Master of Science / Department of Animal Sciences and Industry / Timothy G. Rozell / Several alternatively-spliced mRNA transcripts of the follicle stimulating hormone receptor (FSHR) have been identified in sheep, including FSHR-1 (G protein-coupled form), FSHR-2 (dominant negative form), and FSHR-3 (growth factor type-1 form). Coupling of the FSHR to signaling pathways which activate different downstream effectors leads to speculation that specific splice variants may be transcribed under differing physiological conditions. This is the first study to correlate expression patterns of FSHR-1, FSHR-2, and FSHR-3 and development of follicles in the mature sheep ovary. In Experiment 1, 8 Suffolk-cross ewes were allowed to come into estrus naturally and were euthanized 24 (n=3), 36 (n=3), and 48 (n=2) hours after the onset of estrus. In Experiment 2, 7 Suffolk-cross ewes received CIDRs for 14 days. At CIDR removal, PMSG (500IU) was administered to treatment ewes (n=3), while controls (n=4) received no PMSG. Ewes were euthanized 24 (n=4; 2 CIDR only, 2 PMSG) or 36 (n=3; 2 CIDR only, 1 PMSG) hours later. All visible follicles were aspirated and pooled according to follicular diameter: small (≤ 2.0 mm), medium (2.1-4.0 mm), large (4.1-6.0 mm), and preovulatory (≥ 6.1 mm). Granulosa cells were separated from follicular fluid by centrifugation. Total RNA was extracted from granulosa cells (GC) and reversed transcribed. The resulting cDNA was subjected to qPCR, using primer sets designed to amplify each variant specifically. For Experiment 1, regardless of time after onset of estrus, relative expression of FSHR-3 exceeded that of both FSHR-1 and FSHR-2 in medium follicles (p < 0.01), and tended to be higher in small follicles (p=0.09). For Experiment 2, treatment with PMSG did not significantly alter expression patterns of FSHR variants (p=0.18). The FSHR-3 was expressed higher than FSHR-2 in all follicle sizes (p < 0.01) and was numerically more highly expressed than FSHR-1, although this difference was not significant (p > 0.11). These experiments show that in addition to the well characterized G protein-coupled form of the FSHR, alternatively spliced variants of the FSHR may participate in follicular dynamics during the first follicular wave of the sheep estrous cycle. Furthermore, these results would indicate that an “alternatively” spliced form of the FSHR (FSHR-3) is the predominant form of the FSHR in the sheep.

Age-related declines of androgens and libido in males have been observed for decades. This dissertation sought to elucidate the mechanisms by which hormones may act differentially upon their receptors in the hypothalamus of aging compared to young males. I also examined how sexual experience modulates the ability of hormones to facilitate sexual behavior with aging. Experiment one measured androgen receptors (AR) and estrogen receptor α (ERα) cells in male rats at young, middle-aged and old age. I found that AR cell numbers in hypothalamic regions studied underwent significant age-related increases. Numbers of heavily ERα labeled cells, but not total ERα cells, increased with age. This study demonstrates that the aging brain has the capacity to synthesize hormone receptors which is increased possibly due to decreased testosterone concentrations. Experiment two examined the effect of sexual experience on serum hormones and cells of AR and ERα in hypothalamic regions in young and middle-aged males. The results showed that AR cell numbers increased with aging but did not change with experience. No age- or experience-related alteration in ERα expression occurred. However, serum testosterone increased and estradiol decreased with age. Experience increased total and free testosterone. Interactions of age and experience on total testosterone, estradiol, and luteinizing hormone were found. These results show long-lasting effects of sexual experience on hormones, but not on their receptors in the hypothalamus. Experiment three investigated effects of exogenous testosterone on sexual behavior in young and middle-aged males. The results showed a decline in sexual behavior parameters with age. After castration with testosterone treatment, there were few differences in sexual behavior measures between young and middle-aged males. AR cell numbers were higher and ERα cell numbers lower in testosterone compared to vehicle-treated males of both ages, and few effects of age occurred. These findings indicate that testosterone and aging interact in a complex manner to control numbers of cells expressing hormone receptors in the brain and on the subsequent control of sexual behavior. This insight provides a better understanding of the relationship between molecular changes in the brain and behavior, and suggests new therapeutic targets to human testosterone treatment. / text

This diploma thesis deals with the analysis of structural and dynamic organization of thyrotropin releasing hormone receptor (TRH-R) and δ-opioid receptor (DOR) within plasma membrane (PM) in relation to the specific sub-compartments of PM denominated as domains or membrane rafts. Modern fluorescence microscopy techniques FLIM, FRAP and RICS were used for this purpose. The experiments were performed on the live cells derived from HEK293 cell line. To reach the main goal of this work, the integrity of PM structure was altered by depletion of cholesterol which was performed by incubation of cells with β cyclodextrin. Results clearly support our previously suggested idea that the vast majority of TRH-R is localized in non-raft regions of plasma membrane. This work also compared different modes of performance of FRAP and results obtained by FRAP and RICS because these methods are to some extent analogous. This is one of the first works that used the RICS approach to characterize the G protein-coupled receptors. In the second part of this work, the setup of transient transfection of the HEK293 cells with DOR-ECFP and DOR EYFP constructs was established. Simultaneously, the functionality of these constructs, i.e. the ability of DOR to activate the cognate G protein was determined. Powered by TCPDF (www.tcpdf.org)

Ratas adultas foram tratadas com 2.5, 5, 10, 20 e 40x a dose fisiológica de triiodotironina (T3) por 10 semanas. Houve redução, dose-dependente, da massa óssea, volume e espessura trabecular (Tb.Th), e espessura de osso cortical. Até a dose de 10xT3, houve aumento na taxa de formação óssea (BFR). Doses maiores de T3 reduziram a BFR, e a reabsorção óssea. Esses dados mostram que, até um certo grau de tireotoxicose, a osteopenia é resultado de aumento da formação e reabsorção ósseas, com predomínio da última. Em graus mais elevados, a osteopenia é causada por redução no remodelamento ósseo, com predomínio da atividade reabsortiva. Em seguida, comparamos os efeitos do T3 e GC-1, um tireomimético seletivo pelo receptor b de T3 (TRb), no osso de camundongos fêmeas adultas. Na tíbia, o T3 reduziu a resistência, rigidez e resiliência, enquanto o GC-1 aumentou esses parâmetros e a Tb.Th, o que mostra que o GC-1 melhora a qualidade óssea. Considerando-se a seletividade do GC-1 pelo TRb, esses achados sugerem que o TRb medeia predominantemente ações positivas do T3 no osso. / Adult rats were treated with 2.5, 5, 10, 20 and 40x the physiological dose of triiodothyronine (T3) for 10 weeks. There was a dose-dependent reduction in bone mass, trabecular volume and thickness (Tb.Th), and cortical thickness. Up to 10xT3, there was an increase in the bone formation rate (BFR). Higher doses of T3 decreased BFR and bone resorption. These data show that, up to a certain degree of thyrotoxicosis, osteopenia is the result of an increase in bone formation and resorption, with a prevalence of resorption. In more severe thyrotoxicosis, the osteopenia is caused by a reduction in bone remodeling, with a predominance of bone resorption. Then, we compared the effects of T3 and GC-1, a thyromimetic that is selective for T3 receptor b (TRb), on bone of adult female mice. T3 treatment decreased tibial resistance, stiffness and resilience, while GC-1 increase these parameters and Tb.Th, showing that GC-1 improves bone quality. Considering the selectiveness of GC-1 for TRb, these findings suggest that TRb mediates mainly positive actions of T3 in the bone.

Le pronostic de certains cancers s’est considérablement amélioré avec l’arrivée sur le marché des anticorps thérapeutiques. Devant l’essor de ces nouveaux médicaments associé à l’identification de nouveaux biomarqueurs, de nouvelles perspectives émergent pour l’imagerie moléculaire in vivo. En effet, disposer de nouveaux traceurs moléculaires spécifiques de ces biomarqueurs permettra de caractériser l’hétérogénéité des cellules cancéreuses, de suivre l’expression de ces marqueurs au cours de l’évolution de la tumeur, mais également de suivre l’efficacité d’un traitement sur la régression tumorale du patient. Pour répondre à cette évolution de diagnostic moléculaire in vivo, il convient de développer de nouvelles sondes moléculaires. L'objectif de ma thèse répond à ce nouveau besoin avec l'ingénierie et le marquage d'un format d'anticorps recombinant adapté à l'imagerie in vivo : le diabody 12G4 dirigé contre le récepteur de l’hormone antimüllérienne (AMH), marqueur de certains cancers de la sphère génitale. / Prognosis of cancers dramatically improved with the development on the market of therapeutic antibodies. With the increase of these new biodrugs, associated with the identification of new biomarkers, new opportunities emerge for the in vivo molecular imaging.. Indeed, to use new molecular tracers specific of tumoral biomarkers will allow to study and characterize the cancer heterogeneity, to monitor the expression of these markers during the tumor evolution, but also to check the treatment effectiveness on patients’ tumoral regression. To answer this evolution of in vivo molecular diagnosis, it is favorable to develop new molecular probes. The aim of this thesis answers this new need with the engineering and labeling of a new recombinant antibody format suitable to in vivo imaging : the 12G4 diabody directed against the type II human receptor for the anti-Müllerian hormone, a biomarker of some cancers of the genital area.

The glycoprotein hormone family comprising of Luteinizing Hormone (LH), Chorionic Gonadotropin (hCG), Follicle Stimulating Hormone (FSH) and Thyroid Stimulating Hormone (TSH) plays important role in reproduction and overall physiology of the organism. These hormones are heterodimeric molecules consisting of an identical α subunit non-covalently associated with the hormone-specific β subunit. Both subunits of all these hormones are N-glycosylated. In addition, hCGβ subunit also has four O-linked oligosaccharides located at the C-terminus of the polypeptide(1). The α and β subunits of all these hormones contain five and six disulfide bonds respectively and the crystal structures of hCG and hFSH indicate that both subunits of the hormones belong to the cystine knot family of proteins(2-4). Although the β subunits are hormone specific, there are distinct similarities in these subunits with the 12 cysteines conserved in all these subunits (1). These hormones, because of their unique structural features have proved to be important models for structure–function relationship studies of complex dimeric glycoproteins. Folding of subunits during biosynthesis, role of glycosylation in folding pathways and in vitro and in vivo bioactivity of the hormone, as well as, identification of domains important for subunit association, receptor binding and subsequent signal transduction have been topics of active investigations. The receptors of these hormones belong to the family of G-protein coupled receptors (GPCR) and have unique hormone specific exodomain not present in other members of the GPCR family and characteristic seven transmembrane domains followed by a C terminal domain(5). Primary structure analysis of Glycoprotein hormone receptors family revealed sequence conservation, maximum homology being observed in the transmembrane domain (TMD)(6). Significant homologies could be observed in the hormone specific extracellular domains (ECD) also (7). Despite these homologies, the receptors exhibit exquisite specificity with very low cross reactivity with other members of the hormone family (8). Elucidation of the molecular details of the contacts between the hormone and the receptors has not been achieved so far. Various approaches have been employed to delineate the residues or domains of both hormone and receptors involved in interaction. These include testing of chimeras or mutants of hormones or receptors for changes in activity (9-12), chemical modifications(13) and competition with peptides from either hormones (14) or receptors (15). Polyclonal and monoclonal antibodies against glycoprotein hormones and various fragments of their receptors have been used to determine the role of different domains of both in binding and response (6, 16, 17). However, till date there is no consensus on the specific mechanisms by which the glycoprotein hormone docks onto its receptor. It was proposed that the initial contact between the hormone and the receptor occurs through high affinity binding of the hormone specific β subunit to the Leucine rich regions of the ECD that results in conformational changes in both hormone, as well as, the receptor and brings hormone/ECD complex closer to the TMD of the receptor. The secondary, relatively lower affinity interactions between the hormone and the receptor then take place through common α subunit and exoloops of TMD of the receptor resulting in signal generation (18, 19). Recently a different kind of model has been proposed which suggests that the hormone does not make any direct contacts with the TMD of the receptor. The signal is transduced by the change in contacts between ECD and TMD brought about by hormone’s interaction with ECD(8, 20). The present study was initiated with an overall objective of understanding the molecular details of the hormone receptor interactions of this family, particularly hCG- LH receptor interactions. Two different approaches were employed for this purpose; the first, direct approach being structure elucidation of the members of the glycoprotein hormone family while the second approach uses antibodies against hCG as tools to probe into hormone-receptor interactions. The results obtained using these two approaches have been consolidated in the present thesis and are organized as follows. Chapter 1 is an extensive review of the literature and it builds background for the present work while the exact aim and scope of the present work have been defined in Chapter 2. Chapter 3 describes cloning, expression and purification of recombinant glycoprotein hormones hLH, hCG and single chain derivative of hCG. The Chapter 4 gives details of the molecular aspects of hCG-LH receptor interaction dissected using hCG monoclonal antibodies (MAbs). Chapter 5 discusses implications of the observations made in the present study and states the future directions envisaged. There are a number of endocrinopathies associated with abnormal levels of glycoprotein hormones and treatments of such disorders often demand large quantities of either agonists or antagonists of the hormones. The structure-function relationship studies should help in identifying domains/residues important for subunit interaction, receptor binding, and signal transduction, which would also allow engineering of agonists and antagonists of hormone action. However, structure determination of the glycoprotein hormone family using X-ray crystallography has proved to be a difficult task and it is believed that the heterogeneity in glycosylation is the primary reason for this low success rate in the process of crystallization. The first crystal structure of hCG was that of completely deglycosylated hCG but such a molecule displays antagonistic behavior(2, 3). Use of NMR spectroscopy, the alternate method commonly used for structure determination is often limited by the availability of large quantities of biologically active hormones free of any contaminants. Large quantities of LH, hCG and FSH are also required for treatment of infertile patients suffering from gonadotropin deficiency. The first goal of the present study was thus to produce and purify biologically active recombinant hCG and hLH. Owing to the inherent features of glycoprotein hormones and their potential therapeutic applications, the recombinant expression of these hormones is an important goal from both basic research, as well as, commercial point of view. Considering the above mentioned features it is clear that the expression system used for the hyperexpression of these glycoprotein hormones should also serve as a model system for investigating structure–function relationships and folding of subunits during biosynthesis, in addition to providing sufficient quantities of the hormones for clinical applications. It has been demonstrated that N-linked glycosylation during biosynthesis facilitates protein folding and conformational maturation of glycoprotein hormone subunits into an assembly-competent, biologically active form (21). Therefore, the ideal recombinant expression system should also be able to glycosylate the protein during biosynthesis. The Pichia pastoris yeast expression system was chosen for hyperexpression of glycoprotein hormones as it blends the advantages of both bacterial and mammalian expression systems. Earlier, expression of biologically active hCG and the subunits of hCG and bovine FSH using Pichia pastoris expression system has been reported from the laboratory (22, 23). Chapter 3 (section 3.3.1) of the thesis describes hyperexpression of hLH. The expression of these heterologous proteins was scaled up using fermentation procedures to fulfill the requirements of large quantities of hormones for various applications. Purification of Pichia expressed hormones turned out to be a complex task as large quantity of the hormone was secreted out in the fermentation medium (10litre volume) that was of high ionic strength. Of several different strategies attempted for concentration and partial purification of recombinant hCG, hydrophobic interaction chromatography (HIC) using Phenyl Sepharose matrix emerged as the most efficient technique as a first step of purification. Subsequently, cation exchange chromatography using SP- Sepharose matrix yielded completely purified biologically active recombinant hCG (section 3.3.2). The preliminary data also suggested that Pichia cells express a biologically active form of hCG which appeared to be less glycosylated and of lower molecular weight. Using the same protocol purification of hLH, as well as, single chain derivative of hCG, phCGαβ was achieved (section 3.3.3). These recombinant proteins were characterized extensively using various biochemical, as well as, immunological criteria and were shown to be similar to their natural counterparts with respect to their ability to bind LH receptor and to transduce signal as judged by radioreceptorassays and in vitro bioassays respectively. The hydrophobic interaction chromatography proved an important starting point for purification of all the other members of the glycoprotein hormone family expressed using Pichia pastoris expression system. With the availability of purified, biologically active recombinant hCG in large quantities it was now possible to make attempts towards structure elucidation using NMR spectroscopy. The structure determination of such complex proteins by NMR spectroscopy is made relatively easier by labeling the proteins with magnetically more active, stable isotopes of carbon and nitrogen, 13C and 15N respectively however the cost is often prohibitively high. The Pichia pastoris expression system offers simple means of labeling the proteins as the cells can be grown on simple salts of carbon and nitrogen such as 13C labeled methanol, 15N labeled ammonium chloride or ammonium sulphate. The Chapter 3 also gives a brief account of the preliminary attempts made to label the recombinant hCG with 15N and the structural studies carried out with the carbohydrate moieties of the recombinant hCG using solution NMR spectroscopy. This work was carried out in collaboration with the laboratory of Prof. J.P Kamerling of the University of Utrecht, Netherlands and the efforts are currently underway to elucidate the complete structure of the Pichia expressed hCG. The common feature of receptors and antibodies against the ligand is that both display very specific, high affinity binding towards the ligand. Hence, it is logical to speculate that the antigen binding regions of the antibodies that inhibit hormone binding and/or response, exhibit homology with distinct domains of the receptor. By identifying the epitopes recognized by such antibodies, it should be possible to predict contact points between the hormone and the receptor. In the present study, this hypothesis has been tested using monoclonal antibodies (MAbs) against hCG recognizing different epitopes in the hormone molecule and having different effects on hormone binding and response (Chapter 4). These MAbs were classified as α subunit specific, β subunit specific or heterodimer specific depending on their abilities to bind either subunit in addition to the hormone itself. Interestingly, it was observed that the hCGβ subunit specific MAbs, as well as, heterodimer specific MAbs inhibited hCG receptor binding and hence the response generated by hCG, while the hCGα subunit specific MAbs inhibited only response to the hormone without interfering in binding (Section 4.3.1). To dissect out these interactions further the epitopes recognized by these antibodies on hCG molecule were determined (Section 4.3.2), single chain fragment variable (ScFv) were generated from each of these antibodies and it was shown that these ScFv retain the functionality of the original antibody (Section 4.3.3). Further, the amino acid sequence of each antibody was determined (Section 4.3.4) and finally shown that the antigen binding domains of antibodies show homology to the distinct regions of the LH receptor on sequence alignments between the two using three different programs (4.3.5). The hCGβ subunit specific MAb 52/28' displayed distinct homology with the ECD of LH receptor while the α subunit specific MAb C10 showed regions homologous to TMD of the receptor and the heterodimer specific MAb E12 was found to be similar to the hinge region of the receptor. This clearly indicates that the β subunit of hCG is in close contact with ECD of the receptor while the α subunit makes contacts with the TMD of receptor. The present study thus supports the existing model of hormone receptor interactions, which states that the hormone first binds to the exodomain of the receptor mainly through its β subunit while the integrity of the α subunit is critical for signaling. (24, 25). Also, the observations made in the present study exhibit an interesting possibility of antigen antibody complexes being used as surrogate models for gaining insights into hormone receptor complex. Further, it has been reported that hCG has immunocontraceptive potential(26). Active and passive immunization studies with hCG in primates and humans have demonstrated the possibility of controlling fertility by the antibodies capable of neutralizing hCG. This forms the basis for female contraceptive vaccine that has undergone Phase II clinical trials in India. The MAb E12 characterized in the present study displayed highly specific binding to heterodimeric hCG exclusively without showing any cross reactivity with hLH (Section 4.3.1). The epitope mapping analysis revealed that this antibody recognizes a unique discontinuous epitope present only in the heterodimeric hCG and is distinct from the unique C-terminal extension of hCGβ absent in hLHβ (Section 4.3.2). The MAb, IgG or its recombinant single chain fragment variable (ScFv), inhibited response to hCG, but not to hLH (4.3.3). Thus, the epitope recognized by this MAb is an ideal candidate antigen for immunocontraception. The MAb E12 can also be used for passive immunization in case of emergency contraception. Another potential application of hCG specific antibodies is in homing and the treatment of tumors that secrete hCGβ subunit. The hCGβ subunit specific MAbs used in the present study 52/12 and 52/28' that inhibited hCG receptor binding as well as response generated by hCG can be used in treating such tumors. The functional ScFvs generated from these MAbs in the present study can be made use of on humanization. Thus, the present study has yielded some important molecules for therapeutic applications besides providing a new platform for structure-function relationship studies of the complex glycoprotein hormones.

Two fundamental problems of developmental biology are the understanding of cell fate specification, and the integration of broader environmental contexts into developmental programs. While cell fate specification is largely achieved by differential gene expression programs, environmental integration relies on cellular receptors. A predominant mechanism to mediate both processes utilizes nuclear hormone receptors (NHRs). However, it remains unclear how diverse the NHR’s modes of action are in regulating gene expression. This thesis utilizes the development of the C. elegans germ line as a model system to study a novel link that integrates cell fate specification and the nutritional environment. In C. elegans, germ cell fate specification is chiefly controlled by posttranscriptional mechanisms. Furthermore, overall germline development is influenced by the animal’s nutritional status. However, it remains unknown whether germline posttranscriptional control mechanisms and germ cell fate decisions are linked to nutrition, and if so, how this link may operate in molecular terms. This thesis reports the characterization of the nuclear hormone receptor nhr-114 and its crucial functions for germline development and fertility. Depending on the tissue of expression, nhr-114 regulates overall germline organization, germ cell proliferation and oogenesis. Importantly, all aspects of nhr-114 function are linked to diet. Feeding nhr-114 mutants with a specific E. coli strain, or a tryptophan-supplemented diet significantly reduces germline development defects and sterility. Based on mutant analysis, nhr-114 was found to have overlapping functions with gld-4 cytoplasmic poly(A) polymerase (cytoPAP). This thesis provides evidence that nhr-114 may function in germ cells in a posttranscriptional manner linked to gld-4 cytoPAP. Further evidence shows that NHR-114 interacts with GLD-4 cytoPAP. Together these findings suggest that NHR-114 may control gene expression by transcriptional and posttranscriptional mechanisms in a tissue-specific manner. This thesis proposes that NHR-114 ensures the input of tryptophan to allow germline development; and that this function integrates nutritional information into the germline gene expression programs according to the environment of the worm. Therefore, NHR-114 potentially provides a direct molecular link to how a developmental program is coordinated with the nutritional status of an animal.

Tese(doutorado)-Universidade Federal do Rio Grande, Programa de Pós-Graduação em Aqüicultura, Instituto de Oceanografia, 2011. / Submitted by Cristiane Silva (cristiane_gomides@hotmail.com) on 2012-06-23T16:27:42Z No. of bitstreams: 1 tese corrigida marcio figueiredo.pdf: 4782449 bytes, checksum: df5bb424fe5c9736a80fdc4a63db03a1 (MD5) / Approved for entry into archive by Bruna Vieira(bruninha_vieira@ibest.com.br) on 2012-07-27T20:22:46Z (GMT) No. of bitstreams: 1 tese corrigida marcio figueiredo.pdf: 4782449 bytes, checksum: df5bb424fe5c9736a80fdc4a63db03a1 (MD5) / Made available in DSpace on 2012-07-27T20:22:46Z (GMT). No. of bitstreams: 1 tese corrigida marcio figueiredo.pdf: 4782449 bytes, checksum: df5bb424fe5c9736a80fdc4a63db03a1 (MD5) Previous issue date: 2011 / A aquicultura tem crescido significativamente nas últimas décadas devido ao aumento da demanda de pescado no mundo e à estagnação do setor pesqueiro. Porém, este crescimento depende do desenvolvimento de novos pacotes tecnológicos que visem o aumento da produtividade. Uma alternativa é a manipulação genética (transgenia), sendo que o hormônio do crescimento (GH) tem sido o principal alvo das pesquisas com peixes transgênicos. Entretanto, está comprovado que o excesso de GH acarreta uma série de efeitos adversos devido a sua ação pleiotrópica. A ativação do eixo somatotrófico de forma tecido-específica e independente do excesso de hormônio circulante pode contornar estes problemas. Neste contexto, o objetivo desta tese foi superexpressar o gene do receptor do GH (GHR) no tecido muscular esquelético do zebrafish (Danio rerio) e estudar os efeitos desta manipulação sobre os mecanismos envolvidos na regulação gênica do crescimento muscular. A linhagem transgênica estável obtida expressa o GHR especificamente no tecido muscular 100 vezes mais do que os não transgênicos. Estes transgênicos não apresentaram aumento significativo no crescimento, provavelmente devido à queda na expressão do fator de crescimento tipo insulina I (IGF-I). Esta queda foi, provavelmente, relacionada à ação dos principais moduladores da sinalização do GH (SOCS1 e 3), os quais apresentaram-se aumentados nos transgênicos. Ainda, foi observada uma queda na expressão das principais proteínas musculares estruturais (Acta1, myhc4 e mylz2), o que explica a ausência de hipertrofia nos transgênicos. Por outro lado, o aumento na expressão dos principais fatores reguladores miogênicos (myod, myf5 e myog) explica a hiperplasia observada nas análises histológicas. Para verificar como a superexpressão do GHR ativou a transcrição dos fatores reguladores miogênicos (MRFs) e, por consequência a hiperplasia, foram estudados os possíveis mecanismos envolvidos neste processo. Dentre estes, tanto a via proliferativa (MEK/ERK) quanto à via relacionada com a síntese protéica (PI3K/Akt), não tiveram alteração na expressão de seus genes. Entretanto, foi observado aumento na expressão das proteínas de transporte para o núcleo (importinas 1, 3 e 1), podendo-se concluir que a ativação dos MRFs está relacionada ao transporte do GHR para o núcleo das células musculares. Desta forma, pode se concluir que hiperplasia e hipertrofia seguem duas vias de sinalização intracelular distintas, ambas desencadeadas pelo GH, mas reguladas por mecanismos diferentes. / Aquiculture practice has been significantly increasing during the last decades due to the fish rising demand and to fishery activity stagnation. However, such increase depends on new technological packages development aiming to productivity rises. Genetic manipulation is an alternative, once growth hormone (GH) has been the main target on transgenic fish researches. Nevertheless, it has been proved that GH excess leads to many adverse effects due to its pleiotropic action. The somatotropic axis activation in a tissue-specific manner and independent on the circulating hormone excess may bypass these problems. Following these ideas, the present thesis objective was overexpressing GH receptor’s gene (GHR), in zebrafish (Danio rerio) skeletal muscular tissue, and studying such manipulation effects over the mechanisms involved in muscular growth gene regulation. The stable transgenic lineage obtained expresses GHR, specifically in muscular tissue, 100 times more than non-transgenic. These transgenic did not present significant growth, possibly due to a gene expression fall in insulin-like growth factor I (IGF-I). The mentioned fall is, probably, related to GH main signaling modulators (SOCS1 and 3) action, which were increased in transgenic. Also, a gene expression fall from the main structural muscle proteins (Acta1, myhc4 and mylz2) was observed, explaining the transgenic hypertrophy absence. However, the main myogenic regulatory factors (myod, myf5 and myog) expression rising explains the observed hyperplasia in histological analysis. Intending to verify how GHR overexpression has activated the myogenic regulatory factors (MRFs) transcription and, consequently, the hyperplasia, the mechanisms possibly involved in this process were studied. Among these, even the proliferative pathway (MEK/ERK) or the pathway related to protein synthesis (PI3K/Akt), did not presented gene expression altering. However, a gene expression rise in transporting proteins into nucleus (importins 1, 3 and 1) was observed, which may be understood as a correlation between MRFs activation and GHR transport into muscular cell’s nucleus. Therefore, it may be understood that hyperplasia and hypertrophy follow two distinct intracellular signaling pathways, both triggered by GH, but regulated by different mechanisms. These data may be important for aquiculture new transgenic lineages development.

Ratas adultas foram tratadas com 2.5, 5, 10, 20 e 40x a dose fisiológica de triiodotironina (T3) por 10 semanas. Houve redução, dose-dependente, da massa óssea, volume e espessura trabecular (Tb.Th), e espessura de osso cortical. Até a dose de 10xT3, houve aumento na taxa de formação óssea (BFR). Doses maiores de T3 reduziram a BFR, e a reabsorção óssea. Esses dados mostram que, até um certo grau de tireotoxicose, a osteopenia é resultado de aumento da formação e reabsorção ósseas, com predomínio da última. Em graus mais elevados, a osteopenia é causada por redução no remodelamento ósseo, com predomínio da atividade reabsortiva. Em seguida, comparamos os efeitos do T3 e GC-1, um tireomimético seletivo pelo receptor b de T3 (TRb), no osso de camundongos fêmeas adultas. Na tíbia, o T3 reduziu a resistência, rigidez e resiliência, enquanto o GC-1 aumentou esses parâmetros e a Tb.Th, o que mostra que o GC-1 melhora a qualidade óssea. Considerando-se a seletividade do GC-1 pelo TRb, esses achados sugerem que o TRb medeia predominantemente ações positivas do T3 no osso. / Adult rats were treated with 2.5, 5, 10, 20 and 40x the physiological dose of triiodothyronine (T3) for 10 weeks. There was a dose-dependent reduction in bone mass, trabecular volume and thickness (Tb.Th), and cortical thickness. Up to 10xT3, there was an increase in the bone formation rate (BFR). Higher doses of T3 decreased BFR and bone resorption. These data show that, up to a certain degree of thyrotoxicosis, osteopenia is the result of an increase in bone formation and resorption, with a prevalence of resorption. In more severe thyrotoxicosis, the osteopenia is caused by a reduction in bone remodeling, with a predominance of bone resorption. Then, we compared the effects of T3 and GC-1, a thyromimetic that is selective for T3 receptor b (TRb), on bone of adult female mice. T3 treatment decreased tibial resistance, stiffness and resilience, while GC-1 increase these parameters and Tb.Th, showing that GC-1 improves bone quality. Considering the selectiveness of GC-1 for TRb, these findings suggest that TRb mediates mainly positive actions of T3 in the bone.

Master of Science / Department of Animal Sciences and Industry / Timothy G. Rozell / Follicle stimulating hormone (FSH) is known as the key hormone capable of causing proliferation of granulosa cells in the ovary. The classical receptor belongs to the G protein-coupled superfamily and is designated FSHR-1. A variant in the FSH receptor has been shown to be functional in mouse ovaries. The variant receptor is designated as FSHR-3, and when bound by FSH activates a pathway that shares similar characteristics to the growth factor type I receptor pathway, with no increase in cAMP. The FSHR-3 variant activates MAPK upon binding to FSH, and causes proliferation of cells on which it is known to be expressed. For example ID8 mouse ovarian surface epithelium cells (MOSEC), a cell line that when introduced in immunocompetent mice causes tumors similar to human ovarian cancer and which also express FSHR-3, proliferated in response to FSH. The present study explored the potential for decreasing expression of FSHR-3 protein. The RNA interference (RNAi) technique was used to insert small inhibitory RNA(siRNA) segments corresponding specifically to the R3 variant of the FSH receptor in ID8 MOSEC. Transfected cells were lysed and FSHR-3 protein was visualized using SDS Page and Western blotting analysis. A reduction in expression of FSHR-3 was observed in two of the transfection groups, with the greatest down-regulation of FSHR-3 being 30.1%. From these preliminary results we conclude that the FSHR-3 is expressed on ID8 cells, and that siRNA may be useful to reduce its expression. Thus, it may be possible to slow the growth of FSH-responsive tumors using siRNA to target the FSHR-3 receptor.