OBJECTIVE: Ocular function is fundamental for environmental adaptation and survival capacity. Growth factors are necessary for a mature eyeball, needed for adequate vision. However, the consequences of the deficiency of circulating growth hormone (GH) and its effector insulin-like growth factor I (IGF-I) on the physical aspects of the human eye are still debated. A model of untreated isolated GH deficiency (IGHD), with low but measurable serum GH, may clarify this issue. The aim of this study was to assess the ocular aspects of adult IGHD individuals who have never received GH therapy. DESIGN: Cross sectional study. METHODS: Setting University Hospital, Federal University of Sergipe, Brazil. Patients: Twenty-five adult (13 males, mean age 50.1 years, range 26 to 70 years old) IGHD subjects homozygous for a null mutation (c.57+1G>A) in the GHRH receptor gene, and 28 (15 males, mean age 51.1 years, range 26 to 67 years old) controls were submitted to an endocrine and ophthalmological assessment. Forty-six IGHD and 50 control eyes were studied. Main outcome measures: Visual acuity, intraocular pressure (IOP), refraction (spherical equivalent), ocular axial length (AL), anterior chamber depth (ACD),lens thickness (LT), vitreous depth (VD), mean corneal curvature (CC) and central corneal thickness (CCT). RESULTS: IGHD subjects exhibited unmeasurable serum IGF-I levels, similar visual acuity, intraocular pressure and LT, higher values of spherical equivalent and CC, and lower measures of AL, ACD, VD and CCT in comparison to controls, but within their respective normal ranges. While mean stature in IGHD group was 78 % of the control group, mean head circumference was 92 % and axial AL was 96 %. CONCLUSIONS: These observations suggest mild ocular effects in adult subjects with severe IGF-I deficiency due to non-treated IGHD. / OBJETIVO: A função ocular é fundamental para a adaptação ambiental e a capacidade de sobrevivência. Fatores de crescimento são julgados necessáriospara alcançar um globo ocular maduro, e conseqüente visão adequada. No entanto, as consequências da deficiência isoladadohormônio de crescimento circulante (GH) edo seu efetor, o fator de crescimento semelhante à insulina I (IGF-I) nos aspectos físicos do olho humano ainda são debatidas. Um modelo de deficiência isolada de GH não tratada (DIGH) pode esclarecer esta questão. O objetivo deste estudo foi avaliar os aspectos físicos do globo ocular de indivíduos adultos com DIGH que nunca receberam terapia com GH. DESENHO: Estudo transversal. MÉTODOS: Ambiente: Hospital Universitário, Universidade Federal de Sergipe, Brasil. Pacientes: 25 indivíduosadultos (13 homens,com média de idade de 50,1 anos, entre 26 e 70 anos), com DIGH homozigotos para uma mutação nula (c.57 + 1G> A) no gene do receptorGHRH do grupo DIGH e 28 controles (15 homens, com média de idade de 51,1 anos, entre 26 e 67 anos), pareados, foram submetidos à avaliação endócrina e oftalmológica. Principais medidas: acuidade visual(AV), pressão intraocular(PIO),refração (equivalente esférico, EE), comprimento axial ocular (CA), profundidade da câmara anterior(PCA), medida da espessura do cristalino(EC), profundidade do vítreo(PV), curvatura corneana média(CCM) e espessura central corneana(ECC). RESULTADOS:Indivíduos com DIGH apresentaram IGF-I sérico não mensurável, similarAV, PIO e EC, valores mais altos doEEe CCM, e menores valores do CA, PCA, PV e ECC em comparação com os controles, mas dentro das respectivas faixas normais. Enquanto a estaturamédia no grupo DIGH foi de 78% do grupo de controle, a média da circunferência da cabeça foi de 92% e a média docomprimento axial foi de 96%. CONCLUSÃO: Essas observações sugerem efeitos oculares discretosem indivíduos adultos com grave deficiência de IGF-I devido à DIGH não tratada.

OBJECTIVE: Ocular function is fundamental for environmental adaptation and survival capacity. Growth factors are necessary for a mature eyeball, needed for adequate vision. However, the consequences of the deficiency of circulating growth hormone (GH) and its effector insulin-like growth factor I (IGF-I) on the physical aspects of the human eye are still debated. A model of untreated isolated GH deficiency (IGHD), with low but measurable serum GH, may clarify this issue. The aim of this study was to assess the ocular aspects of adult IGHD individuals who have never received GH therapy. DESIGN: Cross sectional study. METHODS: Setting University Hospital, Federal University of Sergipe, Brazil. Patients: Twenty-five adult (13 males, mean age 50.1 years, range 26 to 70 years old) IGHD subjects homozygous for a null mutation (c.57+1G>A) in the GHRH receptor gene, and 28 (15 males, mean age 51.1 years, range 26 to 67 years old) controls were submitted to an endocrine and ophthalmological assessment. Forty-six IGHD and 50 control eyes were studied. Main outcome measures: Visual acuity, intraocular pressure (IOP), refraction (spherical equivalent), ocular axial length (AL), anterior chamber depth (ACD),lens thickness (LT), vitreous depth (VD), mean corneal curvature (CC) and central corneal thickness (CCT). RESULTS: IGHD subjects exhibited unmeasurable serum IGF-I levels, similar visual acuity, intraocular pressure and LT, higher values of spherical equivalent and CC, and lower measures of AL, ACD, VD and CCT in comparison to controls, but within their respective normal ranges. While mean stature in IGHD group was 78 % of the control group, mean head circumference was 92 % and axial AL was 96 %. CONCLUSIONS: These observations suggest mild ocular effects in adult subjects with severe IGF-I deficiency due to non-treated IGHD. / OBJETIVO: A função ocular é fundamental para a adaptação ambiental e a capacidade de sobrevivência. Fatores de crescimento são julgados necessáriospara alcançar um globo ocular maduro, e conseqüente visão adequada. No entanto, as consequências da deficiência isoladadohormônio de crescimento circulante (GH) edo seu efetor, o fator de crescimento semelhante à insulina I (IGF-I) nos aspectos físicos do olho humano ainda são debatidas. Um modelo de deficiência isolada de GH não tratada (DIGH) pode esclarecer esta questão. O objetivo deste estudo foi avaliar os aspectos físicos do globo ocular de indivíduos adultos com DIGH que nunca receberam terapia com GH. DESENHO: Estudo transversal. MÉTODOS: Ambiente: Hospital Universitário, Universidade Federal de Sergipe, Brasil. Pacientes: 25 indivíduosadultos (13 homens,com média de idade de 50,1 anos, entre 26 e 70 anos), com DIGH homozigotos para uma mutação nula (c.57 + 1G> A) no gene do receptorGHRH do grupo DIGH e 28 controles (15 homens, com média de idade de 51,1 anos, entre 26 e 67 anos), pareados, foram submetidos à avaliação endócrina e oftalmológica. Principais medidas: acuidade visual(AV), pressão intraocular(PIO),refração (equivalente esférico, EE), comprimento axial ocular (CA), profundidade da câmara anterior(PCA), medida da espessura do cristalino(EC), profundidade do vítreo(PV), curvatura corneana média(CCM) e espessura central corneana(ECC). RESULTADOS:Indivíduos com DIGH apresentaram IGF-I sérico não mensurável, similarAV, PIO e EC, valores mais altos doEEe CCM, e menores valores do CA, PCA, PV e ECC em comparação com os controles, mas dentro das respectivas faixas normais. Enquanto a estaturamédia no grupo DIGH foi de 78% do grupo de controle, a média da circunferência da cabeça foi de 92% e a média docomprimento axial foi de 96%. CONCLUSÃO: Essas observações sugerem efeitos oculares discretosem indivíduos adultos com grave deficiência de IGF-I devido à DIGH não tratada.

A l'interface de la génétique et de l'environnement, l'épigénétique contribue à la diversité phénotypique. Déterminer l'impact de la variation épigénétique sur les caractères quantitatifs (QT) est un nouveau défi. La croissance staturale fournit l’opportunité d’étudier la variabilité de plusieurs traits phénotypiques liés entre eux : des QT cliniques (la taille, l’accélération de la vitesse de croissance en réponse à l'hormone de croissance, GH) et des QT biologiques tels que la concentration d’IGF1 et la réponse de cette concentration à la GH. L’ « Insulin-like Growth Factor 1 » (IGF1) contrôle la croissance postnatale chez les mammifères, y compris l'homme. Nous l’avons choisi comme locus candidat pour nos études épigénétiques. Nous avons quantifié la méthylation des deux promoteurs P1 et P2 de ce gène, qui régulent son expression. Notre objectif était d’évaluer la contribution de la méthylation d’ADN de ces promoteurs i) à la taille des enfants en croissance, ii) à l’IGF1 circulant, iii) et à la réponse de ces paramètres à un traitement par la GH. Taille et IGF1 circulant. La relation entre la méthylation des promoteurs d’IGF1 et la taille a été étudiée au sein de deux cohortes du service d'endocrinologie pédiatrique, totalisant 216 enfants prépubères de différentes statures. Nous avons montré que la méthylation d'un groupe de six CGs situés dans la partie proximale du promoteur P2 du gène IGF1 présentait une corrélation inverse avec la croissance et l'IGF1 circulant. Les enfants les plus grands sont ainsi moins méthylés sur ces CGs que les enfants de petite taille. La contribution de la méthylation à la variance de la taille a été évaluée à environ 13%, et à 10% pour la variance de l'IGF1 sérique. Pour montrer que l’association observée reflète une causalité biologique, nous avons étudié le lien entre la méthylation des promoteurs P1 et P2 et l'activité transcriptionnelle du gène IGF1 in vivo et in vitro. Nous avons montré que les quantités de transcrits de classe II, issus du promoteur P2, sont inversement corrélés à la méthylation du promoteur P2 dans les cellules sanguines mononucléées. In vitro, nous avons cloné le promoteur P2 déméthylé ou méthylé dans un plasmide rapporteur (luciferase) transfecté dans la lignée HEK293 : le promoteur déméthylé s’est révélé nettement plus actif (+57%). Finalement, nous suggérons que l’hyperméthylation de certains CGs du P1 et du P2 d’IGF1 pourrait être un des nombreux mécanismes moléculaires responsables d’une moindre expression du gène et d’un phénotype de petite taille. La réponse au traitement par la GH. Une fraction des enfants de petite taille est traitée par l'hormone de croissance (GH) pour accélérer sa croissance, mais l’efficacité du traitement est très variable entre les individus. Les causes de cette variabilité sont partiellement comprises : la génétique joue un rôle, mais il reste une place possible pour la variabilité épigénétique. Dans ce but, nous avons étudié l'effet direct de la variabilité épigénétique sur la transcription du gène IGF1 et l’IGF1 circulant, dans un test aigu d’administration de GH, puis sur la réponse thérapeutique à un traitement d’un an par la GH. Après une injection de GH, nous avons constaté une augmentation variable du nombre de transcrits d’IGF1 chez les enfants étudiés. L'augmentation des transcrits de la classe II était inversement corrélée à la méthylation des CGs du P2. La variabilité de méthylation au CG-137 contribuait pour 20% à 67% de l’expression d’IGF1 en réponse à la GH. Chez 136 enfants de petite taille, nous avons montré que la méthylation de l'ADN du promoteur P2 était associée à la réponse au traitement par la GH au cours de la première année. Cette association est observée pour l'augmentation de la vitesse de croissance et pour les taux d’IGF1. (...) / At the interface of genetics and environment, epigenetics contributes to phenotypic diversity. Quantifying the impact of epigenetic variation on quantitative traits (QT), an emerging challenge in humans. Growth provides a handset of quantitative traits to epigenetic studies. We studied the variability of several inter-related QTs: clinical QTs (height, height reponse to growth hormone and biological QT (serum IGF1 and serum IGF1 response to GH). Since insulin-like growth factor 1 (IGF1) controls postnatal growth in mammals including human, we tested whether the CG methylation of the two promoters (P1 and P2) of the IGF1 gene could be a epigenetic contributor to the individual variation i) in circulating IGF1 and stature in growing children. ii) on response of these parameters to treatment with (GH). Child height and circulating IGF1. To explore the relation between IGF1 promoter methylation and height, we studied two cohorts of pedriatric endocrinology department, totalling 216 prepubertal children with various statures. The methylation of a cluster of six CGs located within the proximal part of the IGF1 P2 promoter showed a strong negative association with serum IGF1 and growth. These correlations were observed in two cohorts of growing children. Tall children show lower levels of methylation in several CGs in P2 and P1 promoters of IGF1 gene than short children with idiopathic short stature. CG methylation contributed 13% to the variance of height and 10% to the variance of serum IGF1. To test if the found association reflected biological causality, we tested if methylation at the P2 promoter affects the transcriptional activity of the IGF1 gene. The transcriptional activity of the P2 promoter was inversely correlated with the CG methylation in mononuclear blood cells. We established that high levels of CG methylation at the two promoters of IGF1 contributed to the many molecular mechanisms responsible for “idiopathic” short stature. Response to treatment with (GH). Short children using growth hormone (GH) to accelerate their growth respond to this treatment with a variable efficacy. The causes of this individual variability are partially understood and could involve epigenetics. In this aim, we investigated the contribution of DNA methylation to the response to GH at two levels: direct effect of GH on transcription of IGF1 gene, on circulating IGF1 and on the growth response to GH. Following a GH injection, we found a variable increase in IGF1 transcripts across the studied children. The increase in P2-driven transcripts showed a strong inverse correlation with 4/8 of P2 CGs. Among the CGs of P1 promoter, only CG-611 showed an inverse correlation with P1-driven transcripts. Variability of DNA methylation in these CGs contributes with 27% to 67% of increase in transcripts. In 136 children with idiopatic short stature, we showed that DNA methylation of the P2 promoter is associated with growth response to GH during the first year of GH administration, for both increment in growth rate and circulating IGF1. CG-137 methylation of P2 promoter contributes 25% to variance of growth response to GH. The link between DNA methylation and the response to a treatment in humans illustrating the role of epigenetic marks as potent contributors to conclusion « pharmacoepigenetics». Our work can find application in growth physiology and therapeutics, as well as for studies in aging, longevity or cancer where IGF1 has a prominent role.

A l'interface de la génétique et de l'environnement, l'épigénétique contribue à la diversité phénotypique. Déterminer l'impact de la variation épigénétique sur les caractères quantitatifs (QT) est un nouveau défi. La croissance staturale fournit l’opportunité d’étudier la variabilité de plusieurs traits phénotypiques liés entre eux : des QT cliniques (la taille, l’accélération de la vitesse de croissance en réponse à l'hormone de croissance, GH) et des QT biologiques tels que la concentration d’IGF1 et la réponse de cette concentration à la GH. L’ « Insulin-like Growth Factor 1 » (IGF1) contrôle la croissance postnatale chez les mammifères, y compris l'homme. Nous l’avons choisi comme locus candidat pour nos études épigénétiques. Nous avons quantifié la méthylation des deux promoteurs P1 et P2 de ce gène, qui régulent son expression. Notre objectif était d’évaluer la contribution de la méthylation d’ADN de ces promoteurs i) à la taille des enfants en croissance, ii) à l’IGF1 circulant, iii) et à la réponse de ces paramètres à un traitement par la GH. Taille et IGF1 circulant. La relation entre la méthylation des promoteurs d’IGF1 et la taille a été étudiée au sein de deux cohortes du service d'endocrinologie pédiatrique, totalisant 216 enfants prépubères de différentes statures. Nous avons montré que la méthylation d'un groupe de six CGs situés dans la partie proximale du promoteur P2 du gène IGF1 présentait une corrélation inverse avec la croissance et l'IGF1 circulant. Les enfants les plus grands sont ainsi moins méthylés sur ces CGs que les enfants de petite taille. La contribution de la méthylation à la variance de la taille a été évaluée à environ 13%, et à 10% pour la variance de l'IGF1 sérique. Pour montrer que l’association observée reflète une causalité biologique, nous avons étudié le lien entre la méthylation des promoteurs P1 et P2 et l'activité transcriptionnelle du gène IGF1 in vivo et in vitro. Nous avons montré que les quantités de transcrits de classe II, issus du promoteur P2, sont inversement corrélés à la méthylation du promoteur P2 dans les cellules sanguines mononucléées. In vitro, nous avons cloné le promoteur P2 déméthylé ou méthylé dans un plasmide rapporteur (luciferase) transfecté dans la lignée HEK293 : le promoteur déméthylé s’est révélé nettement plus actif (+57%). Finalement, nous suggérons que l’hyperméthylation de certains CGs du P1 et du P2 d’IGF1 pourrait être un des nombreux mécanismes moléculaires responsables d’une moindre expression du gène et d’un phénotype de petite taille. La réponse au traitement par la GH. Une fraction des enfants de petite taille est traitée par l'hormone de croissance (GH) pour accélérer sa croissance, mais l’efficacité du traitement est très variable entre les individus. Les causes de cette variabilité sont partiellement comprises : la génétique joue un rôle, mais il reste une place possible pour la variabilité épigénétique. Dans ce but, nous avons étudié l'effet direct de la variabilité épigénétique sur la transcription du gène IGF1 et l’IGF1 circulant, dans un test aigu d’administration de GH, puis sur la réponse thérapeutique à un traitement d’un an par la GH. Après une injection de GH, nous avons constaté une augmentation variable du nombre de transcrits d’IGF1 chez les enfants étudiés. L'augmentation des transcrits de la classe II était inversement corrélée à la méthylation des CGs du P2. La variabilité de méthylation au CG-137 contribuait pour 20% à 67% de l’expression d’IGF1 en réponse à la GH. Chez 136 enfants de petite taille, nous avons montré que la méthylation de l'ADN du promoteur P2 était associée à la réponse au traitement par la GH au cours de la première année. Cette association est observée pour l'augmentation de la vitesse de croissance et pour les taux d’IGF1. (...) / At the interface of genetics and environment, epigenetics contributes to phenotypic diversity. Quantifying the impact of epigenetic variation on quantitative traits (QT), an emerging challenge in humans. Growth provides a handset of quantitative traits to epigenetic studies. We studied the variability of several inter-related QTs: clinical QTs (height, height reponse to growth hormone and biological QT (serum IGF1 and serum IGF1 response to GH). Since insulin-like growth factor 1 (IGF1) controls postnatal growth in mammals including human, we tested whether the CG methylation of the two promoters (P1 and P2) of the IGF1 gene could be a epigenetic contributor to the individual variation i) in circulating IGF1 and stature in growing children. ii) on response of these parameters to treatment with (GH). Child height and circulating IGF1. To explore the relation between IGF1 promoter methylation and height, we studied two cohorts of pedriatric endocrinology department, totalling 216 prepubertal children with various statures. The methylation of a cluster of six CGs located within the proximal part of the IGF1 P2 promoter showed a strong negative association with serum IGF1 and growth. These correlations were observed in two cohorts of growing children. Tall children show lower levels of methylation in several CGs in P2 and P1 promoters of IGF1 gene than short children with idiopathic short stature. CG methylation contributed 13% to the variance of height and 10% to the variance of serum IGF1. To test if the found association reflected biological causality, we tested if methylation at the P2 promoter affects the transcriptional activity of the IGF1 gene. The transcriptional activity of the P2 promoter was inversely correlated with the CG methylation in mononuclear blood cells. We established that high levels of CG methylation at the two promoters of IGF1 contributed to the many molecular mechanisms responsible for “idiopathic” short stature. Response to treatment with (GH). Short children using growth hormone (GH) to accelerate their growth respond to this treatment with a variable efficacy. The causes of this individual variability are partially understood and could involve epigenetics. In this aim, we investigated the contribution of DNA methylation to the response to GH at two levels: direct effect of GH on transcription of IGF1 gene, on circulating IGF1 and on the growth response to GH. Following a GH injection, we found a variable increase in IGF1 transcripts across the studied children. The increase in P2-driven transcripts showed a strong inverse correlation with 4/8 of P2 CGs. Among the CGs of P1 promoter, only CG-611 showed an inverse correlation with P1-driven transcripts. Variability of DNA methylation in these CGs contributes with 27% to 67% of increase in transcripts. In 136 children with idiopatic short stature, we showed that DNA methylation of the P2 promoter is associated with growth response to GH during the first year of GH administration, for both increment in growth rate and circulating IGF1. CG-137 methylation of P2 promoter contributes 25% to variance of growth response to GH. The link between DNA methylation and the response to a treatment in humans illustrating the role of epigenetic marks as potent contributors to conclusion « pharmacoepigenetics». Our work can find application in growth physiology and therapeutics, as well as for studies in aging, longevity or cancer where IGF1 has a prominent role.

Einleitung: Wachstumsprozesse beim gesunden Menschen sind weitgehend untersucht und bekannt. Über die Auswirkungen von stark erniedrigtem bzw. fehlendem Wachstumshormon auf das Längen- und Breitenwachstum einzelner auxiologischer Parameter und die Folgen einer Wachstumshormonsubstitutionstherapie auf die Körperproportionen im Einzelnen weiß man außer den Kenntnissen über die Körperhöhenveränderungen wenig. Fragestellung: Ziel dieser Arbeit war die auxiologische Untersuchung von Kindern mit Wachstumshormonmangel vor und unter einer Substitutionstherapie mit Wachstumshormon. im Vordergrund stand die Erkennung bestimmter auxiologischer Muster vor und die Frage nach Veränderungen der Körperproportionen unter der Therapie. Methode: Grundlage der Daten sind 62 Kinder mit idiopathischem und 20 Kindern mit organischem Wachtumshormonmangel, bei denen über einen maximalen Zeitraum von fünf Jahren halbjährlich 22 verschiedene Parameter vermessen und drei weitere berechnet worden sind. Ergebnisse: Die phänotypischen Merkmale des hypophysären Kleinwuchses vor Substitutionsbeginn (Puppengesicht, Akromikrie, pyknomorpher Körperbau) sind Ausdruck bestimmter auxiologischer Konstellationen, die Längenparameter weichen signifikant stärker als die Breitenparameter vom Altersnormwert ab. In Abhängigkeit von der Therapiedauer kommt es durch unterschiedlich stark ausgeprägte Größenzuwachsraten im Verhältnis zum Längenwachstum insbesondere bei der Unterarmlänge, den Breiten- und Tiefenmaßen des Thorax und den Kopfmaßen zu Veränderungen der Körperproportionen. In der Regel verbessern sich die Ausgangsproportionen. Der relative Körperfettgehalt, sowie die Umfänge (Brust-, Taillen- und Hüftumfang) nehmen unter der Therapie in Relation zur Körperhöhe ab. Schlussfolgerung: Anhand der vorliegenden Daten wird gezeigt, dass die Substitutionstherapie mit Wachstumshormon beim hypophysären Kleinwuchs Auswirkungen auf das Längen- und Breitenwachstum einzelner auxiologischer Parameter hat und sich von physiologischem Wachstum unterscheidet. Für die Bestätigung unserer Beobachtungen sind weitere prospektive Untersuchungen in größeren Kollektiven notwendig. / Introduction: Processes of growth in healthy children are well established. However, little is known about the effects of strongly degraded or missing growth hormone on the lengths and breadths growth of single axiological parameters. In particular, changes of body proportions under hormone replacement therapy are not known. Objective: The aim of the study was to estimate the influence of growth hormone deficiency of single axiological parameters and changes in body proportions during replacement therapy with growth hormone. Patients/Material and Methods: Subjects studied include 62 children with idiopathic growth hormone deficiency and 20 children with organic reason of growth hormone deficiency. 22 anthropometric measurements per person were taken by the same trained examiner prior to the start of GH-treatment and then every six months over a maximal period of 5 years. Results: Results give a distinct anthropometric picture before start of therapy. All linear parameters were significantly below average, while width measurements differed less. During treatment changes of the body proportions occur due to different growth rates of measured distances compared to height growth. The most positive changes were seen in the upper arm, hand and feet growth under GH-therapy. A comparable low growth was seen in forearm length, chest width and depth and head measures. The growth dynamics of the other parameters correspond to that of height itself. Conclusion: It is shown that the replacement therapy with growth hormone has different effects on the length and width growth of different bones. The growth is different from physiological growth processes in patient with growth hormone deficiency. Further prospective examinations are necessary to confirm our observations in larger collectives.

A l'interface de la génétique et de l'environnement, l'épigénétique contribue à la diversité phénotypique. Déterminer l'impact de la variation épigénétique sur les caractères quantitatifs (QT) est un nouveau défi. La croissance staturale fournit l’opportunité d’étudier la variabilité de plusieurs traits phénotypiques liés entre eux : des QT cliniques (la taille, l’accélération de la vitesse de croissance en réponse à l'hormone de croissance, GH) et des QT biologiques tels que la concentration d’IGF1 et la réponse de cette concentration à la GH. L’ « Insulin-like Growth Factor 1 » (IGF1) contrôle la croissance postnatale chez les mammifères, y compris l'homme. Nous l’avons choisi comme locus candidat pour nos études épigénétiques. Nous avons quantifié la méthylation des deux promoteurs P1 et P2 de ce gène, qui régulent son expression. Notre objectif était d’évaluer la contribution de la méthylation d’ADN de ces promoteurs i) à la taille des enfants en croissance, ii) à l’IGF1 circulant, iii) et à la réponse de ces paramètres à un traitement par la GH. Taille et IGF1 circulant. La relation entre la méthylation des promoteurs d’IGF1 et la taille a été étudiée au sein de deux cohortes du service d'endocrinologie pédiatrique, totalisant 216 enfants prépubères de différentes statures. Nous avons montré que la méthylation d'un groupe de six CGs situés dans la partie proximale du promoteur P2 du gène IGF1 présentait une corrélation inverse avec la croissance et l'IGF1 circulant. Les enfants les plus grands sont ainsi moins méthylés sur ces CGs que les enfants de petite taille. La contribution de la méthylation à la variance de la taille a été évaluée à environ 13%, et à 10% pour la variance de l'IGF1 sérique. Pour montrer que l’association observée reflète une causalité biologique, nous avons étudié le lien entre la méthylation des promoteurs P1 et P2 et l'activité transcriptionnelle du gène IGF1 in vivo et in vitro. Nous avons montré que les quantités de transcrits de classe II, issus du promoteur P2, sont inversement corrélés à la méthylation du promoteur P2 dans les cellules sanguines mononucléées. In vitro, nous avons cloné le promoteur P2 déméthylé ou méthylé dans un plasmide rapporteur (luciferase) transfecté dans la lignée HEK293 : le promoteur déméthylé s’est révélé nettement plus actif (+57%). Finalement, nous suggérons que l’hyperméthylation de certains CGs du P1 et du P2 d’IGF1 pourrait être un des nombreux mécanismes moléculaires responsables d’une moindre expression du gène et d’un phénotype de petite taille. La réponse au traitement par la GH. Une fraction des enfants de petite taille est traitée par l'hormone de croissance (GH) pour accélérer sa croissance, mais l’efficacité du traitement est très variable entre les individus. Les causes de cette variabilité sont partiellement comprises : la génétique joue un rôle, mais il reste une place possible pour la variabilité épigénétique. Dans ce but, nous avons étudié l'effet direct de la variabilité épigénétique sur la transcription du gène IGF1 et l’IGF1 circulant, dans un test aigu d’administration de GH, puis sur la réponse thérapeutique à un traitement d’un an par la GH. Après une injection de GH, nous avons constaté une augmentation variable du nombre de transcrits d’IGF1 chez les enfants étudiés. L'augmentation des transcrits de la classe II était inversement corrélée à la méthylation des CGs du P2. La variabilité de méthylation au CG-137 contribuait pour 20% à 67% de l’expression d’IGF1 en réponse à la GH. Chez 136 enfants de petite taille, nous avons montré que la méthylation de l'ADN du promoteur P2 était associée à la réponse au traitement par la GH au cours de la première année. Cette association est observée pour l'augmentation de la vitesse de croissance et pour les taux d’IGF1. (...) / At the interface of genetics and environment, epigenetics contributes to phenotypic diversity. Quantifying the impact of epigenetic variation on quantitative traits (QT), an emerging challenge in humans. Growth provides a handset of quantitative traits to epigenetic studies. We studied the variability of several inter-related QTs: clinical QTs (height, height reponse to growth hormone and biological QT (serum IGF1 and serum IGF1 response to GH). Since insulin-like growth factor 1 (IGF1) controls postnatal growth in mammals including human, we tested whether the CG methylation of the two promoters (P1 and P2) of the IGF1 gene could be a epigenetic contributor to the individual variation i) in circulating IGF1 and stature in growing children. ii) on response of these parameters to treatment with (GH). Child height and circulating IGF1. To explore the relation between IGF1 promoter methylation and height, we studied two cohorts of pedriatric endocrinology department, totalling 216 prepubertal children with various statures. The methylation of a cluster of six CGs located within the proximal part of the IGF1 P2 promoter showed a strong negative association with serum IGF1 and growth. These correlations were observed in two cohorts of growing children. Tall children show lower levels of methylation in several CGs in P2 and P1 promoters of IGF1 gene than short children with idiopathic short stature. CG methylation contributed 13% to the variance of height and 10% to the variance of serum IGF1. To test if the found association reflected biological causality, we tested if methylation at the P2 promoter affects the transcriptional activity of the IGF1 gene. The transcriptional activity of the P2 promoter was inversely correlated with the CG methylation in mononuclear blood cells. We established that high levels of CG methylation at the two promoters of IGF1 contributed to the many molecular mechanisms responsible for “idiopathic” short stature. Response to treatment with (GH). Short children using growth hormone (GH) to accelerate their growth respond to this treatment with a variable efficacy. The causes of this individual variability are partially understood and could involve epigenetics. In this aim, we investigated the contribution of DNA methylation to the response to GH at two levels: direct effect of GH on transcription of IGF1 gene, on circulating IGF1 and on the growth response to GH. Following a GH injection, we found a variable increase in IGF1 transcripts across the studied children. The increase in P2-driven transcripts showed a strong inverse correlation with 4/8 of P2 CGs. Among the CGs of P1 promoter, only CG-611 showed an inverse correlation with P1-driven transcripts. Variability of DNA methylation in these CGs contributes with 27% to 67% of increase in transcripts. In 136 children with idiopatic short stature, we showed that DNA methylation of the P2 promoter is associated with growth response to GH during the first year of GH administration, for both increment in growth rate and circulating IGF1. CG-137 methylation of P2 promoter contributes 25% to variance of growth response to GH. The link between DNA methylation and the response to a treatment in humans illustrating the role of epigenetic marks as potent contributors to conclusion « pharmacoepigenetics». Our work can find application in growth physiology and therapeutics, as well as for studies in aging, longevity or cancer where IGF1 has a prominent role.

Em nosso laboratório foi demonstrado que ratos submetidos à restrição crônica de sal na dieta apresentaram maior peso corpóreo (PC), menor sensibilidade à insulina e alterações pressóricas em comparação com ratos submetidos à sobrecarga crônica de sal na dieta. No presente estudo, o objetivo foi avaliar alguns mecanismos de obesidade e alterações hormonais associados à dieta hipossódica. Foram utilizados ratos Wistar machos submetidos à dieta hipo (HO: 0,15% NaCl), normo (NO: 1,27% NaCl) ou hipersódica (HR: 7,94% NaCl) desde o desmame até 12 semanas de idade. Nestes animais foram realizadas medidas de pressão arterial (PA), freqüência cardíaca (FC), consumo de ração, PC, perfil dos hormônios [leptina (LEP), GH, insulina (INSh - anticorpo anti-insulina humana), T3, T4 e TSH] e decaimento da 125I-insulina (DEC-INS). O consumo de ração foi avaliado durante sete dias consecutivos com um (1G) ou quatro (4G) ratos por gaiola de plástico e com um rato por gaiola metabólica (1GM). O PC também foi medido neste período. Glicemia (GLI) e insulinemia (INSr - anticorpo anti-insulina de rato) basais foram quantificadas antes da realização do estudo do DEC-INS. Para avaliar o DEC-INS, foi injetado 1mCi de 125I-insulina humana pelo cateter implantado na veia jugular, após 6 a 8 horas de jejum fisiológico. As amostras de sangue foram coletadas pelo cateter implantado na carótida a cada 30 segundos durante 2 minutos e depois a cada 1 minuto até 10 minutos de experimento. Os resultados obtidos demonstraram que ratos submetidos à dieta HR apresentaram maior PA em comparação a ratos em dieta NO e HO. Os ratos submetidos à dieta HO apresentaram menor FC cardíaca em relação ao grupo NO. A avaliação de ingestão alimentar revelou que ratos em dieta HR consumiram maiores quantidades de ração (1G e 4G) em comparação com ratos em dieta NO e HO. O grupo HO apresentou maior PC em comparação com os grupos de ratos NO e HR. Os ratos em dieta HR ou HO apresentaram níveis diminuídos de LEP em comparação com ratos submetidos à dieta NO. Os ratos que receberam dieta HO apresentaram níveis elevados de INSr em comparação com ratos em dieta HR e NO. Os ratos em dieta HR apresentaram níveis elevados de T4 total e níveis reduzidos de TSH em comparação com animais em dieta NO e HO. INSr e GLI basais foram maiores nos ratos em dieta HO do que nos ratos que receberam dieta HR e NO. O DEC-INS (decaimento exponencial) foi mais rápido no grupo HR demonstrado por meio de menor meia-vida da 125I-insulina. Os resultados sugerem que maior PC nos ratos sob restrição salina crônica é devido a maior eficiência metabólica (maior PC e menor consumo de dieta) que pode estar relacionada com alterações hormonais e com a dieta HO. Palavras-chave: Pressão arterial, sal, sódio, ingestão alimentar e perfil hormonal. / Previous studies from our laboratory have shown that chronic salt restriction decreases blood pressure, increases insulin resistance, and body weight (BW) in Wistar rats. The aim of this study was to evaluate some mechanisms of obesity and hormonal alterations associated with chronic salt restriction. Male Wistar rats were fed a low (LSD: 0,15% NaCl), normal (NSD: 1,27% NaCl), or high salt diet (HSD: 7,94% NaCl) from weaning. At the 12th week of age, tail-cuff blood pressure (TCBP), intra arterial blood pressure (BP), heart rate ((HR), food intake, BW, hormonal levels [leptin (LEP), growth hormone (GH), insulin (INSh - antibody anti-human insulin), T3, T4 and TSH] and 125I insulin decay study (DECAY-INS) were measured. To evaluate the food intake and body weight, each dietary group was divided in 3 subgroups, according to housing conditions: one (1C) or four (4C) rats per cage and one rat per metabolic cage (1MC) for daily food consumption and BW determinations during 7 days. Fasting plasma glucose (GLU) and insulin (INSr - antibody anti-rat insulin) were measured before the DECAY-INS. To evaluate the DECAY-INS, 1mCi human 125I-insulin was injected through the jugular catheter after 6-8 hours of food restriction. Blood samples were withdrawn through the carotid catheter every 30 sec during 2min, and in sequence, every 1 min for additional 10 minutes. 125Iinsulin was determined by RIA for human insulin. Rats on HSD had higher intra arterial BP and TCBP compared to rats on NSD and LSD. Heart rate was lower on LSD than on NSD. In all housing conditions, BW was higher on LSD than on NSD and HSD. Food intake was higher on HSD (1C and 4C) than on NSD and LSD. Plasma GH and LEP were higher on NSD than on the 68 other two groups. Plasma INSh was higher in LSD compared to HSD and NSD rats. Plasma total T4 was higher on HSD than on the NSD and LSD, TSH was lower on HSD than on NSD and HSD, and T3 was not different among all groups. Fasting GLU and INSr were higher on LSD compared to HSD and NSD rats. The exponential insulin decay was faster on HSD demonstrated by a lower 125I-INS half-life. These results suggest that obesity in rats on chronic LSD is due to a higher metabolic efficiency (higher body weight and lower diet consumption), that may be related to the hormonal consequences of LSD. Key Words: blood pressure, salt, sodium, food intake and hormonal profile.

Made available in DSpace on 2016-12-23T14:20:37Z (GMT). No. of bitstreams: 1 Reginaldo Alex Calcavara.pdf: 2029583 bytes, checksum: 1eb772d96a68609843a4414a83ede253 (MD5) Previous issue date: 2013-08-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A pesquisa articula os temas da ontologia do espaço e a dicotomia entre a Geografia Física e a Geografia Humana no âmbito do movimento de renovação crítica da geografia brasileira. Busca demonstrar que há um nexo entre a posição assumida por esta vertente da geografia brasileira frente à referida dicotomia e a reflexão ontológica acerca do objeto da disciplina, diretamente influenciada pelo pensamento marxista. Indica como campo alternativo de reflexão ontológica a abordagem do filósofo alemão Martin Heidegger em torno da questão do ser, destacando, dentro da amplitude e centralidade deste assunto na obra do referido filósofo, a perspectiva da questão da técnica que, redimensionando a noção de produção, permitiria oferecer um horizonte alternativo para a ontologia na geografia capaz, sugere-se, de abrir a questão do ser enquanto questão a ser pensada / The study articulates the themes of ontology of space and the dichotomy between Physical Geography and Human Geography in the renewal movement critical of Brazilian geography. Seeks to demonstrate that there is a link between, on the one hand, the position taken by this part of Brazilian geography forward that dichotomy, and on the other hand, the ontological reflection about the object of discipline, directly influenced by Marxism. Indicates how alternative field of ontological thinking approach of the German philosopher Martin Heidegger on the question of being, especially within the scope and centrality of this issue in the work of that philosopher s perspective the question of technique, redefining the notion of production, would provide a horizon alternative ontology in geography capable, it is suggested, opening the question of being as a question to be thought

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-06-06T15:05:37Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AvaliacaoPicosHormonio.pdf: 2169494 bytes, checksum: 0e4cd9b130002323eb3e56d35597841a (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2017-06-12T13:22:25Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AvaliacaoPicosHormonio.pdf: 2169494 bytes, checksum: 0e4cd9b130002323eb3e56d35597841a (MD5) / Made available in DSpace on 2017-06-12T13:22:25Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AvaliacaoPicosHormonio.pdf: 2169494 bytes, checksum: 0e4cd9b130002323eb3e56d35597841a (MD5) Previous issue date: 2016-12-28 / A baixa estatura (BE) é uma importante causa de encaminhamento para avaliação na endocrinologia pediátrica. A deficiência do hormônio do crescimento (DGH) precisa ser considerada quando outras causas de BE são excluídas, porém há limitações quanto ao estabelecimento de seu diagnóstico definitivo, sendo assunto de vários debates e controvérsias. Apesar de muito questionados, os testes de estímulo de GH ainda são considerados o padrão para a confirmação diagnóstica de DGH. O presente estudo objetivou avaliar a sensibilidade, especificidade e acurácia dos diferentes pontos de corte de pico de GH utilizados para o diagnóstico de DGH, sob o estímulo do teste de tolerância à insulina (TTI) e do teste da clonidina, além de identificar o melhor nivel de pico de GH para confirmar o diagnóstico por meio da abordagem da curva ROC (Receiver Operating Characteristics). Para este fim, foi realizado um estudo retrospectivo e de caráter observacional, a partir da coleta de dados clínicos e laboratoriais de 62 pacientes do serviço de endocrinologia do Hospital Universitário João de Barros Barreto (HUJBB). O padrão-ouro considerado para análise de desempenho dos pontos de corte em ambos os testes de estímulo de GH foi a resposta terapêutica. Assim, 26 pacientes que obtiveram acréscimo de altura de pelo menos 0,3 desvio-padrão ao final de um ano de tratamento com o GH recombinante humano (rhGH) foram classificados como DGH. Os pacientes que não obtiveram esse ganho foram classificados como não-DGH. Ambos os grupos partiram de estaturas médias semelhantes (p = 0,8155) e obtiverem ganho de estatura ao final do acompanhamento, porém esse ganho foi maior no grupo DGH em comparação ao não-DGH (20,5 ± 14,8 cm vs. 9,2 ± 6,7 cm, respectivamente; p = 0,0064). O grupo DGH apresentou pico mediano de GH significativamente menor em relação ao grupo não-DGH em ambos os testes (p < 0,0001). Foram definidas sensibilidade, especificidade e acurácia dos pontos de corte 3, 5, 7 e 10 ng/mL no TTI e no teste da clonidina, não sendo observada superioridade de um teste sobre o outro. Adicionalmente, foram encontrados os pontos de corte 7,92 ng/mL e 6,78 ng/mL para o TTI e teste da clonidina, respectivamente, a partir da construção da curva ROC, representando os níveis de pico de GH mais sensíveis e específicos para o diagnóstico de DGH. Concluímos que os pontos de corte encontrados neste estudo poderão representar uma ferramenta emergente na seleção de pacientes que provavelmente se beneficiariam do tratamento com rhGH, sendo eles DGH por uma causa conhecida ou mesmo DGHI. / Short stature (SS) is an important referral cause for evaluation in pediatric endocrinology. Growth hormone deficiency (GHD) needs to be considered when other causes of BE are excluded, but there are limitations in establishing its definitive diagnosis, being the subject of several debates and controversies. Although highly questioned, GH stimulation tests are still considered the standard for the diagnostic confirmation of GHD. The present study aimed to evaluate the sensitivity, specificity and accuracy of the different GH peak cut-off points for diagnosis of GHD, in response to stimulus by insulin tolerance test (ITT) and clonidine test, in addition to identifying the best GH peak level to confirm diagnosis using a Receiver Operating Characteristics (ROC) curve analysis. For this purpose, a retrospective and observational study was carried out. Clinical and laboratory data from 62 patients at the endocrinology department of the Hospital Universitário João de Barros Barreto (HUJBB) were collected. The gold standard considered for performance analysis of cut-off points in both GH stimulation tests was the therapeutic response. Thus, 26 patients who achieved a height increase of at least 0,3 standard-deviation at the end of one year of treatment with recombinant human GH (rhGH) were classified as GHD. The remaining patients who did not obtain this gain formed the group called non-GHD. Both groups had similar mean height (p = 0,8155) and gained height at the end of follow-up, but this gain was higher in the GHD group compared to the non-GHG group (20,5 ± 14,8 cm vs. 9,2 ± 6,7 cm, respectively, p = 0,0064). GHD group had a significantly lower meddle GH peak than the non-GHG group in both tests (p <0,0001). Sensitivity, specificity and accuracy of cut-off points 3, 5, 7 and 10 ng/mL were defined in the TTI and in the clonidine test, and there was no superiority of one test over the other. In addition, the cut-off points found were 7,92 ng/mL and 6,78 ng/mL in the TTI and clonidine test, respectively, based on the construction of the ROC curve, representing the most sensitive and specific GH peak levels for the diagnosis of GHD. We conclude that the cut-off points found in this study may represent an emerging tool in the selection of patients who would probably benefit from treatment with rhGH, both in cases of GHD for a known cause and in cases of IGHD.

L'hormone de croissance (GH) est une hormone paradoxale. Historiquement reconnue comme responsable de la croissance post-natale, elle est actuellement considérée comme une véritable cytokine, synthétisée en de nombreux sites extra-hypophysaires et impliquée, lorsque dérégulée, dans les processus de tumorigénèse. Le travail présenté dans cette thèse a permis de caractériser et localiser par RT-PCR in situ, les cellules capables de synthétiser la GH dans le système immunitaire du fœtus et du rat adulte, puis dans les différents systèmes de prolifération cellulaire du carcinome canalaire mammaire humain montrant ainsi que la GH, par son action autocrine / paracrine est non seulement impliquée dans le développement embryonnaire mais participe à la progression tumorale. Nos travaux in vitro montrent que l'internalisation et la translocation nucléaire de la GH complexée à son récepteur sont indépendantes de l'activation de JAK2 « Janus Kinase 2 », cependant indispensable à son exportation hors du noyau. L'étude du système de régulation négative du signal induit par la GH nous a permis de mettre en évidence une surexpression de la protéine CIS « Cytokine-Inducible SH2-containing protein », dans les zones de prolifération tumorale des différents carcinomes étudiés et dans 8 lignées tumorales mammaires. La surexpression de CIS, in vitro, inhibe la voie de signalisation JAK/STAT « Signal Transducer and Activator of Transcription » et active la voie des MAPK « Mitogen Activated Protein Kinases ». Nous avons pour finir, corrélé l'activation prédominante de CIS à la synthèse de GH « autocrine » dans les cellules tumorales mammaires MCF-hGH. La localisation tant nucléaire que cytoplasmique de la GH et de toutes les molécules informatives laisse entrevoir des mécanismes de régulation encore inconnus. Les travaux futurs tenteront de répondre à la question maintenant cruciale : la GH, hormone de jouvence ou véritable oncogène ?