Atualmente, a maioria dos endocrinologistas pediátricos usa uma dose fixa de GH, calculada por quilo de peso ou área de superfície corporal, para todos os pacientes. Algumas crianças com DGH, tratadas com as doses atuais, não atingem uma estatura normal e outras não atingem a estatura-alvo geneticamente prevista pela altura dos pais. Além disso, algumas crianças com DGH desenvolvem características acromegalóides após o uso por longo prazo da medicação. A existência de um marcador preciso e eficiente seria útil para a individualização da dose de rGH. Esse marcador deveria ser mensurado em um período de tempo menor que a velocidade de crescimento (VC). Neste estudo usamos as concentrações de IGF-I como esse marcador. Durante 12 meses, acompanhamos trinta crianças portadoras de DGH grave, em tratamento prévio por cerca de quatro anos, divididas em dois grupos de 15, para comparar o tratamento com a dose de rGH baseada no peso versus o tratamento com a dose de rGH ajustada pelas concentrações de IGF-I para mantê-las em uma faixa alvo (entre 0 a +2 DP). Foi possível manter concentrações de IGF-I dentro de valores predeterminados pelo ajuste da dose de rGH em 13 dos 15 pacientes, mas a VC não foi diferente entre os grupos (6,8±2,6 vs. 6,9±2,7 cm/ano; p=NS); porém, quando considerados apenas os pacientes prépúberes que mantiveram concentrações de IGF-I entre 0 e +2DP em pelo menos 75% das dosagens, sua velocidade de crescimento foi maior em comparação com as crianças que mantiveram concentrações de IGF-I abaixo de 0DP em 50% ou mais das dosagens (8,8±1,8 vs. 6,3±2,9 cm/ano; p<0,05). Em paralelo, comparamos as concentrações de IGF-I de diferentes genótipos do exon 3 do receptor de GH (GHR), nessas trinta crianças, durante o tratamento. Duas das mais comuns isoformas em seres humanos são geradas pela retenção (full length GHR - GHRfl) e exclusão do exon 3 (exon 3 deleted GHR - GHRd3). A influência desse polimorfismo na resposta ao tratamento com rGH em pacientes com DGH tem sido alvo de controvérsia. No presente estudo, apesar de receberem a mesma dose de rGH (41±10 vs. 41±8 ug/kg d; p=NS), pacientes carreando ao menos um alelo-d3 GHR, como grupo, tiveram maiores níveis de IGF-I que aqueles homozigotos para o alelo GHR full-length (0,9±0,9 vs. -0,3±1,2 DP; p<0,05) , indicando uma melhor sensibilidade ao GH para o primeiro genótipo. A VC não foi diferente entre os grupos GHRd3 vs. GHRfl (7,3±1,9 vs. 6,4±3,1cm/ano; p=NS). / Currently, most pediatric endocrinologists use a fixed rGH dose calculated according to the weight or body surface area for all patients. Some children with GHD, treated with the present doses, do not achieve normal height, and some, even reaching normal height, do not achieve the genetic target height determined by their parents\' heights. At the same time, some children with GHD develop acromegalic characteristics after long-term treatment with rGH. The existence of a specific and effective marker to individualize rGH dose would be useful to control therapy of children with GHD. This marker ideally should be obtained in a shorter time interval than the growth velocity (GV). In the present study, we measured insulin-like growth factor (IGF-I) as this marker. During one year, we followed 30 children with severe GHD, treated previously with rGH for an average of 4yrs, divided in two groups of 15, to compare weight-based versus IGF-I-based rGH dosing to reach IGF-I levels between a target range (0 and +2 SDS). It was feasible to maintain IGF-I levels in this predetermined range by adjusting the rGH dose in 13 of 15 patients, but growth velocity was not different in groups with weight-based or IGF-I based rGH dose (6.8±2.6 vs. 6.9±2.7cm/y; p=NS); however, in prepubertal children who reached predetermined target IGF-I levels in 75% of the measurements GV was higher than in those who did not (8.8±1.8 vs. 6.3±2.9 cm/y; p<0.05). In parallel, we compared IGF-I levels of different GH receptor (GHR) exon 3 genotypes in these 30 children during treatment. Two of the more common GHR isoforms in humans are generated by retention (full length GHR - GHRfl) and by exclusion of exon 3 (exon 3 deleted GHR - GHRd3). The influence of this polymorphism on the response to rGH treatment in patients with GH deficiency has been controversial. In the present study, despite receiving similar rGH doses (41±10 vs. 41±8 ug/kg d; p=NS), patients carrying at least one GHR d3-allele, as a group, had higher IGF-I levels than those homozygous for the GHR full-length allele (0.9±0.9 vs. -0.3±1.2 SDS; p<0.05), indicating a greater GH sensitivity with the former genotype. GV was not different between groups GHRd3 vs. GHRfl (7.3±1.9 vs. 6.4±3.1cm/y; p=NS).

L'hormone thyroïdienne (T3) contrôle à la fois les processus développementaux et physiologiques. Elle agit via les récepteurs de l'hormone thyroïdienne (TR), membres de la famille des récepteurs hormonaux nucléaires. Ils agissent comme des facteurs de transcription dépendants du ligand. La méthylation de l'ADN en position 5 de la cytosine est une modification épigénétique importante qui affecte la structure de la chromatine et l'expression des gènes. Des études récentes ont établi un rôle important des protéines de la famille TET (Ten-eleven translocation) dans la régulation de la dynamique de la méthylation de l'ADN. Elles convertissent la 5-méthyl-cytosine (5mC) en 5-hydroxyméthylcytosine (5hmC). D’autres études ont démontré que les protéines TET (TET1, TET2 et TET3) possèdent des fonctions de régulation transcriptionnelle dépendantes et indépendantes de leur activité catalytique. Notre étude a identifié TET3 comme une nouvelle protéine interagissant avec TR. Le domaine AF2 de TR ainsi que le domaine catalytique et le domaine CXXC de TET3 sont responsables de cette interaction. Celle-ci permet la stabilisation de TR lié à la chromatine, entraînant une potentialisation de son activité transcriptionnelle. L'effet de modulation de TET3 sur TR présenté ici est indépendant de son activité hydroxylase de TET3. Ainsi, cette étude met en évidence un nouveau mode d'action de TET3 en tant que régulateur non classique de TR, modulant sa stabilité et son accès à la chromatine plutôt que son activité de transcription intrinsèque. Des mutations du gène codant pour TRα provoquent le symptôme RTHα dont la gravité varie en fonction de la mutation. Les différentes capacités d’interaction des mutants TRα, pertinents pour la maladie de RTHα humaine, avec TET3 pourraient expliquer les différences d’effet dominant négatif. La fonction de régulation de TET3 pourrait s’appliquer plus généralement aux facteurs de transcription des récepteurs nucléaires, car différents membres de la superfamille des récepteurs nucléaires présentent la même interaction avec TET3, tels que AR (récepteur des androgènes), ERR (récepteur des œstrogènes) et RAR (récepteur de l'acide rétinoïque). L'interaction entre TET3 et RAR implique le domaine de liaison ADN de RAR. La pertinence fonctionnelle de l'interaction TET3 / RAR a été étudiée plus en détail dans les cellules souches embryonnaire (cellules ES). L’absence combinée des trois TET a entraîné la diminution de 5hmC et la dérégulation des gènes impliqués dans la différenciation des cellules ES. Parmi les gènes dérégulés, nous avons identifié un sous-ensemble de gènes cibles de l’acide rétinoïque, suggérant que les RAR (récepteurs d'acide rétinoïque) et les TET pourraient travailler ensemble pour réguler la différenciation des cellules ES. Une étude supplémentaire a révélé que les protéines TET peuvent jouer un rôle dans la facilitation du recrutement de RAR aux régions promotrices de ses gènes cibles. En outre, nos résultats montrent un rôle potentiel de l'activité hydroxylase des protéines TET dans la modulation de l'activité transcriptionnelle des RAR. En conclusion, notre travail a identifié les protéines TET comme nouveaux régulateurs des récepteurs nucléaires. Les mécanismes exacts impliqués doivent être étudiés plus avant. / Thyroid hormone (T3) controls both developmental and physiological processes. Its nuclear receptors, thyroid hormone receptors (TRs), are members of the nuclear hormone receptor family which act as ligand-dependent transcription factors. DNA methylation at the fifth position of cytosine is an important epigenetic modification that affects chromatin structure and gene expression. Recent studies have established a critical function of the Ten-eleven translocation (TET) family proteins in regulating DNA methylation dynamics by converting 5-methyl-cytosine (5mC) into 5-hydroxymethylcytosine (5hmC). Studies demonstrated that TETs proteins (including TET1, TET2 and TET3) possess catalytic activity dependent and independent transcriptional regulatory functions. Our study identified TET3 as a new TR interacting protein. The AF2 domain of TR and the catalytic domain and CXXC domain of TET3 are responsible for their interaction. This interaction allows the stabilization of chromatin bound TR, resulting in a potentiation of its transcriptional activity. The modulation effect of TET3 on TR presented here is independent of its hydroxylase activity. Thus this study evidences a new mode of action for TET3 as a non-classical regulator of TR, modulating its stability and access to chromatin rather that its intrinsic transcriptional activity. Mutations in TR cause the RTH symptom which severity varies with the particular mutation. The differential ability of different TRα mutants, relevant for the human RTHα disease, to interact with TET3 might explain their differential dominant negative activity. The regulatory function of TET3 might be more general towards the nuclear receptor transcriptional factors since different members of the superfamily present the same interaction with TET3, such as AR (androgen receptor), ERR (Estrogen-related receptor) and RAR (retinoic acid receptor). The interaction between TET3 and RAR involves the DNA binding domain of RAR. The functional relevance of TET3/RAR interaction was further studied in ES cells. Combined deficiency of all three TETs led to depletion of 5hmC and deregulation of genes involved in ES differentiation. Among the deregulated genes, a subset of RA response genes was identified, suggesting that RARs (retinoic acid receptors) and TETs might work together to regulate ES cell differentiation. Further dissection revealed that TET proteins may have a role in facilitating RAR recruitment to the promoter regions of these RAR target genes. Moreover, our results indicated a potential role of the hydroxylase activity of TET proteins in modulating RAR transcriptional activity. Altogether, our work identified TET proteins as new regulators of NR (Nuclear Receptors). The exact mechanisms involved need to be further studied.

The duplication of genes followed by selection is perhaps the most prominent way in which molecular biological systems gain multiplicity, diversity and functional complexity in evolution. Whole genome duplications (WGDs) therefore have the potential of generating an extraordinary amount of evolutionary innovation. It is now accepted that the vertebrate lineage has gone through two rounds of WGD in its early stages, after the divergence of invertebrate chordates and before the emergence of jawed vertebrates. These basal vertebrate WGDs are called 2R for two rounds of whole genome duplication. An additional WGD called 3R occurred early in the evolution of teleost fishes, before the radiation of this species-rich group. This thesis describes the evolution of several endocrine and neuronal gene families in relation to the vertebrate WGDs, through a comparative genomic approach including both phylogenetic analyses and chromosomal location data across a wide range of vertebrate taxa. These results show that numerous endocrine gene families have expanded in 2R and in several cases also in 3R. These include the gene families of oxytocin and vasopressin receptors (OT/VP-R), somatostatin receptors (SSTR) and insulin-like growth factor binding proteins (IGFBP). For the OT/VP-R and SSTR families, previously undescribed subtypes were identified. The protein hormone family that includes growth hormone (GH), prolactin (PRL) and somatolactin (SL) acquired a new PRL gene in 2R, however the origins of GH, PRL and SL likely predate 2R. The corresponding family of receptors diversified during different time periods through a combination of local duplications and 3R. Neuronal gene families of the visual system have also expanded in 2R and 3R. The results presented here demonstrate that the vertebrate repertoire of visual opsin genes arose in 2R as part of chromosomal blocks that also include the OT/VP-R genes. The gene families including the transducin alpha, beta and gamma subunits also arose in 2R, hinting at the importance of these events in the diversification and specialization of phototransduction cascades for rods and cones. Thus, the whole genome duplications have been important contributors to the evolution of both vision and endocrine regulation in the vertebrates.

Dans les cellules somatotropes, la liaison du facteur de libération de l’hormone de croissance (GHRH) à son récepteur (GHRH-R) stimule la synthèse et la sécrétion de l’hormone de croissance (GH) ainsi que la prolifération cellulaire. Chez les mammifères, le vieillissement est caractérisé par une diminution de la sécrétion de GH, liée à une perte de sensibilité des somatotropes au GHRH. Chez le rat âgé, des modifications de niveaux d'ARNm du GHRH-R et une diminution d'affinité et de capacité de liaison du GHRH sont rapportés. Au cours du vieillissement, une augmentation des niveaux de glucose et d’acides gras libres sérique suggère qu’une gluco- ou lipotoxicité puisse contribuer au dysfonctionnement de la fonction somatotrope. À ce jour, la restriction calorique modérée de longue durée (RCMLD) constitue l’intervention la plus efficace pour prévenir ou retarder les détériorations liées à l’âge. Des études ont montré des effets bénéfiques de la RCMLD sur l’axe somatotrope au cours du vieillissement via un maintien des paramètres de liaison du GHRH-R. Compte tenu de l’importance de cet axe, la compréhension des mécanismes menant à la somatopause ainsi que ceux associés aux effets bénéfiques de la RCMLD s’avère importante. Les objectifs principaux de la présente thèse étaient : 1) de déterminer les effets de la RCMLD chez le rat, sur le GHRH-R hypophysaire et la sensibilité des somatotropes au GHRH, 2) d’identifier les mécanismes associés à la somatopause et aux effets bénéfiques de la RCMLD, et 3) de préciser les effets d’une gluco-ou lipotoxicité sur l’axe somatotrope de rats et leur implication dans la somatopause. Des rats de 8 mois ont été soumis à une restriction calorique de 40% jusqu’à l’âge de 18-20 mois et ont été comparés à des rats jeunes et âgés nourris ad libitum. Cette étude a permis de mettre en évidence des effets bénéfiques de la RCMLD sur la régulation et la fonctionnalité du GHRH-R et de proposer que le glucose et les acides gras libres (AGL) circulants soient impliqués dans le vieillissement de la somatotrope. Une étude de micro-puce à ADN à permis d’identifier des gènes associés à des mécanismes de protection et de réparation des dommages cellulaires mis en place dans l’hypophyse antérieure au cours du vieillissement et par la RCMLD. Finalement, les effets d’un stress gluco- ou lipotoxique sur la fonction somatotrope ont été étudiés chez des rats de 2 et 6 mois, infusés 72 h avec une solution de glucose ou d’Intralipides, mimant les niveaux circulants de glucose et d’AGL retrouvés chez le rat âgé. Les résultats obtenus montrent que la glucotoxicité affecte la régulation de certains gènes de la somatotrope, dont le GHRH-R, et suggèrent que la capacité de réponse à ce type de stress est altérée. Les mécanismes par lesquels la glucotoxicité exerce ces effets pourraient inclure la génération de stress oxydant. L’ensemble de ces résultats proposent de nouvelles pistes mécanistiques qui pourraient contribuer au retardement de la somatopause et, ultimement, à l’élaboration de nouvelles stratégies d’intervention nutritionnelles ou pharmacologiques ciblant les mêmes voies que la RCMLD, avec une efficacité similaire ou supérieure. / In somatotroph cells, growth hormone-releasing hormone (GHRH) binding to its receptor (GHRH-R) stimulates growth hormone (GH) secretion and cell proliferation. In mammals, aging is characterized by a decrease of GH, associated with a decline of GHRH somatotroph sensitivity. In the aged rat, changes in GHRH-R mRNA levels and decrease of GHRH affinity and capacity were reported. In the course of aging, significant increase of glucose and free fatty acid (FFA) serum levels are observed, suggesting that gluco- or lipotoxicity could contribute to somatotroph dysfunction. Up to now, long-term moderate caloric restriction (LTMCR) has been the most efficient intervention to prevent or delay age-related deteriorations. Studies have shown beneficial effects of LTMCR on somatotroph axis during aging, through the maintenance of GHRH-R binding parameters. Knowing the importance of this axis, an understanding of the mechanisms associated with the effects of somatopause and benefits of LTMCR is important. Therefore, the main objectives of this thesis were: 1) to determine the effects of LTMCR on rat pituitary GHRH-R and somatotroph sensitivity to GHRH, 2) to identify the mechanisms associated to somatopause and the beneficial effects of LTMCR and 3) to specify the effect of gluco- or lipotoxicity on the rat somatotroph axis and their implications in somatopause. Eight-month-old rats were submitted to a 40% LTMCR until the age of 18 to 20-months and where compared to young and old rats fed ad libitum (AL). This study highlighted beneficial effects of LTMCR on GHRH-R regulation and functionality and suggested that high circulating levels of glucose and FFA could be involved in somatotroph aging. A microarray study was also performed, allowing the identification of genes associated to cellular protection and damage repair mechanisms regulated by aging and LTMCR in the anterior pituitary. Finally, effects of a gluco- or lipotoxic stress on the somatotroph function was assessed in 2- and 6-month-old rats submitted to a 72-h glucose and/or Intralipid infusion, to mimic the levels of glucose and FFA found in aged rats. The results showed that glucotoxicity affects the regulation of specific genes in the somatotroph, such as the GHRH-R gene, and suggest that the response capacity against this type of stress is altered with age very early on. Mechanisms by which glucotoxicity exerts these effects might include oxidative stress production. Altogether, these results proposed novel mechanisms that could contribute to delay somatopause and, ultimately, to the development of new nutritional or pharmacologic interventions targeting the same pathways as LTMCR, with a similar or greater efficiency.

No description available.

This study was aimed on analysis of polymorphism at position 2141 in exon 5 of the growth hormone gene (GH) and polymorphism at position 257 in exon 10 of the growth hormone receptor gene (GHR) and to determine the influence of these polymorphisms on meat tenderness. To analysis has been used 333 meat samples of Czech Pied bulls. Genotypization of GH and GHR loci was performed by PCR-RFLP. For detection of single nucleotide polymorphism in both genes was used restriction endonuclease AluI. To determine meat tenderness was used the method of measuring shear force by Warner and Bratzler. The shear force values of raw meat samples were measured at day 14 post mortem. Then was statistically evaluated the association between genotypes and shear force. In the study population occurred for GH locus 166 individuals with genotype LL, 161 heterozygotes LV and 6 homozygotes VV. The relative genotype frequencies were thus 0.499 (LL), 0.483 (LV) and 0.018 (VV). The frequency of L allele was 0,74 and of V allele was 0,26. For GHR locus was found in the study population 178 homozygotes AA, 105 heterozygotes AG and 50 homozygotes GG. The relative frequencies of genotypes were 0.535 (AA), 0.315 (AG) and 0.15 (GG). The frequency of A allele was 0,692 and of G allele was 0,308. Using statistical analysis revealed a significant effect of genotype GH gene on shear force, tenderness resp. (P<0,05). For GHR locus showed no effect of genotype on shear force (P>0,05).

Atualmente, a maioria dos endocrinologistas pediátricos usa uma dose fixa de GH, calculada por quilo de peso ou área de superfície corporal, para todos os pacientes. Algumas crianças com DGH, tratadas com as doses atuais, não atingem uma estatura normal e outras não atingem a estatura-alvo geneticamente prevista pela altura dos pais. Além disso, algumas crianças com DGH desenvolvem características acromegalóides após o uso por longo prazo da medicação. A existência de um marcador preciso e eficiente seria útil para a individualização da dose de rGH. Esse marcador deveria ser mensurado em um período de tempo menor que a velocidade de crescimento (VC). Neste estudo usamos as concentrações de IGF-I como esse marcador. Durante 12 meses, acompanhamos trinta crianças portadoras de DGH grave, em tratamento prévio por cerca de quatro anos, divididas em dois grupos de 15, para comparar o tratamento com a dose de rGH baseada no peso versus o tratamento com a dose de rGH ajustada pelas concentrações de IGF-I para mantê-las em uma faixa alvo (entre 0 a +2 DP). Foi possível manter concentrações de IGF-I dentro de valores predeterminados pelo ajuste da dose de rGH em 13 dos 15 pacientes, mas a VC não foi diferente entre os grupos (6,8±2,6 vs. 6,9±2,7 cm/ano; p=NS); porém, quando considerados apenas os pacientes prépúberes que mantiveram concentrações de IGF-I entre 0 e +2DP em pelo menos 75% das dosagens, sua velocidade de crescimento foi maior em comparação com as crianças que mantiveram concentrações de IGF-I abaixo de 0DP em 50% ou mais das dosagens (8,8±1,8 vs. 6,3±2,9 cm/ano; p<0,05). Em paralelo, comparamos as concentrações de IGF-I de diferentes genótipos do exon 3 do receptor de GH (GHR), nessas trinta crianças, durante o tratamento. Duas das mais comuns isoformas em seres humanos são geradas pela retenção (full length GHR - GHRfl) e exclusão do exon 3 (exon 3 deleted GHR - GHRd3). A influência desse polimorfismo na resposta ao tratamento com rGH em pacientes com DGH tem sido alvo de controvérsia. No presente estudo, apesar de receberem a mesma dose de rGH (41±10 vs. 41±8 ug/kg d; p=NS), pacientes carreando ao menos um alelo-d3 GHR, como grupo, tiveram maiores níveis de IGF-I que aqueles homozigotos para o alelo GHR full-length (0,9±0,9 vs. -0,3±1,2 DP; p<0,05) , indicando uma melhor sensibilidade ao GH para o primeiro genótipo. A VC não foi diferente entre os grupos GHRd3 vs. GHRfl (7,3±1,9 vs. 6,4±3,1cm/ano; p=NS). / Currently, most pediatric endocrinologists use a fixed rGH dose calculated according to the weight or body surface area for all patients. Some children with GHD, treated with the present doses, do not achieve normal height, and some, even reaching normal height, do not achieve the genetic target height determined by their parents\' heights. At the same time, some children with GHD develop acromegalic characteristics after long-term treatment with rGH. The existence of a specific and effective marker to individualize rGH dose would be useful to control therapy of children with GHD. This marker ideally should be obtained in a shorter time interval than the growth velocity (GV). In the present study, we measured insulin-like growth factor (IGF-I) as this marker. During one year, we followed 30 children with severe GHD, treated previously with rGH for an average of 4yrs, divided in two groups of 15, to compare weight-based versus IGF-I-based rGH dosing to reach IGF-I levels between a target range (0 and +2 SDS). It was feasible to maintain IGF-I levels in this predetermined range by adjusting the rGH dose in 13 of 15 patients, but growth velocity was not different in groups with weight-based or IGF-I based rGH dose (6.8±2.6 vs. 6.9±2.7cm/y; p=NS); however, in prepubertal children who reached predetermined target IGF-I levels in 75% of the measurements GV was higher than in those who did not (8.8±1.8 vs. 6.3±2.9 cm/y; p<0.05). In parallel, we compared IGF-I levels of different GH receptor (GHR) exon 3 genotypes in these 30 children during treatment. Two of the more common GHR isoforms in humans are generated by retention (full length GHR - GHRfl) and by exclusion of exon 3 (exon 3 deleted GHR - GHRd3). The influence of this polymorphism on the response to rGH treatment in patients with GH deficiency has been controversial. In the present study, despite receiving similar rGH doses (41±10 vs. 41±8 ug/kg d; p=NS), patients carrying at least one GHR d3-allele, as a group, had higher IGF-I levels than those homozygous for the GHR full-length allele (0.9±0.9 vs. -0.3±1.2 SDS; p<0.05), indicating a greater GH sensitivity with the former genotype. GV was not different between groups GHRd3 vs. GHRfl (7.3±1.9 vs. 6.4±3.1cm/y; p=NS).

A plethora of naturally-produced steroid hormones, or artificial homologues of them, are being introduced into the aquatic and terrestrial environments each year. Two examples of these are the natural oestrogen 17-oestradiol (E2) and the oestrogen receptor antagonist, Bisphenol A (BPA), both of which target the ribonucleoprotein telomerase through upregulation of its telomerase reverse transcriptase component, TERT. The main objectives of this study were firstly to isolate and characterize the actual mRNA sequence for the telomerase catalytic subuninit, Tert, in rainbow trout (Oncorhynchus mykiss) (Walbaum, 1792) and European purple sea urchin (Paracentrotus lividus) (Lamarck, 1816), with the aim of developing qPCR assays for the amplification and quantification of Tert. Further objectives were to use these assays in controlled exposure studies to establish whether and to what extent the aforementioned chemicals regulate Tert transcription and by doing so further understand the mechanism of Telomerase gene expression and the extent to which environmental oestrogen can interfere. The initial step of sequence characterization and assay devlopment was successful in the case of rainbow trout where two possible splice variants of Tert mRNA are identified, omTertShort and omTertLong. Two qPCR assays were developed for the relative quantification of both of these splice variants in rainbow trout samples, the latter of these successfully amplifying its target in test samples. In order to demonstrate in vitro and in vivo modulation of telomerase activity and mRNA expression, early life-stages of rainbow trout and purple sea urchin, as well as rainbow trout hepatocytes, were exposed to a range of concentrations of E2 and BPA. Purple sea urchin embryos were exposed to 200, 20 and 2 ng E2/ml for 28 hours until they had reached the stage of pluteus larvaes. Rainbow trout embryos were exposed to 500, 20 and 0.1 ng E2/ml and 600 and 150 ng BPA/ml for 167 days from immediately after fertilization. Rainbow trout hepatocytes were exposed to 20 and 2 ng E2/ml for 48 hours. The results from this study show that telomerase activity as well as TERT mRNA expression can be significantly modulated by exposure to oestrogens and other oestrogenic chemicals. E2 concentrations as low as 20 ng/ml lead to an increase in telomerase activity early-life stages of purple sea urchin and upregulation in the transcription of Tert mRNA in unhatched rainbow trout embryos. BPA induced similar response (600 ng/ml) in hatched rainbow trout alevins larvae. Very high exposures to E2 (500 ng/ml) do however lead to downregulation of Tert mRNA in hatched alevins larvae. Differential regulatory response can be observed between different tissue types of 167 day old fry, with an upregulatory response observed at 0.1 ng E2/ml in liver and muscle tissues, but not in brain. Similarly, brain tissues were observed expressing significantly less mRNA than liver and muscle samples when exposed to BPA (150 ng/ml). It is evident that the previously observed link between environmental oestrogens and telomerase is also present in the two test species examined; purple sea urchin and rainbow trout.

Dispatched 3 (Disp3), a thyroid hormone-regulated gene, is studied extensively in our laboratory. Phenotype of cells with overexpressed Disp3 and its expression pattern make it a perfect candidate for a molecular link between thyroid hormone action and cholesterol homeostasis in the brain. Moreover, we hypothesize that it might play a role in certain neurodegenerative disorders and brain tumours. This thesis is aimed at the process of regulation of this gene via thyroid hormone receptor (TR), specifically identification of responsive elements of the thyroid hormone receptor that are necessary for the regulation. Also, we searched for elements recognized by liver X receptor (LXR), as LXR binds to the same arrangement of repeats as TR and there are a number of genes regulated by both of them. We combined in silico analysis of the Disp3 locus with reporter luciferase assays. A cluster of six elements identified around the first exon with two of them being conserved among human and mice draw our attention. In order to analyze this sequence in more detail, reporter vectors of various truncations of 3 kb region around exon 1 were constructed and tested in reporter assays. Reporter assays did not reveal any substantial element activated by TR or LXR; on the other hand, region containing repressor element(s)...

No description available.