INTRODUÇÃO: A crescente incidência de câncer de mama, que cada vez mais acomete mulheres jovens, tem despertado muito interesse no diagnóstico precoce e na busca do tratamento mais eficaz e minimamente agressivo. Da mesma forma, vem aumentando a parcela da população com alto risco para câncer de mama, para a qual as atenções têm se voltado no sentido de se encontrar um caminho que permita prevenir o surgimento da neoplasia. Inspirados por Peyrat, que primeiro associou o câncer de mama com a presença de fator de crescimento insulina like tipo 1 (IGF-1), vários autores se lançaram nesta procura e, apesar de controversa, a literatura internacional tende a mostrar uma relação direta entre IGF-1 e câncer de mama na pré-menopausa. A proteína carreadora de IGF do tipo 3, ou IGFBP-3, também foi adicionada ao rol de sustâncias com possibilidade de promover câncer, porém, não tem demonstrado clara regularidade em seu mecanismo de ação. Tendo como alvo a população com alto risco para câncer de mama, quer histopatológico, quer familiar, e direcionada pelos cálculos matemáticos de Gail e Tyrer-Cuzick, essa tese objetivou avaliar a relação entre IGF-1 e IGFBP-3 em mulheres brasileiras atendidas pelo Setor de Mastologia da Disciplina de Ginecologia do Departamento de Obstetrícia e Ginecologia da Faculdade de Medicina da Universidade de São Paulo. MÉTODOS: estudo transversal em que foram comparados os níveis séricos de IGF-1 e IGFBP-3, dosados pelo método imunométrico quimioluminescente, em 3 diferentes grupos: mulheres com câncer de mama não tratado (n= 51), mulheres com risco populacional (n= 66) e mulheres com alto risco para câncer de mama (n= 108). Foram considerados fatores de comparação: idade, estado menopausal e índice de massa corpórea. RESULTADOS: 1) não houve diferenças entre os grupos com respeito ao IMC; 2) foi estatisticamente significativa a diferença das médias de idades dos grupos, sendo as mulheres com alto risco para câncer de mama mais jovens (p<0,001); 3) quanto ao estado menopausal, houve significativa diferença entre os grupos, com predomínio de mulheres pós-menopausa no grupo de pacientes com câncer de mama (58,8%), com p <0,001; 4) não foi encontrada relação estatisticamente significante entre percentual de IGF-1 em relação à mediana, percentual de IGFBP-3 em relação à mediana e razão dos percentuais de IGF-1 e IGFPB-3 em relação à mediana para os 3 grupos estudados;4) não houve variações nas dosagens de acordo com o tipo de situação determinante do alto risco para câncer de mama (familiar ou histopatológico); 5) não houve alteração estatisticamente significativa das variáveis estudadas, mesmo após subdivisão do grupo de alto risco (risco vitalício entre 20 e 29% X risco vitalício superior a 29%). CONCLUSÃO: Não foi observada variação dos níveis séricos de IGF-I e IGFBP-3 em mulheres com câncer de mama, com alto risco para câncer de mama ou com risco populacional / INTRODUCTION: The rising incidence of breast cancer, a disease that is increasingly affecting young women, has aroused a great deal of interest in early diagnosis and the search for a more efficient and minimally aggressive treatment. Likewise, the population at high risk for breast cancer has been growing, and it is now the focus of research efforts in the struggle to prevent neoplasia. Inspired by Peyrat, who was the first to associate breast cancer with insulin-like growth factor type 1 (IGF-1), several authors have taken on the challenge, and the international literature now leans towards a direct relationship, albeit still controversial, between IGF-1 and breast cancer in premenopause. The IGF binding protein 3 (IGFBP-3) has also been added to the list of cancer-causing agents, but its mechanism of action has not shown to be clearly regular. Targeting a population at high risk for breast cancer for histopathologic or familial reasons, and guided by the mathematical calculations of Gail and Tyrer-Cuzick, this thesis aimed at evaluating the relationship between IGF-1 and IGFB-3 in Brazilian female outpatients at the Mastology Sector of the Gynecology Discipline of the Department of Obstetrics and Gynecology of the School of Medicine of the University of São Paulo. METHODS: Transverse study to compare the serum levels of IGF-1 and IGFBP- 3 measured by the chemiluminescent immunometric method. The patients were divided into 3 different groups: women with untreated breast cancer (n=51), women with a population-based risk of breast cancer (n=66), and women at high risk for breast cancer (n=108). The comparison factors were age, menopausal state, and body mass index. RESULTS: 1) there were no differences among the groups with respect to BMI; 2) but there were statistically significant differences among the groups regarding mean age, and the younger women were those at a higher risk for breast cancer (p<0.001); 3) as for menopausal state, the groups were significantly different, and the postmenopausal women were prevalent among the patients with breast cancer (58.8%; p<0.001); 4) no statistically significant relationship was found between the IGF-1 percentage and the median, the IGFBP-3 percentage and the median, or the IGF-1 and IGFBP-3 ratio and the median in any of the groups; 5) measurements did not vary according to the determinant reason for high breast cancer risk (familial or histopathologic); 6) statistically significant changes in the variables under consideration did not take place, even after subdivision of the high-risk group (lifelong risk between 20% and 29% X lifelong risk over 29%). CONCLUSION: The serum levels of IGF-I and IGFBP-3 showed no variation among women with breast cancer, at high risk for cancer, or with populationbased risk

INTRODUÇÃO: A crescente incidência de câncer de mama, que cada vez mais acomete mulheres jovens, tem despertado muito interesse no diagnóstico precoce e na busca do tratamento mais eficaz e minimamente agressivo. Da mesma forma, vem aumentando a parcela da população com alto risco para câncer de mama, para a qual as atenções têm se voltado no sentido de se encontrar um caminho que permita prevenir o surgimento da neoplasia. Inspirados por Peyrat, que primeiro associou o câncer de mama com a presença de fator de crescimento insulina like tipo 1 (IGF-1), vários autores se lançaram nesta procura e, apesar de controversa, a literatura internacional tende a mostrar uma relação direta entre IGF-1 e câncer de mama na pré-menopausa. A proteína carreadora de IGF do tipo 3, ou IGFBP-3, também foi adicionada ao rol de sustâncias com possibilidade de promover câncer, porém, não tem demonstrado clara regularidade em seu mecanismo de ação. Tendo como alvo a população com alto risco para câncer de mama, quer histopatológico, quer familiar, e direcionada pelos cálculos matemáticos de Gail e Tyrer-Cuzick, essa tese objetivou avaliar a relação entre IGF-1 e IGFBP-3 em mulheres brasileiras atendidas pelo Setor de Mastologia da Disciplina de Ginecologia do Departamento de Obstetrícia e Ginecologia da Faculdade de Medicina da Universidade de São Paulo. MÉTODOS: estudo transversal em que foram comparados os níveis séricos de IGF-1 e IGFBP-3, dosados pelo método imunométrico quimioluminescente, em 3 diferentes grupos: mulheres com câncer de mama não tratado (n= 51), mulheres com risco populacional (n= 66) e mulheres com alto risco para câncer de mama (n= 108). Foram considerados fatores de comparação: idade, estado menopausal e índice de massa corpórea. RESULTADOS: 1) não houve diferenças entre os grupos com respeito ao IMC; 2) foi estatisticamente significativa a diferença das médias de idades dos grupos, sendo as mulheres com alto risco para câncer de mama mais jovens (p<0,001); 3) quanto ao estado menopausal, houve significativa diferença entre os grupos, com predomínio de mulheres pós-menopausa no grupo de pacientes com câncer de mama (58,8%), com p <0,001; 4) não foi encontrada relação estatisticamente significante entre percentual de IGF-1 em relação à mediana, percentual de IGFBP-3 em relação à mediana e razão dos percentuais de IGF-1 e IGFPB-3 em relação à mediana para os 3 grupos estudados;4) não houve variações nas dosagens de acordo com o tipo de situação determinante do alto risco para câncer de mama (familiar ou histopatológico); 5) não houve alteração estatisticamente significativa das variáveis estudadas, mesmo após subdivisão do grupo de alto risco (risco vitalício entre 20 e 29% X risco vitalício superior a 29%). CONCLUSÃO: Não foi observada variação dos níveis séricos de IGF-I e IGFBP-3 em mulheres com câncer de mama, com alto risco para câncer de mama ou com risco populacional / INTRODUCTION: The rising incidence of breast cancer, a disease that is increasingly affecting young women, has aroused a great deal of interest in early diagnosis and the search for a more efficient and minimally aggressive treatment. Likewise, the population at high risk for breast cancer has been growing, and it is now the focus of research efforts in the struggle to prevent neoplasia. Inspired by Peyrat, who was the first to associate breast cancer with insulin-like growth factor type 1 (IGF-1), several authors have taken on the challenge, and the international literature now leans towards a direct relationship, albeit still controversial, between IGF-1 and breast cancer in premenopause. The IGF binding protein 3 (IGFBP-3) has also been added to the list of cancer-causing agents, but its mechanism of action has not shown to be clearly regular. Targeting a population at high risk for breast cancer for histopathologic or familial reasons, and guided by the mathematical calculations of Gail and Tyrer-Cuzick, this thesis aimed at evaluating the relationship between IGF-1 and IGFB-3 in Brazilian female outpatients at the Mastology Sector of the Gynecology Discipline of the Department of Obstetrics and Gynecology of the School of Medicine of the University of São Paulo. METHODS: Transverse study to compare the serum levels of IGF-1 and IGFBP- 3 measured by the chemiluminescent immunometric method. The patients were divided into 3 different groups: women with untreated breast cancer (n=51), women with a population-based risk of breast cancer (n=66), and women at high risk for breast cancer (n=108). The comparison factors were age, menopausal state, and body mass index. RESULTS: 1) there were no differences among the groups with respect to BMI; 2) but there were statistically significant differences among the groups regarding mean age, and the younger women were those at a higher risk for breast cancer (p<0.001); 3) as for menopausal state, the groups were significantly different, and the postmenopausal women were prevalent among the patients with breast cancer (58.8%; p<0.001); 4) no statistically significant relationship was found between the IGF-1 percentage and the median, the IGFBP-3 percentage and the median, or the IGF-1 and IGFBP-3 ratio and the median in any of the groups; 5) measurements did not vary according to the determinant reason for high breast cancer risk (familial or histopathologic); 6) statistically significant changes in the variables under consideration did not take place, even after subdivision of the high-risk group (lifelong risk between 20% and 29% X lifelong risk over 29%). CONCLUSION: The serum levels of IGF-I and IGFBP-3 showed no variation among women with breast cancer, at high risk for cancer, or with populationbased risk

<p>The interest for the growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis and its role in the central nervous system (CNS) has grown during the past decade. GH has been associated with psychological functions as sleep, mood, general well-being and learning and memory. The present thesis is a contribution to clarify the functions and mechanisms involved in the actions of GH and IGF-1 in the CNS. A variant of the GH receptor (GHR) gene transcript lacking exon 3 (GHR3-) was cloned from ovine choroid plexus epithelial cells and tissue. The GHR3- transcript has previously only been identified in human tissue. Further, an anatomical study of the localization of GHR mRNA in the rat brain stem and spinal cord was carried out by the use of in situ hybridization. High densities of GHRs were found in areas associated with the regulation of food intake, sleep and nociception, functions known to be influenced by the GH/IGF-1 axis. The interaction with the opioid system was studied by an acute treatment with morphine. The levels of the transcripts for GHR and GHBP in the rat hippocampus and spinal cord were decreased 4 h after the injection of the opiate and restored to normal levels after 24 h. Young and aged rats injected with GH or IGF-1 showed differential gene regulation of subunits of the NMDA subtype of glutamate receptor in the hippocampus. This indicates an age-related difference in the sensitivity to GH/IGF-1 mediated effects on memory functions. Moreover, hypophysectomized rats treated with GH showed improved performance in the Morris water maze, a spatial memory task. The effect was accompanied with an increase in transcripts for NMDA receptor subunits and its associated membrane anchoring PSD-95 protein. Taken together, the results suggest that GH and/or IGF-1 play important roles in mechanisms associated with cognitive functions.</p>

The objective of this thesis was to investigate the effects of the somatotrope GH/IGF-1 axis upon the thymus. This work included two parts: 1. Translational research study: Thymus function in adult GH deficiency (AGHD) with and without GH treatment Background: Despite age-related adipose involution, T cell generation in the thymus (thymopoiesis) is maintained beyond puberty in adults. In rodents, growth hormone (GH), insulin-like growth factor-1 (IGF-1), and GH secretagogues reverse agerelated changes in thymus cytoarchitecture and increase thymopoiesis. GH administration also enhances thymic mass and function in HIV-infected patients. Until now, thymic function has not been investigated in adult GH deficiency (AGHD). The objective of this clinical study was to evaluate thymic function in AGHD, as well as the repercussion upon thymopoiesis of GH treatment for restoration of GH/IGF-1 physiological levels. Methodology/Principal Findings: Twenty-two patients with documented AGHD were enrolled in this study. The following parameters were measured: plasma IGF-1 concentrations, signal-joint T-cell receptor excision circle (sjTREC) frequency, and sj/b TREC ratio. Analyses were performed at three time points: firstly on GH treatment at maintenance dose, secondly one month after GH withdrawal, and thirdly one month after GH resumption. After 1-month interruption of GH treatment, both plasma IGF-1 concentrations and sjTREC frequency were decreased (p,0.001). Decreases in IGF-1 and sjTREC levels were correlated (r = 0.61, p,0.01). There was also a decrease in intrathymic T cell proliferation as indicated by the reduced sj/b TREC ratio (p,0.01). One month after reintroduction of GH treatment, IGF-1 concentration and sjTREC frequency regained a level equivalent to the one before GH withdrawal. The sj/b TREC ratio also increased with GH resumption, but did not return to the level measured before GH withdrawal. Conclusions: In patients with AGHD under GH treatment, GH withdrawal decreases thymic T cell output, as well as intrathymic T cell proliferation. These parameters of thymus function are completely or partially restored one month after GH resumption. These data indicate that the functional integrity of the somatotrope GH/IGF-1 axis is important for the maintenance of a normal thymus function in human adults. 2. Fundamental study: intrathymic expression of members of the GH/IGF-1 axis and effects of GH on T-cell differentiation in murine fetalthymic organ cultures (FTOC). We here address the question of expression and role of GH/IGF axis in the thymus. Methods: Using RT-qPCR, the expression profile of various components of the somatotrope GH/IGF axis was measured in different thymic cell types and during thymus embryogenesis in Balb/c mice. Effect of GH on T-cell differentiation was explored through thymic organotypic culture. Results: Transcription of Gh, Igf1, Igf2 and their related receptors predominantly occurred in thymic epithelial cells (TEC), while a low level of Gh and Igf1r transcription was also evidenced in thymic T cells (thymocytes). Gh, Ghr, Ins2, Igf1, Igf2, and Igfr1, displayed distinct expression profiles depending on the developmental stage. The protein concentration of IGF-1 and IGF-2 were in accordance with the profile of their gene expression. In fetal thymus organ cultures (FTOC) derived from Balb/c mice, treatment with exogenous GH resulted in a significant increase of double negative CD4-CD8- T cells and CD4+ T cells, with a concomitant decrease in double positive CD4+CD8+ T cells. These changes were inhibited by concomitant treatment with GH and GHR antagonist pegvisomant. However, GH treatment also induced a significant decrease in FTOC Gh, Ghr and Igf1 expression. Conclusion: These data show that the thymotropic properties of the somatotrope GH/IGF-1 axis involve an interaction between exogenous GH and GHR expressed by TEC. Since thymic IGF-1 is not increased by GH treatment, the effects of GH upon T-cell differentiation could implicate a different local growth factor or cytokine.

The interest for the growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis and its role in the central nervous system (CNS) has grown during the past decade. GH has been associated with psychological functions as sleep, mood, general well-being and learning and memory. The present thesis is a contribution to clarify the functions and mechanisms involved in the actions of GH and IGF-1 in the CNS. A variant of the GH receptor (GHR) gene transcript lacking exon 3 (GHR3-) was cloned from ovine choroid plexus epithelial cells and tissue. The GHR3- transcript has previously only been identified in human tissue. Further, an anatomical study of the localization of GHR mRNA in the rat brain stem and spinal cord was carried out by the use of in situ hybridization. High densities of GHRs were found in areas associated with the regulation of food intake, sleep and nociception, functions known to be influenced by the GH/IGF-1 axis. The interaction with the opioid system was studied by an acute treatment with morphine. The levels of the transcripts for GHR and GHBP in the rat hippocampus and spinal cord were decreased 4 h after the injection of the opiate and restored to normal levels after 24 h. Young and aged rats injected with GH or IGF-1 showed differential gene regulation of subunits of the NMDA subtype of glutamate receptor in the hippocampus. This indicates an age-related difference in the sensitivity to GH/IGF-1 mediated effects on memory functions. Moreover, hypophysectomized rats treated with GH showed improved performance in the Morris water maze, a spatial memory task. The effect was accompanied with an increase in transcripts for NMDA receptor subunits and its associated membrane anchoring PSD-95 protein. Taken together, the results suggest that GH and/or IGF-1 play important roles in mechanisms associated with cognitive functions.

Philosophiae Doctor - PhD / The aim of this thesis is first, to evaluate various culture conditions to improve embryo development, and secondly, to analyze spent culture media for any biomarkers that may be predictive of embryo health. Single-step and sequential culture media were compared in both Planer and EmbryoScope™ incubators. Single-step media resulted in better blastocyst development compared to sequential media and the EmbryoScope™ incubation system showed slight improvements in embryo development than the Planer system. The benefits of supplementing the culture medium with either insulin or insulin-like growth factor 1 (IGF-1) or culturing in a 2% O2 environment, using two different strains of mice (hybrid and C57), as well as the suitability of these strains for quality control were compared. In insulin, hybrid embryos were slower to blastulate and had a lower blastocyst rate, whereas C57 embryos were slower to the morula and faster to blastocyst stages, and lower blastocyst rate than the controls. IGF-1 showed no difference in time-lapse morphokinetics (TLM) or blastocyst rates compared to controls in both hybrid and C57 embryos. Under 2% O2, hybrid embryos showed no significant difference in TLM up to the 8-cell stage, but slowed down afterwards, resulting in blastocysts with significantly lower cell counts than the 6% O2 group. The C57 embryos were slower to reach morula and expanded blastocyst, and had lower blastocyst rates in 2%O2 vs 6%O2. The C57 strain had significant slower overall embryo development for all time points than hybrid embryos in insulin, IGF-1 and ultra-low O2, as well as lower blastocyst rates. Measurement of growth differentiation factor 9 (GDF-9) and oxidation-reduction potential (ORP) in spent media as markers for embryo health were evaluated. Day 5 human blastocysts yielded higher pregnancy rates and GDF-9 levels in spent media compared to Day 6 blastocysts, but TLM parameters showed no impact on pregnancy outcome. In Day 6 blastocysts, the non-pregnant group showed significantly faster embryo development compared to the clinically pregnant group up to the 8-cell stage and start of blastulation. GDF-9 did not show any significant differences between non-pregnant and pregnant groups of Day 5 or Day 6 embryo transfers. ORP in spent media from good quality Day 3 embryos that developed into blastocysts were significantly higher than from those that did not, with no difference in control medium ORP. Spent media from arrested embryos showed lower ORP than their corresponding controls. Arrested embryos had slower development at syngamy, morula, blastulation and blastocyst stages. The single step medium in the EmbryoScope™ is the preferred choice for embryo culture. Insulin or IGF-1 media supplementation or 2% O2 culture did not provide any benefit to embryo development. The C57 mouse strain is more sensitive and may be better to detect changes in culture conditions, and therefore better model for quality control assays. GDF-9 values decrease from Day 5 to Day 6 which gives new insight to understanding the role of GDF-9 during embryogenesis. ORP in spent media indicate that embryos that developed into blastocysts did not contribute to ROS, but maintained ORP balance.

No description available.

Le récepteur V2 (V2R) de la vasopressine est un récepteur couplé aux protéines G (RCPG), jouant un rôle fondamental dans le maintien de l’homéostasie hydrosodique. À l’instar de nombreux RCPGs, il est capable d’interagir avec plusieurs types de protéines G hétérotrimériques et possède des voies de signalisation peu explorées aux mécanismes mal compris. Ces voies non canoniques font l’objet des travaux exposés dans ce mémoire. Il s’agit d’explorer les caractéristiques et mécanismes de la signalisation de V2R via G12, et de la voie d’activation d’ERK 1/2 par transactivation du récepteur de l’insulin-like growth factor 1, IGF1R. Par des études de transfert d’énergie de résonance de bioluminescence (BRET), nous exposons la capacité de V2R à interagir avec la sous-unité Gα12 ainsi que la modulation de la conformation de l’hétérotrimère G12 par l’agoniste de V2R, l’arginine-vasopressine. Ces travaux dévoilent également la modulation de l’interaction entre Gα12 et son effecteur classique RhoA, suggérant un engagement de RhoA, ainsi que la potentialisation via Gα12 de la production d’AMP cyclique. À l’aide de diverses méthodes d’inhibition sélective, nos résultats précisent les mécanismes de la transactivation. Ils supportent notamment le rôle initiateur de l’activation de Src par V2R et l’absence d’implication des ligands connus d’IGF1R dans la transactivation. La métalloprotéase MMP 3 apparaît par ailleurs comme un bon candidat pour réguler la transactivation. Ce projet met en lumière des modes de signalisation peu explorés de V2R, dont l’implication physiologique et physiopathologique pourrait s’avérer significative, au-delà d’un apport fondamental dans la compréhension de la signalisation des RCPGs. / Vasopressin V2 receptor is a G protein coupled receptor (GPCR) responsible for the homeostatic regulation of water and sodium recapture from the urine to the bloodstream. Akin to numerous GPCRs, this receptor can interact with more than one heterotrimeric G protein subtype, and is still associated with some poorly explored signaling pathways with indefinite mechanisms. These non-canonical pathways are the focus of this project. This work aims at unveiling the characteristics and mechanisms underlying G12 mediated signaling by V2R and ERK 1/2 activation through the transactivation of the tyrosine kinase Insulin-like growth factor 1 receptor (IGF1R). Using bioluminescence resonance energy transfer (BRET) experiments, we reveal V2R’s ability to interact with the Gα12 subunit, as well as the modulation of G12 heterotrimer’s conformation in response to V2R agonist arginine vasopressin (AVP). AVP-induced modulation of Gα12’s interaction with its classical effector RhoA upon stimulation with AVP suggests the engagement of RhoA, and our data also reveals that Gα12 potentiates AVP-induced cAMP production. Using diverse selective inhibition strategies, our results further define the mechanism of transactivation. Our data support a starter position of AVP-induced Src activation and discard IGF1R known agonists as the potential autocrine/paracrine factor responsible for IGF1R activation. Furthermore, our results suggest that the metalloproteinase MMP 3 is a good candidate for IGF1R transactivation. This project sheds light on lesser known signaling pathways involving V2R, which could reveal important on a physiological and pathophysiological scale, besides bringing a better understanding of the principles of GPCR signaling.

Sabe-se que algumas alterações nutricionais maternas durante o período perinatal estão associadas com doenças metabólicas na vida adulta das proles, tais como diabetes melito tipo 2, resistência à insulina, obesidade e hipertensão arterial. O período da gestação em que estas alterações nutricionais influenciam a prole na idade adulta ainda não está elucidado. Modificações epigenéticas têm sido propostas como mecanismos responsáveis por estas desordens metabólicas. Ratas Wistar de doze semanas de idade foram alimentadas com dieta com conteúdo baixo (HO - 0,15% NaCl) ou normal (NR - 1,3% NaCl) de sódio desde o primeiro dia de gestação até o nascimento da prole ou HO durante a primeira (HO10) ou segunda (HO20) metade da gestação. O peso corpóreo e a ingestão de água e ração foram avaliados semanalmente durante a gestação. Teste de tolerância à insulina (ITT) e à glicose (GTT) e HOMA-IR foram realizados nas proles adultas. Expressão gênica por qRT-PCR e metilação do DNA na região promotora dos genes foram mapeadas utilizando tratamento com bissulfito de sódio e avaliadas por pirosequenciamento. O ganho de peso materno foi menor no HO e HO20 na terceira semana de gestação em comparação com NR e HO10. O peso ao nascimento da prole foi menor em machos e fêmeas dos grupos HO e HO20 em relação ao NR e HO10. O HOMA-IR foi maior nos machos com 12 semanas de idade do grupo HO em comparação com NR e com 20 semanas de idade do grupo HO10 em comparação com NR e HO20. Nas fêmeas com 12 semanas de idade o HOMA-IR foi maior no HO10 comparado com HO. Os níveis de insulina no soro foram maiores tanto nos machos com 20 semanas de idade do grupo HO10 comparado com NR quanto nas fêmeas com 12 semanas de idade do grupo HO10 comparado com HO. A área sob a curva do GTT indicou intolerância à glicose nos machos do grupo HO. A porcentagem de metilação das ilhas CpG no promotor dos genes de Igf1, Igf1r, Ins1, Ins2 e Insr no fígado de machos e fêmeas neonatais e no fígado, tecido adiposo branco e músculo em machos com 20 semanas de idade foi influenciada pela baixa ingestão de sal durante a gestação. Nenhuma destas alterações foi identificada nas fêmeas com 20 semanas de idade. Em conclusão, a baixa ingestão de sal na segunda metade da gestação é responsável pelo baixo peso ao nascimento em ambos os sexos. A intolerância à glicose observada na prole adulta ocorreu somente se a dieta hipossódica é dada durante a gestação inteira. Por outro lado, a resistência à insulina em resposta ao consumo de dieta hipossódica durante a gestação está relacionada com o momento em que ocorre este insulto e com o envelhecimento da prole. Também foi observado que alterações na metilação do promotor do gene Igf1 está correlacionado com o baixo peso ao nascimento em resposta a ingestão de dieta hipossódica durante a gestação / It is known that some maternal nutritional alterations during pregnancy are associated with metabolic disorders in adult offspring, such as insulin resistance, type 2 diabetes mellitus, obesity and arterial hypertension. The period of pregnancy in which these nutritional alterations influence adult offspring remains uncertain. Epigenetic changes are proposed to underlie these metabolic disorders. Twelve-week-old female Wistar rats were fed a low-salt (LS - 0.15% NaCl) or normal-salt (NS - 1.3% NaCl) diet since the first day of gestation until delivery or LS during the first (LS10) or second (LS20) half of gestation. Body weight, food and water intake were weekly evaluated during gestation. Blood glucose, insulin (ITT) and glucose (GTT) tolerance tests, HOMA-IR were performed in adult offspring. Gene expression and DNA methylation were mapped using bisulfite treatment evaluated by pyrosequencing in the male and female neonates and adult offspring. Weight gain was lower in LS and LS20 dams than in NS and LS10 dams in the third week of pregnancy. Birth weights were lower in male and female LS20 and LS rats compared with NS and LS10 neonates. HOMA-IR was higher in 12-week-old LS males compared with NS and in 20-week-old male LS10 rats compared with NS and LS20 rats. In 12-week-old LS10 females, HOMA-IR was higher than in LS. Serum insulin levels were higher in 20 week-old LS10 male compared with NS rats and in 12-week-old LS10 female compared to LS rats. The area under the curve of GTT indicated glucose intolerance in 12- and 20-week-old LS male. Methylation of CpG islands of the Insr, Igf1, Igf1r, Ins1 and Ins2 genes in liver in neonates male and female offspring and liver, white adipose tissue and muscle in 20-week-old male offspring were influenced by low-salt intake during pregnancy. None of these alterations was identified in 20-week-old females. In conclusion, low-salt diet consumption in the second half of pregnancy can result in low birth weights in the males and females offspring. Glucose intolerance observed in adult offspring occurred only if low salt intake was given throughout pregnancy. However, insulin resistance in response to low salt intake during pregnancy is related to the time at which this insult occurs and to the age of the offspring. Alterations in the DNA methylation of Igf1 were observed to be correlated with low birth weight in response to low salt feeding during pregnancy

Parmi les cancers cutanés, le mélanome métastatique est le moins fréquent (5 à 7%) mais le plus meurtrier par sa forte résistante aux thérapies conventionnelles. Bien qu'immunogène, aucun traitement efficace n'existe actuellement pour traiter ce cancer, ce qui fait qu'il est urgent de trouver de nouvelles cibles thérapeutiques. Dans ce contexte, nous avons évalué si l'Insulin-Like Growth Factor-1 (IGF-1) pouvait représenter une cible d'intérêt thérapeutique dans le mélanome en inhibant l'expression du facteur IGF-1, à l'aide d'un épisome antisens, dans deux modèles cellulaires : des cellules de mélanome primaire B16-F0 et métastatique B16-F10 (cellules appelées B16-F0mod et B16-F10mod lorsque l'expression d’IGF-1 est inhibée).Dans des modèles expérimentaux in vivo, nos résultats montrent que la réduction d'expression d'IGF-1 induit une diminution de la tumorigénicité des cellules de mélanome, en générant des tumeurs sous-cutanées plus petites (B16-F0 et B16-F10 dans les souris C57BL/6) et en inhibant totalement (souris C57BL/6) ou fortement (souris NSG) la capacité des B16-F10 à former des métastases pulmonaires. Nous avons cherché à comprendre si cette perte de tumorigénicité, suite à l'inhibition du facteur IGF-1, était due à une modification de l'immunogénicité/antigénicité des cellules tumorales et/ou à une modification du potentiel tumorigène intrinsèque des cellules tumorales métastatiques.1/ L’immunisation de souris C57BL/6 à l'aide de cellules B16-F0mod induit la formation d’effecteurs humoraux lytiques en présence de complément dirigés contre la lignée parentale, mais également d’effecteurs cellulaires CD8+ capables d’induire la lyse des cellules tumorales in vitro et d’inhiber la croissance tumorale in vivo. Bien que l'analyse des voies humorale et cellulaire n'ait pas permis de démontrer les mécanismes IGF-1-dépendants mis en jeu avec les cellules B16-F10, l'immunisation des souris C57BL/6 à l'aide de cellules B16- F0mod conduit à une inhibition de la croissance des tumeurs sous-cutanées et du nombre de métastases pulmonaires, confirmant l'implication du facteur IGF-1 dans des mécanismes d'échappement tumoral au système immunitaire.2/ Nos résultats montrent par ailleurs que le facteur IGF-1 joue un rôle direct sur le potentiel tumorigène intrinsèque des cellules tumorales. Outre son action sur la prolifération des cellules tumorales, IGF-1 est impliqué dans le processus de transition épithélio-mésenchymateuse (augmentation des marqueurs N-cadhérine, vimentine, CD44 et CD29), favorisant le maintien de populations tumorales présentant un caractère souche (Sox2, Oct3/4, CD44, CD24, activité ALDH, side population, capacité à former des sphéroïdes). Par ce mécanisme, IGF-1 favorise à la fois les propriétés migratoires et d'efflux de drogues, comme la mitoxantrone, par les transporteurs ABC, qui explique en partie la forte résistance des mélanomes aux thérapies conventionnelles.Ces travaux montrent que l’inhibition de la voie IGF1/IGF1-R pourrait être une bonne stratégie pour le développement de traitements anti-tumoraux contre le mélanome. Outre le développement de stratégies d'immunothérapie, le blocage de la voie IGF-1 permettrait également de sensibiliser les cellules de mélanome aux traitements conventionnels et de diminuer le potentiel métastatique des cellules tumorales. / Metastatic melanoma is the least common (5-7 %), but is responsible for most skin cancer deaths by its strong resistance to conventional anti-cancer treatments. Although immunogen, no effective treatment currently exists against this aggressive form, making urgent to find new therapeutic targets. In this context, we assessed whether the Insulin-like Growth Factor-1 (IGF-1) could represent a target of therapeutic interest in melanoma inhibiting the expression of IGF-1 by means of an episome-based vector encoding antisense IGF-1, in two cellular models: primary melanoma cells B16-F0 and metastatic B16-F10 (designated B16-F0mod and B16-F10mod when IGF-1 expression is inhibited).In experimental models in vivo, our results show that the reduction of IGF-1 expression induced a decrease of the melanoma cells tumorigenicity, generating smaller tumors under the skin (B16-F0 and B16-F10 in the C57BL/6 mice) and inhibiting totally (C57BL/6) or strongly (NSG mice) the developpment of B16-F10 lung metastases. We sought to understand whether this loss of tumorigenicity, following IGF-1 inhibition, was due to a change of immunogenicity/antigenicity of tumor cells and/or to intrinsic tumorigenic potential modification of metastatic tumor cells.1 / Immunization of mice C57BL/6 mice with B16-F0mod cells induces the formation of humoral lytic effectors in the presence of complement against the parental line, but also CD8+ effector cells capable of inducing tumor cells lysis in vitro and inhibiting tumor growth in vivo. Although the analysis of humoral and cellular pathways did not demonstrate IGF-1- dependent mechanisms involved in B16-F10 cells, immunization of C57BL/6 mice with B16 cells F0mod leads to skin tumor growth inhibition and a reduction in pulmonary metastases number, confirming the involvement of IGF-1 factor in tumor escape mechanisms of the immune system.2 / Our results also show that IGF-1 plays a direct role in the intrinsic tumorigenic potential of tumor cells. In addition to its effect on tumor cells proliferation, IGF-1 is involved in epithelial-mesenchymal transition (increased N-cadherin, vimentin, CD44 and CD29 markers), promoting the maintenance of tumor populations with stemness properties (Sox2, Oct3/4, CD44, CD24, ALDH activity side-population and ability to form spheroids). By this mechanism, IGF-1 promotes both migration properties and drugs efflux such as mitoxantrone, via ABC transporters, which partly explains the strong resistance of melanoma to conventional therapies.This work shows that the inhibition of IGF1/IGF1-R pathway might be a good strategy for the development of anti-tumor treatments against melanoma. In addition to developing immunotherapy strategies, blocking the IGF-1 pathway would also sensitize melanoma cells to conventional therapy and decrease the metastatic potential of tumor cells.