A interação entre as vias de sinalização do ER, do IGF-I e da integrina 1 é essencial para a manutenção da homeostase da glândula mamária normal, e alterações nessas vias de sinalização estão associadas ao processo de tumorigênese da mama. Portanto, o objetivo deste trabalho foi investigar a influência e inter-relação entre as vias de sinalização do IGF-I, do ER e da integrina 1 na regulação da transcrição dos genes PHLDA1 e PAWR. Para isso, células MCF-7 foram tratadas, por diferentes tempos, com 10nM de 17- estradiol (E2), 12,5nM de IGF-I, 30M de LY294002 (inibidor da PI-3K), 30M de SB202190 (inibidor da p38MAPK) e 1M de ICI182780 (antagonista do ER), ou transfectadas com 40nM de siRNA para integrina 1. A expressão gênica foi avaliada por PCR em tempo real e a expressão protéica por Western Blot. A expressão do gene PHLDA1 aumentou após tratamento com E2 por 6h (p=0,05), e esse efeito foi inibido pelo tratamento com ICI (p<0,05). O tratamento com E2 por 24h inibiu a expressão do gene PAWR (p<0,05), e esse efeito foi dependente de ER, pois foi inibido pelo tratamento com ICI (p<0,05). O tratamento com IGF-I por 1,5h causou aumento na expressão do gene PHLDA1 (p<0,05); e o tratamento com IGF-I por 24h causou diminuição na expressão do gene PAWR (p<0,05). Foi observado aumento na expressão protéica de PHLDA1 após tratamento das MCF-7 com E2 ou IGF-I por 1:30h. A regulação da expressão do PAWR pelo IGF-I ocorreu através das vias da PI-3K e p38MAPK. O efeito do IGF-I sobre a expressão dos dois genes foi independente da ativação do ER, mas foi observado sinergismo entre E2 e IGFI na inibição da expressão dos transcritos do PAWR, com diminuição na expressão para nível menor do que o observado após tratamento com E2 ou IGF-I sozinhos (p<0,05). A repressão da expressão da integrina 1 resultou na diminuição dos níveis de expressão dos genes PHLDA1 e PAWR. Não foi observada interação entre o IGF-I e a integrina 1 na regulação dos genes PHLDA1 e PAWR. Portanto, o gene PHLDA1 é regulado positivamente pelo E2 e pelo IGF-I, mas não existe interação entre as vias. O gene PAWR é regulado negativamente pelo E2 e pelo IGF-I; o efeito do IGF-I é dependente da ativação da PI3-K e da p38MAPK, mas não do ER; e existe sinergismo entre E2 e IGF-I na regulação do PAWR / The interactions among ER, IGF-I and 1 integrin signaling pathways are essential for the maintenance of normal mammary gland homeostasis, and alterations in these pathways have been associated with mammary tumorigenesis. Therefore, the goal of this work was to investigate the influence and interaction among IGF-I, ER and 1 integrin signaling pathways on the regulation of PHLDA1 and PAWR transcription regulation. To accomplish that, MCF-7 cells were treated with 10nM 17-estradiol (E2), 12.5nM IGF-I, 30M LY294002 (a PI3-K inhibitor), 30M SB202190 (a p38MAPK inhibitor) and 1M ICI182,780 (ICI an ER antagonist), or transfected with 40nM siRNA aiming 1 integrin. Real time PCR and western blot were used to evaluate gene and protein expression, respectively. PHLDA1 mRNA expression increased after 6h of treatment with E2 (p=0.05), and this effect was inhibited by ICI (p<0.05). Treatment with E2 for 24h repressed PAWR gene expression (p<0.05) and this effect was dependent on ER, since treatment with ICI inhibited it (p<0.05). Treatment with IGF-I for 1.5h resulted in increased PHLDA1 gene expression (p<0,05) and also PHLDA1 protein expression; and treatment with IGF-I for 24h inhibited PAWR mRNA expression (p<0.05). IGF-I regulation of PAWR expression was dependent on PI3-K and p38MAPK activation. The regulation of both genes by IGF-I was independent of ER activation; however, IGF-I acted in synergism with E2 on PAWR expression resulting in lower PAWR expression than the observed after the treatments alone (p<0.05). Repression of 1 integrin expression resulted in downregulation of PHLDA1 and PAWR expression levels. No interaction was observed between IGF-I and 1 integrin on PHLDA1 and PAWR gene expression. In conclusion, PHLDA1 is positively regulated by E2 and IGF-I, but there is no interaction between them on PHLDA1 regulation. On the other hand, PAWR is negatively regulated by E2 and IGF-I; IGF-I effect is dependent on PI3-K and p38MAPK, but not on ER activation; E2 and IGF-I act synergistically on PAWR gene expression regulation

As a functional unit involved in both maintaining endocrine homeostasis and also producing mature eggs, the ovary plays a central role in female reproduction. The development and function of the ovarian follicles are controlled by gonadotropins released from the pituitary. It is widely accepted that the action of gonadotropins on ovarian follicles is mediated by paracrine/autocrine factors produced by the somatic cells surrounding the oocyte. Increasing evidence indicates that the Igf system is involved in mediating the action of gonadotropins in the ovary. Previously, we identified a gonad-specific Igf subtype (Igf3) distinct from Igf1 and Igf2. This fmding further highlights the importance of the Igf system in the fish ovary. In this thesis, efforts were made to understand the role of the Igf system in ovary using zebrafish as the model organism, and attention was focused on Igf3. / Because the expression of Igf3 is correlated with the LH receptor in zebrafish follicles, the regulation of igf3 by gonadotropins was subsequently studied in the ovary. The expression of igf3 was significantly up-regulated in both ovarian fragments and isolated follicles upon treatment with hCG in dose- and time-dependent manners. Treatment with 8-Br-cAMP or IBMX mimicked the effects of hCG on the expression of igf3 in follicles of different stages. / Four Igfs are present in zebrafish, and our results show that all four igfs are expressed in the ovary of zebrafish and exhibit the differential expression profiles during folliculogenesis. Using a primary culture of zebrafish follicle cells, we demonstrated that hCG stimulated igf2b and igf3 expression but suppressed igf2a expression. Moreover, the effect of gonadotropin could be mimicked by IBMX, which increased the intracellular levels of cAMP, suggesting the possible involvement of cAMP in the gonadotropin-based regulation and differential expression of igf2a, igf2b and igf3. These results also show that the Igf3 is the Igf subtype most sensitive to gonadatropin and cAMP. / In addition, the expression patterns of igf1, igf2a, igf2b, igf3, igf1ra and igf1rb were also studied during zebrafish embryogenesis. The unique temporal and spatial expression patterns of igf1, igf2a, igf2b, igf3, igf1ra and igf1rb were revealed by both real-time PCR and whole mount in situ hybridization, the results suggest divergent functions for these Igfs in early zebrafish development. / Taken together, the present studies provide substantial information about the Igf system, especially that of Igf3 in the zebrafish ovary. Data were gathered regarding Igf3 expression, regulation and functions, which is not only helpful for the understanding of the role of the Igf system in fish reproduction, but also contributes toward uncovering the ovarian signaling network involved in oocyte maturation across vertebrates. This study of igfs gene expression provides direct information to the study of Igf signaling in zebrafish. / To study the function of Igf3, bioactive recombinant Igf3 proteins were prepared using a bacterial expression system. Incubation of follicles with recombinant zebrafish Igf3 significantly enhanced oocyte maturation in time-, dose- and stage-dependent manners. The potential mechanisms of Igf3-induced oocyte maturation were then investigated. Igf3 stimulated oocyte maturation via a steroid-independent manner. Igf3 induced oocyte maturation through Igf1rs and the PI3 kinase, PDE3 and MAP kinase were necessary for Igf3-mediated oocyte maturation in zebrafish. / We first examined the gene expression patterns of Igf3 in the ovary. The igf3 gene in zebrafish was found to be alternatively spliced into two transcripts, with transcript variant 1 exclusively expressed in the gonads and transcript variant 2 only expressed during early development. Using specific antibodies developed for zebrafish Igf3, both the prepropeptide and the mature peptide forms of Igf3 were found to be predominantly expressed in the zebrafish ovary. Real-time PCR and in situ hybridization revealed that igf3 mRNA levels were relatively low in the early follicles but significantly increased after the mid vitellogenic stage (midstage III) and were high in the full grown follicles. In the full grown follicles, igf3 mRNA was detected primarily in the somatic follicular cells, with a low level of expression in the oocytes. Igf3 immunoreactivity was confined to the follicular cells alone. / Li, Jianzhen. / Advisers: Hui Zhao; Hon Ki Christopher Cheng. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 122-150). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Mong, Lok Yee. / Thesis submitted in: December 2007. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 124-145). / Abstracts in English and Chinese; some text in appendix also in Chinese. / Acknowledgements --- p.i / Abstract (English version) --- p.iii / Abstract (Chinese version) --- p.v / Table of Contents --- p.vii / List of Tables --- p.ix / List of Figures --- p.xi / List of Abbreviations --- p.xiii / Chapter Chapter 1 - --- Introduction / Chapter 1.1 --- Childhood obesity: a worldwide epidemic --- p.1 / Chapter 1.2 --- Health consequences of childhood obesity --- p.3 / Chapter 1.3 --- Determinants of childhood obesity --- p.5 / Chapter 1.4 --- Hormonal dysregulation and obesity --- p.9 / Chapter 1.5 --- Project objectives and long term significance --- p.14 / Chapter Chapter 2 - --- Research Plan and Methodology / Chapter 2.1 --- Study cohort / Chapter 2.1.1 --- Subject recruitment --- p.15 / Chapter 2.1.2 --- Ethics --- p.16 / Chapter 2.1.3 --- Measurements and blood sample collections --- p.16 / Chapter 2.1.4 --- Subgroup for dietary assessment --- p.18 / Chapter 2.1.5 --- Cohort re-visits in 2006 --- p.19 / Chapter 2.2 --- Genetic study / Chapter 2.2.1 --- Sample size estimation and research subjects --- p.21 / Chapter 2.2.2 --- DNA samples --- p.22 / Chapter 2.2.3 --- Candidate genes --- p.24 / Chapter 2.2.4 --- SNP tagging and prioritizing --- p.25 / Chapter 2.2.5 --- Genotyping methods & quality control --- p.28 / Chapter 2.2.6 --- Statistical analysis --- p.31 / Chapter 2.3 --- Dietary assessment / Chapter 2.3.1 --- Three-day 24-hour dietary recalls --- p.36 / Chapter 2.3.2 --- Lifestyle questionnaire --- p.37 / Chapter 2.3.3 --- Data management --- p.38 / Chapter 2.3.4 --- Statistical methods --- p.39 / Chapter Chapter 3 - --- Results Page / Chapter 3.1 --- Study cohort --- p.41 / Chapter 3.2 --- Genetic study / Chapter 3.2.1 --- Subjects --- p.41 / Chapter 3.2.2 --- SNPs selection --- p.41 / Chapter 3.2.3 --- Factor analysis of adiposity in the study population --- p.44 / Chapter 3.2.4 --- Genotyping and association testing in stage1 --- p.50 / Chapter 3.2.5 --- Genotyping and association testing in stage2 --- p.52 / Chapter 3.2.6 --- Association of the CART gene with adiposity --- p.55 / Chapter 3.2.7 --- Association of the GHR gene with adiposity --- p.60 / Chapter 3.2.8 --- Association of the GHRHR gene with adiposity --- p.69 / Chapter 3.2.9 --- Association of the IGFBP3 gene with adiposity --- p.75 / Chapter 3.2.10 --- Association of the POMC gene with adiposity --- p.83 / Chapter 3.2 --- Dietary assessment / Chapter 3.3.1 --- Nutrient intakes of the subgroup in2004 --- p.87 / Chapter 3.3.2 --- Nutrient intakes of the subgroup in2006 --- p.92 / Chapter 3.3.3 --- Lifestyle pattern of the cohort in2006 --- p.97 / Chapter Chapter 4 - --- Discussion / Chapter 4.1 --- The role of GH-related genes with adolescent adiposity --- p.102 / Chapter 4.2 --- Nutrient intakes and lifestyle pattern of the adolescents --- p.120 / Chapter 4.3 --- Conclusion of this study --- p.123 / References --- p.124 / Appendices / Chapter A --- Information of the SNPs selected --- p.146 / Chapter B --- Comparison of SNPs minor allele frequency (MAF) among two genotyping stages and HapMap data --- p.154 / Chapter C --- Hardy-Weinberg Equilibrium (HWE) of SNPs in two genotyping stages --- p.162 / Chapter D --- Factor score coefficient matrix --- p.170 / Chapter E --- Association of SNPs with factors scores --- p.172 / Chapter F1 --- Consent form (English version) --- p.207 / Chapter F2 --- Consent form (Chinese version) --- p.209 / Chapter G1 --- 24-hour dietary recall forms (English version) --- p.211 / Chapter G2 --- 24-hour dietary recall forms (Chinese version) --- p.218 / Chapter H --- Food photo booklet --- p.225 / Chapter I1 --- Lifestyle questionnaire (English version) --- p.236 / Chapter I2 --- Lifestyle questionnaire (Chinese version) --- p.238

Diss. (sammanfattning) Linköping : Univ., 2004. / Härtill 6 uppsatser.