produtos assados, doces, bebidas e gomas de mascar, e/ou agente antimicrobiano com atividades contra bactérias, fungos e leveduras. Estudos têm relatado efeito antidepressivo e ansiolítico do carvacrol em camundongos, assim como proteção contra a radiação UVB diminuindo a peroxidação lipídica, estresse oxidativo e danos no DNA em células linfocitárias humanas e atividade antioxidante em diferentes sistemas de lipídios. Nós avaliamos a viabilidade celular e parâmetros de citotoxicidade do carvacrol em células de neuroblastoma humano SH-SY5Y. Este parece modificar levemente a morfologia das células, sem modificar significativamente a biomassa celular, parecendo ser tóxico na concentração de 100 μg/mL. Nas demais concentrações (1 a 50 μg/mL) não houve citotoxicidade. No ensaio de DCFH-DA o carvacrol reduziu a produção de ERO intracelular e diminui significativamente a produção de radicais peroxil no ensaio TRAP. Esses dados reforçam a ideia do carvacrol ser um potencial antioxidante, sendo necessários mais estudos para avaliar o mecanismo de ação deste composto. / Reactive oxygen species (ROS) are produced through aerobic respiration and during inflammation. Besides, external aggressions such as radiation, pollution, stress, alcoholism and smoking increase their production. Elevated levels of ROS can cause oxidative damage to lipids, proteins and DNA, compromising the normal cell function, and may be involved in the pathogenesis and progression of several diseases. There is suggesting that natural antioxidants found in foods provide additional health benefits, acting as anticarcinogenic, anti-inflammatory agents or antimutagenic. Oregano (Oreganum sp) is a Mediterranean spice used in food as a condiment and in popular medicine to treat several types of diseases. The essential oil has strong antimicrobial activity, due to the high content of monoterpenes, the main ones being carvacrol, thymol and para-cymene. Carvacrol (5-isopropyl-2metilfenol) is a phenol monoterpene with spicy taste and odor and has been widely used in food industry as additive to preserve foods, as flavoring agent in baked goods, candy, drinks and chewing gums, and/or antimicrobial agent with activity against bacteria, fungi and yeasts. Studies have reported antidepressant and anxiolytic effects of carvacrol in mice, as well as protection against UVB radiation, decreased lipid peroxidation, oxidative stress and DNA damage in human lymphocyte cells, and antioxidant activity in different lipid systems. We evaluated cell viability and cytotoxicity parameters of carvacrol in human neuroblastoma cells SH-SY5Y. Carvacrol induced morphology changes in cells without significant modification of the cellular biomass content and it was toxic at the concentration of 100 μg/mL. In other concentrations (1-50 μg/mL) it showed no cytotoxicity. In DCFH-DA assay carvacrol reduced the intracellular ROS production and significantly decreased the production of peroxyl radicals in the TRAP assay. These data reinforce the idea of carvacrol as a potential antioxidant and more research is needed to evaluate the mechanism of action of this compound.

Les récepteurs à dépendance ont la particularité de présenter deux types de signalisation cellulaire : en présence de leur ligand, ils activent des voies positives de survie, de différenciation et de migration, mais en l'absence de leur ligand, ils activent au contraire l'apoptose de la cellule. Cette dualité fonctionnelle des récepteurs à dépendance est impliquée à la fois lors du développement embryonnaire, mais également dans le maintien de l'homéostasie chez l'adulte. En effet, elle permet le contrôle de la prolifération cellulaire, mais aussi l'élimination de cellules migrant en dehors de leur territoire défini, dans un environnement où la quantité de ligand est limitée. De ce fait, les récepteurs à dépendance ont été caractérisés comme des suppresseurs de tumeur conditionnels dans certaines pathologies cancéreuses. TrkC, le récepteur de la neurotrophine-3 (NT-3) appartient à cette famille fonctionnelle. En l'absence de NT-3, TrkC, sous sa forme monomérique, est clivé par les caspases en deux sites : ce clivage induit la libération d'un fragment hautement apoptotique, appelé TrkC-KF pour « Killer Fragment ». Dans ce manuscrit, nous décrivons les mécanismes moléculaires par lesquels TrkC-KF induit l'apoptose. Nous avons en effet pu déterminer que TrkC-KF s'associe dans le cytoplasme avec la protéine Cobral, qui permet son transport à la mitochondrie : TrkC-KF permet ainsi l'activation de Bax, relarguant le cytochrome C nécessaire à la formation de l'apoptosome. TrkC-KF est également transporté dans le noyau des cellules par les importines : cette localisation nucléaire permet son interaction avec le facteur de transcription Heyl / Dependence receptors are able to activate two cellular pathways: in the presence of their ligand, they activate positive signaling of survival, differentiation and migration, but in the absence of their ligand, they activate the apoptosis of the cell. This dual function is implied both during the embryonic development, and in the homeostasis maintenance in adults. lndeed, it enables the control of cellular proliferation, and the elimination of cells migrating outside their defined territory, in an environment where the ligand availability is limited. Therefore, dependence receptors have also been caracterised as conditionnal tumor suppressors in various types of cancer. TrkC, the neurotrophin-3 (NT-3) receptor, belongs to this functional family. ln the absence of NT-3, TrkC under its monomeric for mis cleaved by caspases : this cleavage induces the release of a highly apoptotic fragment, called TrkC-KF (Killer Fragment). ln this manuscript, we describe the molecular mecanism by which TrkC-KF induces apoptosis. lndeed, we determined that TrkC-KF associates in the cytoplasm with the Cobral protein, allowing its transport to the mitochondria : TrkC-KF enables Bax activation, which releases the cytochrome C forming the apoptosome. TrkC-KF is also transported by importins into the nucleus, where it directly interacts with the transcription factor Heyl. This complex inhibits MDM2 transcription, allowing p53 stabilisation : p53 activates the transcription of proapoptotic genes, among them Cobral and Bax. TrkC-KF has therefore a dual signalisation, one mitochondrial pathway enabling the apoptosome formation, one nuclear pathway upregulating the transcription of TrkC-KF partners. We then aimed at defining the biological relevance of this apoptotic signaling of TrkC, both during the development and tumorigenesis. lndeed, TrkC expression, in the absence of NT-3, induces in vivo the apoptosis of developing neurons in the nervous system. Moreover, NT-3-KO mice show a loss of 70% of neurons in the forming dorsal root ganglia (DRG), while TrkC-KO mice show only a 30% loss.