Dissertation (PhD)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Gonadotropin-releasing hormone (GnRH) is well known as the central regulator of the reproductive system through its stimulation of gonadotropin synthesis and release from the pituitary via binding to its specific receptor, known as the gonadotropin-releasing hormone receptor type I (GnRHR-I). The gonadotropins, luteinising hormone (LH) and follicle-stimulating hormone (FSH), bind to receptors in the gonads, leading to effects on steroidogenesis and gametogenesis. The recent finding of a second form of the GnRH receptor, known as the type II GnRHR or GnRHR-II, in non-mammalian vertebrates triggered the interest into the possible existence and function of a GnRHR-II in humans. The current study addressed this issue by investigating the presence of transcripts for a GnRHR-II in various human tissues and cells. While it was demonstrated that antisense transcripts for this receptor, containing sequence of only two of the three coding exons, are ubiquitously and abundantly expressed in all tissues examined, potentially full-length (containing all three exons), sense transcripts for a GnRHR-II were detected only in human ejaculate. Further analysis revealed that the subset of cells in the ejaculate expressing these transcripts is mature sperm. These findings, together with the reported role for GnRH in spermatogenesis and reproduction led to the further analysis of the presence of a local GnRH/GnRHR network in human and vervet monkey ejaculate or sperm. Indeed, such a network seems to be present in humans since transcripts for both forms of GnRH present in mammals, as well as transcripts for the GnRHR-I, are expressed in human ejaculate. Furthermore, transcripts for the GnRHR-II are expressed in both human and vervet monkey ejaculate. Thus, it would appear that locally produced GnRH-1 and/or GnRH-2 in the human male reproductive tract might mediate their effects on fertility via a local GnRHR-I, and possibly via GnRHR-II. Remarkably, in the pituitary, LH and FSH are present in the same gonadotropes, yet they are differentially regulated by GnRH under various physiological conditions. While it is well established that post-transcriptional regulatory mechanisms occur, the contribution of transcriptional regulation to the differential expression of the LHβ- and FSHβ-subunit genes is unclear. In this study, the role of GnRH-1 and GnRH-2 via the GnRHR-I and the GnRHR-II in transcriptional regulation of mammalian LHβ- and FSHβ genes was determined in the LβT2 mouse pituitary gonadotrope cell-line. It is demonstrated for the first time that GnRH-1 may affect gonadotropin subunit gene expression via GnRHR-II in addition to GnRHR-I, and that GnRH-2 also has the ability to regulate gonadotropin subunit gene expression via both receptors. Similar to other reports, it is shown that the transcriptional response to GnRH-1 of LHβ and FSHβ is low (about 1.4-fold for bLHβLuc and 1.2-fold for oFSHβLuc). In addition, evidence is supplied for the first time that GnRH-2 transcriptional regulation of the gonadotropin β subunits is also low (about 1.5-fold for bLHβLuc and 1.1-fold for oFSHβLuc). It is demonstrated that GnRH-1 is a more potent stimulator of bLHβ promoter activity as compared to GnRH-2 via the GnRHR-I, yet both hormones result in a similar maximum induction of bLHβ. However, GnRH-2 is a more efficacious stimulator of bLHβ transcription via the GnRHR-II than GnRH-1. No discriminatory effect of GnRH-1 vs. GnRH-2 was observed for oFSHβ promoter activity via GnRHR-I or GnRHR-II. By comparison of the ratio of expression of transfected oFSHβ- and bLHβ promoterreporters via GnRH-1 with that of GnRH-2, it is shown that GnRH-2 is a selective regulator of FSHβ gene transcription. This discriminatory effect of GnRH-2 is specific for GnRHR-I, as it is not observed for GnRHR-II, where GnRH-1 results in a greater oFSHβ- to-bLHβ ratio. These opposite selectivities for GnRHR-I and GnRHR-II on the ratios of oFSHβ:bLHβ promoter activity for GnRH-1 vs. GnRH-2 suggest a mechanism for fine control of gonadotropin regulation in the pituitary by variation of relative GnRHR-I vs. GnRHR-II levels. In addition, a concentration-dependent modulatory role for PACAP on GnRH-1- and GnRH-2-mediated regulation of bLHβ promoter activity, via both GnRHR-I and GnRHR-II, and of oFSHβ promoter activity, via GnRHR-I, is indicated. The concentration-dependent effects suggest the involvement of two different signalling pathways for the PACAP response. Together these findings suggest that transcription of the gonadotropin genes in vivo is under extensive hormonal control that can be finetuned in response to varying physiological conditions, which include changing levels of GnRH-1, GnRH-2, GnRHR-I and GnRHR-II as well as PACAP. / AFRIKAANSE OPSOMMING: Gonadotropien-vrystellingshormoon (GnRH) is bekend as die sentrale reguleerder van die voorplantingsisteem deur die stimulasie van gonadotropiensintese en - vrystelling vanaf die pituïtêre klier via binding aan ‘n spesifieke reseptor, die sogenaamde tipe I gonadotropien-vrystellingshormoonreseptor (GnRHR-I). Die gonadotropiene, lutineringshormoon (LH) en follikel-stimuleringshormoon (FSH), bind aan reseptore in die gonades waar dit steroïedogenese en gametogenese beïnvloed. Die onlangse ontdekking van ‘n tweede vorm van die GnRH-reseptor, bekend as die tipe II GnRHR of GnRHR-II, in nie-soogdier vertebrate het belangstelling in die moontlike bestaan en funksie van ‘n GnRHR-II in die mens gewek. Hierdie kwessie is aangeraak deur die teenwoordigheid van transkripte vir ‘n GnRHR-II in verskeie weefsel- en seltipes van die mens te ondersoek. Daar is aangetoon dat nie-sin transkripte vir hierdie reseptor, wat die DNA-opeenvolgings van slegs twee van die drie koderende eksons bevat het, oormatig uitgedruk word in al die weefseltipes wat ondersoek is. Daarteenoor is potensieel vollengte (bevattende al drie eksons) sin transkripte vir ‘n GnRHR-II in die mens slegs in semen gevind. Verdere analise het getoon dat dit volwasse sperma binne die semen is wat laasgenoemde transkripte uitdruk. Hierdie bevindinge, tesame met die aangetoonde rol vir GnRH in spermatogenese en reproduksie het gelei tot die verdere analise van die teenwoordigheid van ‘n lokale GnRH/GnRHR-netwerk in mens- en blouaapsemen of -sperm. So ‘n netwerk blyk om teenwoordig te wees in die mens, aangesien transkripte vir beide vorme van GnRH wat in soogdiere gevind word, asook transkripte vir die GnRHR-I, in menssemen uitgedruk word. Daarbenewens word transkripte vir die GnRHR-II uitgedruk in beide mens- en blouaapsemen. Dit wil dus voorkom asof lokaalgeproduseerde GnRH-1 en/of GnRH-2 in die manlike voortplantingstelsel van die mens hul effek op vrugbaarheid bemiddel via ‘n lokale GnRHR-I, en moontlik ook via GnRHR-II. Dit is opmerklik dat LH en FSH teenwoordig is in dieselfde gonadotroopselle van die pituïtêre klier en tog verskillend gereguleer word deur GnRH tydens verskeie fisiologiese kondisies. Terwyl dit bekend is dat post-transkripsionele reguleringsmeganismes teenwoordig is, is die bydrae van transkripsionele regulering tot die differensiële uitdrukking van die LHβ- en FSHβ-subeenheidgene minder duidelik. In hierdie studie is die rol van GnRH-1 en GnRH-2 via die GnRHR-I en die GnRHR-II in transkripsionele regulering van soogdier-LHβ- en -FSHβ-gene in die LβT2 muis pituïtêre gonadotroopsellyn bepaal. Dit is vir die eerste keer aangetoon dat GnRH-1 ‘n effek mag hê op gonadotropiensubeenheid-geenuitdrukking via GnRHR-II bykomend tot GnRHR-I, en dat GnRH-2 ook die vermoë besit om gonadotropiensubeenheid-geenuitdrukking via beide reseptore te reguleer. Soos deur ander studies aangetoon is die transkripsionele respons van LHβ en FSHβ tot GnRH-1 klein (ongeveer 1.4-voudig vir bLHβLuc en 1.2- voudig vir oFSHβLuc). Verder is daar vir die eerste keer bewys gelewer dat transkripsionele regulering van die gonadotropien β-subeenhede deur GnRH-2 ook gering is (ongeveer 1.5-voudig vir bLHβLuc en 1.1-voudig vir oFSHβLuc). Daar is aangetoon dat GnRH-1 ‘n sterker stimuleerder van bLHβ-promotoraktiwiteit is in vergelyking met GnRH-2 via die GnRHR-I, hoewel beide hormone tot ‘n soortgelyke maksimum induksie van bLHβ lei. GnRH-2 is egter ‘n meer effektiewe stimuleerder van bLHβ-transkripsie as GnRH-1 via die GnRHR-II. Geen verskille is gevind tussen die effekte van GnRH-1 en GnRH-2 op oFSHβ-promotoraktiwiteit via GnRHR-I of GnRHR-II nie. Wanneer die verhouding van uitdrukking van getransfekteerde oFSHβ- en bLHβ- promotor-verslaggewers via GnRH-1 met dié van GnRH-2 vergelyk is, is aangetoon dat GnRH-2 ‘n selektiewe reguleerder van FSHβ-geentranskripsie is. Hierdie diskriminasieeffek van GnRH-2 is spesifiek vir GnRHR-I aangesien dit nie vir GnRHR-II waargeneem word nie. GnRH-1 lei tot ‘n groter oFSHβ tot bLHβ-verhouding via GnRHR-II. Hierdie teenoorgestelde selektiwiteite van GnRHR-I en GnRHR-II op die verhoudings van oFSHβ tot bLHβ-promotoraktiwiteit vir GnRH-1 teenoor GnRH-2 suggereer dat daar ‘n meganisme bestaan vir die fyn regulering van gonadotropiene in die pituïtêre klier, deurdat die relatiewe vlakke van GnRHR-I teenoor GnRHR-II gevarieer word. Daarbenewens is ‘n konsentrasie-afhanklike moduleringsrol vir PACAP op GnRH-1- en GnRH-2-bemiddelde regulering van bLHβ-promotoraktiwiteit aangetoon, via beide GnRHR-I en GnRHR-II, asook op oFSHβ-promotoraktiwiteit via GnRHR-I. Hierdie konsentrasie-afhanklike effekte dui op die betrokkenheid van twee verskillende seinpadweë vir die PACAP-respons. Tesame suggereer hierdie bevindinge dat transkripsie van die gonadotropiengene in vivo onder ekstensiewe hormonale kontrole is wat verfyn kan word in respons to veranderlike fisiologiese kondisies. Laasgenoemde sluit veranderende vlakke van GnRH-1, GnRH-2, GnRHR-I en GnRHR-II asook PACAP in.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The gonadotropin-releasing hormone (GnRH) receptor is a G-protein-coupled receptor in the pituitary gonadotropes and is an important control point for reproduction. GnRH binds to the GnRH receptor (GnRHR) resulting in the synthesis and release of follicle stimulating hormone (FSH) and luteinizing hormone (LH). The sensitivity of the pituitary to GnRH can be directly correlated with GnRHR levels. The mouse GnRHR promoter contains three cis elements containing binding sites for steroidogenic factor-1 (SF-1), namely site 1 (-15/-7), site 2 (-244/- 236) and site 3 (-304/-296) as well as an activator protein-1 (AP-1)-like consensus sequence (TGAGTCA) at position –336/-330. While sites 1 and 2 and the AP-1 site have been previously shown to be involved in regulation of transcription of the mouse GnRHR (mGnRHR) promoter in some cell lines, the role of site 3 has not been previously investigated. This study investigated whether transcription of the mGnRHR gene is regulated by GnRH and glucocorticoids in the LβT2 gonadotrope pituitary cell line, and the role therein of site 3 and the AP-1 site and their cognate proteins, using a combination of in vitro protein- DNA binding studies and promoter-reporter assays. The role played by site 3 and the AP-1 site in basal transcription of the mGnRHR gene in LβT2 cells was the first area of investigation during this study. Luciferase reporter plasmids containing 600 bp of the mGnRHR promoter were used where the site 3 and AP-1 sites were either wild-type or mutated. Two constructs were prepared from the wild-type construct, i.e. wild type (LG), site 3 mutant (m3) and AP-1 mutant (mAP-1). Transfection of LG, m3 and mAP-1 plasmids into LβT2 cells was carried out to determine the effect of these mutations on the basal expression of the mGnRHR gene. Mutation of site 3 resulted in a 1.5 fold increase in the transcriptional activity of the mGnRHR promoter. This suggests that site 3 plays a role in the inhibition of basal transcriptional levels of the mGnRHR promoter in LβT2 cells. Mutation of the AP-1 site resulted in a 50% decrease in basal transcriptional levels of the mGnRHR promoter in LβT2 cells. This suggests that the AP-1 site is involved in positively mediating the basal transcriptional response of the GnRHR promoter in LβT2 cells. Experiments towards the understanding of the mechanism of the cis elements (site 3 and AP-1 site) on the mGnRHR promoter were carried out along with the role of protein kinase A (PKA) pathways, proteins involved and the effect of varying doses for varying times of GnRH, as well as the overexpression of PKA and the SF-1 protein. It was found that site 3 and the AP-1 site are not involved in the GnRH response. Results suggest that site 3 is partially involved in the PKA response in LβT2 cells. Site 3 can bind SF-1 protein as shown via competitive electrophoretic mobility shift assays (EMSA). When EMSA’s were performed on the AP-1 site the findings were that the c-Fos protein was not involved in the activation of the AP-1 site. A factor was found to bind to the AP-1 site, which did not require the intact AP-1 site, suggesting that it could be the c-Jun protein that binds to the AP-1 site under basal conditions. Another area that was investigated was whether the mGnRHR promoter can be regulated by dexamethasone (dex) either via the AP-1 site or site 3. A dose and time-dependent increase in promoter activity was observed with dex. This effect appears to require site 3 and the AP-1 site, as shown by the complete loss of response when these sites were individually mutated, consistent with a functional interaction between site 3 and the AP-1 site in LβT2 cells. / AFRIKAANSE OPSOMMING: Die gonadotropienvrystellings hormoon (GnRH) reseptor is ‘n G-proteïen-gekoppelde reseptor in die pituitêre gonadotrope en is ’n belangrike beheerpunt vir reproduksie. GnRH bind aan die GnRH reseptor (GnRHR) met die gevolg dat follikel stimulerende hormoon (FSH) en luteïeniserende (LH) gesintetiseer en vrygestel word. Die sensitiwiteit van die pituitêre klier vir GnRH kan direk met GnRHR vlakke gekorreleer word. Die muis GnRHR promotor bevat drie cis elemente met bindingssetels vir steroïedogeniese faktor 1 (SF1), naamlik setel 1 (-15/-7), setel 2 (-244/-236) en setel 3 (-304/-296) sowel as ’n aktiveerder proteïen 1 (AP-1) tipe konsensus sekwens (TGAGTCA) in posisie -336/-330. Terwyl setels 1 en 2 en die AP-1 setel voorheen getoon is om by die regulering van transkripsie van die muis GnRHR (mGnRHR) promotor in party sellyne betrokke te wees, is die rol van setel 3 nog nie vantevore bestudeer nie. In hierdie studie is ondersoek of die transkripsie van die mGnRHR geen deur GnRH en glukokortikoïede in die LβT2 gonadotroop pituitêre sellyn gereguleer word, en die rol van setel 3 en die AP-1 setel en hulle binders, deur gebruik te maak van in vitro proteïen-DNA bindings studies en promotor-verslaggewer essais. Die rol wat setel 3 en die AP-1 setel in basale transkripsie van die mGnRHR gene in LβT2 selle gespeel het, was die eerste onderwerp wat in hierdie studie bestudeer is. Lusiferase verslaggewer plasmiede wat die eerste 600 bp van die mGnRHR promotor bevat het en waarin setel 3 en die AP-1 setels óf wilde tipe óf gemuteer was, is gebruik. Two konstrukte is vanaf die wilde tipe konstruk berei, naamlik wilde tipe (LG), ’n setel 3 mutant (m3) en ’n AP-1 mutant (mAP-1). Transfeksie van LG, m3 en mAP-1 plasmiede in LβT2 selle is deurgevoer om te bepaal wat die effek van hierdie mutasies op die basale ekspressie van die mGnRHR gene was. Mutasie van setel 3 het ’n 1.5-voudige toename in die transkripsionele aktiwiteit van die mGnRHR promotor tot gevolg gehad. Dit suggereer dat setel 3 ’n rol in die inhibisie van die basale transkripsievlakke van die mGnRHR promotor in LβT2 selle speel. Mutasie van die AP-1 setel het tot ‘n 50% verlaging in basale transkripsievlakke van die mGnRHR promotor in LβT2 selle gelei. Dit suggereer dat die AP-1 setel betrokke is in die positiewe bemiddeling van die basale transkriptionele respons van die GnRHR promotor in LβT2 selle. Eksperimente wat gemik was om die meganisme van die cis-elemente (setel 3 en die AP-1 setel) op die mGnRHR promotor te verklaar, asook om die rol van proteïen kinase A (PKA) paaie, proteïene daarby betrokke en die effek van varieende dosisse vir verskillende tye van GnRH, sowel as die oorekspressie van PKA en die SF-1 proteïen, is deurgevoer. Dit is gevind dat setel 3 en die AP-1 setel nie betrokke by die GnRH respons is nie. Die resultate suggereer dat setel 3 gedeeltelik betrokke is by die PKA respons van LβT2 selle. Setel 3 kan SF-1 proteïen bind soos getoon deur kompeterence elektroforetiese mobiliteits verskuiwings essais (EMSA). As EMSA’s deurgevoer is op die AP-1 setel is bevind dat die c-Fos proteïen nie betrokke is in die aktivering van die AP-1 setel nie. ’n Faktor is gevind om aan die AP-1 setel te bind wat nie ’n intakte AP-1 setel vereis het nie, wat gesuggereer het dat dit die c-Jun proteïen kan wees wat aan die AP-1 setel onder basale omstandighede bind. ’n Ander area wat ondersoek is, is of die GnRHR promotor gereguleer kan word deur deksametasoon (dex) óf via die AP-1 setel óf via setel 3. ’n Dosis en tyds-afhanklike toename in promotor aktiwiteit is waargeneem met dex. ’n Vereiste vir hierdie effek blyk om die teenwoordigheid van setel 3 en die AP-1 setel te wees, soos aangetoon deur die totale verlies aan response as hierdie twee setels individueel gemuteer is, en wat weereens in ooreenstemming met die funksionele interaksie tussen setel 3 en die AP-1 setel in LβT2 selle is.

Orientadores: Lucia Helena Simões da Costa Paiva, Luis Otavio Zanatta Sarian / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-11-09T15:59:32Z (GMT). No. of bitstreams: 1 Pinheiro_RosilenedeLima_M.pdf: 979982 bytes, checksum: d2884ebad26e9df7e5560eea68502e27 (MD5) Previous issue date: 2008 / Resumo: Objetivo: Avaliar a relação entre parâmetros corporais usados para definir obesidade central, circunferência da cintura (CC) e relação cintura quadril (RCQ), e a expressão de receptores esteróides em mulheres com carcinoma de mama na pré e pós-menopausa. Métodos: Realizado um estudo seccional com 473 mulheres com doença maligna da mama, estadiamento I a III, tratadas no Instituto Nacional de Câncer, Brasil, em 2004. Dados epidemiológicos e clínicos foram obtidos. Os parâmetros antropométricos usados para definir obesidade foram obtidos na visita de admissão, antes do procedimento cirúrgico, com técnicas recomendadas pela Organização Mundial de Saúde: peso (Kg), altura (cm), circunferência da cintura (cm), Índice de massa corporal (IMC, peso/(altura2)) e relação cintura quadril. A expressão de receptores para estrogênio (RE) e progesterona (RP) foi determinada por imunohistoquimica. O estudo foi aprovado pelo Comitê de Ética da Instituição. Comparações de freqüências foram analisadas através do teste exato de Fisher. Razão de prevalência (PrevR) foi calculada para avaliar as diferenças nas prevalências de RH com as categorias para IMC, CC e RCQ. Resultados: A maior parte das mulheres apresentava sobrepeso (68,9%) e obesidade central (CC > 88cm e RCQ > 0,85 em 64,3% e 73,4%, respectivamente). A maioria (54,1%) apresentou tumores com expressão para RE e RP, sendo 78,6% expressando, pelo menos, um dos RH. Existiu uma proporção significantemente maior de tumores RE+/RP+ em mulheres na pósmenopausa (p<0,001). BMI (p=0,12), WC (p=0,07) e RCQ (p=0,55) não foram relacionados à expressão de RH em mulheres na pré-menopausa. Em mulheres na pós-menopausa, BMI (p=0,30), WC (p=0,35) e RCQ (p=1,00) também não foram relacionados à expressão de RH. Conclusão: O presente estudo reforça o conceito de que a expressão dos receptores hormonais para tumores mamários é dependente do estado menopausal. CC e RCQ podem não ser bons preditores de expressão de RH em doenças malignas da mama para mulheres na pré e pósmenopausa. Palavras-chave: câncer de mama, obesidade, receptor hormonal, circunferência da cintura / Abstract: Objective: to evaluate the relation between body parameters used to define central obesity -waist circumference (WC) and waist to hip ratio (WRH)- and steroid receptor status in breast carcinomas of pre- and postmenopausal women. Subjects and methods: This is a cross-sectional study on 473 women with breast malignancies stage I-III, treated at the National Cancer Institute, Brazil, in 2004. Clinical and epidemiological data were obtained. The anthropometrics used to define obesity were obtained at the admission visit, before the surgical procedure, with the techniques recommended by the World Health Organization: weight (Kg), height (cm), the waist and hip circumferences (cm), body mass index (BMI, weight/(height^2)), and the waist to hip ratio. The expression of estrogen (ER) and progesterone (RP) receptors were determined with imunohistochemistry. The institutons¿ ethics review board has approved the study. Frequency comparisons were analyzed with Fisher¿s exact tests. Prevalence ratios (PrevR) were calculated to assess the differences in prevalence for HR within the categories for BMI, WC and WHR. Results: Most women were overweight or obese (68.9%), and had central obesity (WC>=88 and WHR>=0.85 in 64.3 and 73.4%, respectively). The majority (54.1%) of the women had tumors that expressed ER and PR, being that 78.6% of the sample expressed at least one of the HR. There was a significantly higher proportion of positive RE+/RP+ tumors in postmenopausal women (p < 0.001). BMI (p=0.12), WC (p=0.07) and WHP (p=0.55) were not related to the HR status in premenopausal women. In postmenopausal women, BMI (p=0.30), WC (p=0.35) and WHP (p=1.00) were also not related to the HR status. Conclusions: The present study reinforces the concept that the HR status of breast tumors is dependent upon the menopausal status. WC or WHR may not be good predictors of HR status in breast malignancies of pre- and postmenopausal women. Keywords: breast cancer, obesity, hormonal receptor, waist circumference / Mestrado / Ciencias Biomedicas / Mestre em Tocoginecologia

by Lo Ka-Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 175-197). / Chapter I --- Title page --- p.i / Chapter II --- Thesis committee --- p.ii / Chapter II --- Acknowledgment --- p.iii-iv / Chapter III --- Abstract --- p.v-vi / Chapter IV --- Table of content --- p.vii-xiv / Chapter V --- List of figures --- p.xv-xviii / Chapter VI --- List of tables --- p.xix-xx / Forewords: / Overall objectives --- p.1 / Introduction on the fish used in this research study --- p.2 / Chapter Chapter 1: --- Literature Review on Biomarkers of Stress in Teleosts --- p.5 / Chapter 1.1 --- Definition of stress --- p.7 / Chapter 1.2 --- Classification of stress indicators --- p.8 / Chapter 1.2.1 --- Primary stress indicators --- p.8 / Chapter 1.2.1.1 --- Molecular stress indicators --- p.8 / Chapter 1.2.1.2 --- Hormonal stress indicators --- p.9 / Chapter (I) --- Corticosteroid --- p.9 / Chapter (II) --- Catecholamines --- p.11 / Chapter 1.2.2 --- Secondary stress indicators --- p.12 / Chapter 1.2.2.1 --- Metabolic changes --- p.12 / Chapter (I) --- Glucose metabolism --- p.13 / Chapter (II) --- Lactic acid --- p.14 / Chapter 1.2.2.2 --- Osmoregulatory changes --- p.15 / Chapter 1.2.2.3 --- Haematological changes --- p.16 / Chapter 1.2.2.4 --- Reproductive changes --- p.17 / Chapter 1.2.3 --- Tertiary stress indicators --- p.18 / Chapter 1.2.3.1 --- Histopathological indicators --- p.18 / Chapter 1.2.3.2 --- Ecological indicators --- p.19 / Chapter 1.3 --- Recent trends on the study of biomarkers --- p.20 / Chapter 1.3.1 --- Use of detoxification enzymes for specific indication of toxic pollutants in aquatic environment --- p.20 / Chapter 1.3.1.1 --- Metallothioneins (MTs) --- p.20 / Chapter 1.3.1.2 --- Cytochrome P450 monoxygenase (CYP450) --- p.21 / Chapter 1.3.2 --- Use of HSP 70 as a biomarker in teleost --- p.22 / Chapter 1.4 --- Future perspectives on the study of biomarkers in fish --- p.24 / Chapter Chapter 2: --- Literature Review on Heat Shock Proteins (HSPs) --- p.28 / Chapter 2.1 --- General Characteristics of HSPs --- p.29 / Chapter 2.1.1 --- HSP90 family --- p.30 / Chapter 2.1.2 --- HSP70 family --- p.31 / Chapter 2.1.3 --- HSP60 family (Chaperonin-60) --- p.32 / Chapter 2.1.4 --- Low-molecular weight HSPs (HSP20) --- p.33 / Chapter 2.2 --- Structure of HSP70 encoding gene --- p.33 / Chapter 2.2.1 --- General characteristics of HSP70-encoding gene --- p.33 / Chapter 2.2.2 --- Heat shock transcription factor (HSF) --- p.35 / Chapter 2.2.3 --- Heat shock elements (HSE) --- p.35 / Chapter 2.3 --- Stress-mediated control of HSP70 transcription --- p.36 / Chapter 2.4 --- Characterization of HSP70 expression in teleost --- p.38 / Chapter 2.4.1 --- Tissues-specific expression of HSP70 in teleost --- p.39 / Chapter 2.4.2 --- Inter-relationship of HSP70 expression with seasonal variation and thermotolerance of teleost --- p.40 / Chapter 2.4.3 --- Induction of HSP70 in teleost upon acute thermal stress --- p.41 / Chapter 2.4.4 --- Induction of HSP70 in teleost by non-thermal stressors --- p.43 / Chapter 2.4.4.1 --- Heavy metal-induced HSP70 expression --- p.43 / Chapter 2.4.4.2 --- Handling stress-induced HSP70 expression --- p.43 / Chapter Chapter 3: --- "Induction of HSP70 in blood cells of seabream, Sparus sarba subjected to in vivo and in vitro thermal stress" --- p.48 / Chapter 3.1 --- Introduction --- p.49 / Chapter 3.2 --- Materials and Methods --- p.52 / Chapter 3.2.1 --- Overall experimental design --- p.52 / Chapter 3.2.2 --- Fish --- p.53 / Chapter 3.2.3 --- Blood sampling --- p.53 / Chapter 3.2.4 --- Preparation of blood cells --- p.54 / Chapter 3.2.5 --- Thermal stress regimes --- p.54 / Chapter 3.2.5.1 --- Time couse of HSP70 induction profile in blood cells after in vitro exposure to thermal stress --- p.54 / Chapter 3.2.5.2 --- HSP70 synthesis in blood cells taken from fish subjected to in vivo hyper- thermic stress --- p.55 / Chapter 3.2.5.3 --- Transcriptional inhibitory effect of actinomycin D on the synthesis of HSP70 in blood cells subjected to in vitro thermal stress --- p.55 / Chapter 3.2.6 --- Protein analysis --- p.56 / Chapter 3.2.7 --- Gel electrophoresis --- p.57 / Chapter 3.2.8 --- Immunoblotting (Western blot analysis) --- p.57 / Chapter 3.2.9 --- Autroradiography --- p.58 / Chapter 3.3 --- Results --- p.59 / Chapter 3.3.1 --- Time course of HSP70 induction profile in blood cells subjected to in vitro thermal treatments --- p.59 / Chapter 3.3.1.1 --- Results of immunoblotting from blood cells of fish acclimated to 26°C --- p.59 / Chapter 3.3.1.2 --- Results of immunoblotting in blood cells from 18°C-acclimated fish --- p.60 / Chapter 3.3.1.3 --- Results of immunoblotting in blood cells from fish acclimated to 20°C --- p.60 / Chapter 3.3.1.4 --- 35S-methionine labelling of de novo protein synthesis in blood cells of fish acclimated to 15 and 20°C --- p.61 / Chapter 3.3.2 --- Blood cell HSP70 levels in 20°C-acclimated fish subjected to in vivo hyperthermic stress --- p.61 / Chapter 3.3.3 --- Transcriptional inhibitory effect of actinomycin D on HSP70 induction in blood cells subjected to in vitro thermal stress --- p.62 / Chapter 3.4 --- Discussions --- p.60 / Chapter 3.4.1 --- Characteristics of HSP70 induction in blood cells of seabream subjected to in vitro temperature stress --- p.72 / Chapter 3.4.1.1 --- Induction profile of HSP70 in blood cells --- p.72 / Chapter 3.4.1.2 --- Time course ofHSP70 induction in blood cells --- p.75 / Chapter 3.4.1.3 --- Effect of acclimation temperature of fish on the induction of HSP70 --- p.76 / Chapter 3.4.2 --- Comparison of HSP70 induction in in vitro and in vivo thermal treatment on blood cells --- p.78 / Chapter 3.4.3 --- "Effect of transcriptional inhibitor, actinomycin D, on the de novo synthesis of HSP70" --- p.80 / Chapter 3.5 --- Conclusions --- p.70 / Chapter Chapter 4: --- "Effects of seasonal variation and transportation stress on level of HSP70, serum glucose and serum Cortisol in seabream, Sparus sarba" --- p.86 / Chapter 4.1 --- Introduction --- p.87 / Chapter 4.2 --- Materials and methods --- p.90 / Chapter 4.2.1 --- Overall experimental design --- p.90 / Chapter 4.2.2 --- Fish and blood sampling --- p.91 / Chapter 4.2.3 --- Preparation of blood samples --- p.92 / Chapter 4.2.4 --- Determination of HSP70 levels in blood cells sampled from seabream upon different seasons --- p.92 / Chapter 4.2.5 --- Immunoblotting analysis --- p.92 / Chapter 4.2.6 --- Enzyme-linked Immnosorbent Assay (ELISA) --- p.93 / Chapter 4.2.7 --- Measurement of serum parameter in seabream --- p.95 / Chapter 4.2.7.1 --- Serum glucose --- p.95 / Chapter 4.2.7.2 --- Serum Cortisol --- p.96 / Chapter 4.3 --- Results --- p.96 / Chapter 4.3.1 --- Determination of HSP70 levels in blood cells sampled from seabream in different seasons --- p.96 / Chapter 4.3.1.1 --- Immunoblotting analysis --- p.96 / Chapter 4.3.1.2 --- Enzyme-linked immunosorbent assay (ELISA) --- p.96 / Chapter 4.3.2 --- Serum analysis of seabream sampled from fish farm in different seasons --- p.98 / Chapter 4.3.2.1 --- Serum glucose --- p.98 / Chapter 4.3.2.2 --- Serum Cortisol --- p.99 / Chapter 4.4 --- Discussions --- p.117 / Chapter 4.4.1 --- Characterization of HSP70 expression in blood cells of seabream --- p.117 / Chapter 4.4.2 --- Dynamicity of HSP70 content and thermo- tolerance of fish in different seasons --- p.118 / Chapter 4.4.3 --- Effect of transportation stress on HSP70 induction in blood cells of seabream --- p.121 / Chapter 4.4.4 --- Dynamicity of serum glucose level in seabream subjected to seasonal variations --- p.123 / Chapter 4.4.5 --- Effect of transportation stress on the serum glucose level of seabream in different seasons --- p.124 / Chapter 4.4.6 --- Dynamicity of senam Cortisol level in seabream subjected to seasonal variations --- p.125 / Chapter 4.4.7 --- Effect of transportation stress on the serum Cortisol level of seabream in different seasons --- p.126 / Chapter 4.4.8 --- "Comments on the use of HSP70, serum Cortisol and serum glucose as biomarkersin environmental supervision" --- p.126 / Chapter 4.5 --- Conclusions --- p.129 / Chapter Chapter 5: --- "In vitro and in vivo effects of Cortisol, dexamethasone and adrenaline on the induction of HSP70 in seabream, Sparus sarba" --- p.131 / Chapter 5.1 --- Introduction --- p.132 / Chapter 5.2 --- Materials and methods --- p.133 / Chapter 5.2.1 --- Overall experimental design --- p.133 / Chapter 5.2.2 --- Acclimation of fish and regimes of treatment --- p.133 / Chapter 5.2.3 --- Serum Cortisol and adrenaline analysis --- p.135 / Chapter 5.2.4 --- "Protein analysis, gel electrophoresis, immuno- blotting and ELISA analysis" --- p.136 / Chapter 5.3 --- Results --- p.137 / Chapter 5.3.1 --- "HSP70 level in blood cells treated with Cortisol, dexamethasone and adrenaline in vitro" --- p.137 / Chapter 5.3.2 --- "Serum hormones and HSP70 level in tissues of fish injected with Cortisol, adrenaline and dexamethasone invivo" --- p.137 / Chapter 5.3.2.1 --- Serum Cortisol and adrenaline level of fish after hormone injections --- p.137 / Chapter 5.3.2.2 --- "HSP70 level in blood cells, brain and liver tissue of fish after hormone injections" --- p.138 / Chapter (I) --- Level of HSP70 in blood cells of fish after hormone injections --- p.138 / Chapter 5.4 --- Discussions --- p.156 / Chapter 5.4.1 --- In vitro and in vivo study of the hormonal effect on HSP70 level in blood cells of seabream --- p.156 / Chapter 5.4.2 --- Hypothetical mechanism of hormone-receptor mediated HSP70 regulation --- p.158 / Chapter 5.4.3 --- In vivo study of the hormonal effect on HSP70 level in blood cells of seabream --- p.160 / Chapter 5.4.4 --- In vivo study on the hormonal effect of HSP70 synthesis in liver of seabream --- p.163 / Chapter 5.4.5 --- In vivo study on the hormonal effect of HSP70 synthesis in brain tissue of seabream --- p.165 / Chapter 5.4.6 --- HSP70 level in different tissues of fish in relation to the induction and sensitivity against stress --- p.166 / Chapter 5.5 --- Conclusions --- p.169 / Chapter Chapter 6: --- Summary --- p.171 / References --- p.175

O desenvolvimento embrionário tem inicio a partir da fecundação do oócito pelo espermatozóide no interior do oviduto. O oviduto é um órgão tortuoso que mede de 20 a 30cm e está dividido em três porções: istmo, ampola e infundíbulo. Os hormônios influenciam a atividade das células-alvo pela ligação de moléculas receptoras especificas. A imuno-histoquímica é o conjunto de procedimentos que utiliza anticorpos como reagentes específicos para detecção de antígenos presentes em células ou tecidos, portanto, através desta técnica é possível verificar a presença de receptores hormonais em determinados órgãos. Este estudo teve como objetivo localizar a presença de receptores para o hormônio luteinizante (LH) nas diferentes porções do oviduto utilizando a técnica de imuno-histoquímica. Foram utilizadas 18 éguas que se encontravam em estro, ou seja, apresentavam um folículo maior que 35mm e trato reprodutivo condizente com a fase estrogênica do ciclo estral. Das 18 éguas utilizadas neste trabalho, 16 éguas (88,8 %) apresentaram receptores para hormônio luteinizante (RLH) no oviduto. Destas 16 éguas, 8 (44,4 %) apresentaram RLH no epitélio e 7 (38,8 %) apresentaram RLH no tecido muscular do istmo, 14 (77,7 %) apresentaram RLH no epitélio e 13 (72,2 %) no tecido muscular da ampola, 10 (55.5 %) apresentaram RLH no epitélio e 1 (5,5 %) no tecido muscular do infundíbulo. Nas éguas que apresentaram receptores no epitélio a intensidade verificada foi de 1,5; 2,5 e 2,6 no istmo, ampola e infundíbulo, respectivamente enquanto que na porção muscular foi de 1,14; 2,3 e 3 respectivamente, para cada uma das três porções estudadas. Foi verificada uma maior intensidade de receptores na ampola do oviduto, o que pode relacionar o LH no processo de fecundação do oócito pelo o espermatozóide. / Embryonic development begins with the fertilization of the egg by the sperm in the oviduct. The oviduct is a tortuous organ which extended measures 20 to 30cm and is divided into three parts: the isthmus, ampulla and infundibulum. Hormones influence the activity of target cells by binding to specific receptor molecules. Immunohistochemistry is the set of procedures that use antibodies as reagents for detection of specific antigens present in cells or tissues, therefore, using this technique it is possible to verify the presence of hormone receptors in certain organs. This study aimed to verify the presence of hormone receptors for luteinizing hormone (LH) in different portions of the oviduct using the technique of immunohistochemistry. We used 18 mares were in estrus that had a follicle greater than 35mm and reproductive tract consistent with the estrogen phase of the estrous cycle. From the 18 mares that were part of that study, 16 mares (88.8 %) had receptors for luteinizing hormone (RLH) in the oviduct. From these 16 mares, 8 (44.4 %) had RLH in the epithelium and 7 (38.8 %) had RLH in the muscle of the isthmus, 14 (77.7 %) had RLH epithelium and 13 (72.2 %) in the muscle of the ampulla, 10 (55.5 %) had RLH in the epithelium m and 1 (5.5 %) in the muscle of the infundibulum. In mares that had receptors in the epithelium the intensity verified was 1,5 ; 2,5 and 2,6 on the isthmus, ampulla and infundibulum, respectively while in the muscular portion was 1,14 ; 2,3 and 3 respectively, for each of the three portions studied. It was verified a greater intensity of receptors in the ampulla of the oviduct, which may relate the LH in the process of fertilization of the oocyte by the sperm.

Estrogen-receptor-related receptor gamma, ERRγ, is highly expressed in cartilage and upregulates the chondrogenic transcription factor, Sox9, in a chondrocytic cell line. To assess the effect of increasing ERRγ activity on cartilage in vivo, we generated transgenic animals driving ERRγ expression with a chondrocyte-specific promoter. I verified that one transgenic line exhibited 26% increased ERRγ protein at E14.5. No major morphological defects were seen at this stage, but I observed significant reduction in the size of the appendicular skeleton in P7 mice, such that all elements of the appendicular skeleton were significantly reduced by 4 – 10%. I continued the phenotype analysis at the histological level and found that the P7 animals displayed significantly reduced growth plate height, caused by deficiencies in the size of the proliferative and hypertrophic zones of the growth plate. This suggests a previously unknown role for ERRγ in regulating endochondral ossification in growth plate chondrocytes.

Estrogen-receptor-related receptor gamma, ERRγ, is highly expressed in cartilage and upregulates the chondrogenic transcription factor, Sox9, in a chondrocytic cell line. To assess the effect of increasing ERRγ activity on cartilage in vivo, we generated transgenic animals driving ERRγ expression with a chondrocyte-specific promoter. I verified that one transgenic line exhibited 26% increased ERRγ protein at E14.5. No major morphological defects were seen at this stage, but I observed significant reduction in the size of the appendicular skeleton in P7 mice, such that all elements of the appendicular skeleton were significantly reduced by 4 – 10%. I continued the phenotype analysis at the histological level and found that the P7 animals displayed significantly reduced growth plate height, caused by deficiencies in the size of the proliferative and hypertrophic zones of the growth plate. This suggests a previously unknown role for ERRγ in regulating endochondral ossification in growth plate chondrocytes.

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Vaginal atrophy symptoms such as dryness, irritation, and itching, are common after menopause. Vaginal estrogen therapy is the most effective treatment but not appropriate for all women. Women with estrogen-responsive breast cancer treated with aromatase inhibitor (AI) treatment, suppressing estrogen levels, often suffer from more pronounced vaginal atrophy symptoms. However, vaginal estrogen treatment is not recommended, leaving them without effective treatment options. The aim of this thesis was to study the effect of long-term anti-estrogen therapy on circulating estrogen levels and biochemical factors in vaginal mucosa in relation to morphological changes and clinical signs of vaginal atrophy. Circulating estrogen levels were analyzed by use of mass spectrometry and radioimmunoassay. Immunohistochemistry was used to study vaginal proliferation and steroid hormone receptors in vaginal mucosa. Vaginal gene expression was studied by use of microarray technology and bioinformatic tools, and validated by use of quantitative real-time PCR and immunohistochemistry. An estrogenic regulation of aquaporins and a possible role in vaginal dryness was investigated in vaginal mucosa and in Vk2E6E7 cells. Aromatase inhibitor-treated women had higher than expected estradiol and estrone levels but still significantly lower than other postmenopausal women. Aromatase was detected in vaginal tissue, the slightly stronger staining in vaginal mucosa from AI-treated women, suggest a local inhibition of vaginal aromatase in addition to the systemic suppression. Vaginal mucosa from AI-treated women had weak progesterone receptor, and strong androgen receptor staining intensity. Low estrogen levels lead to low expression of genes involved in cell adhesion, proliferation, and differentiation as well as weak aquaporin 3 protein immunostaining. The higher than expected estrogen levels in AI-treated women suggest that estrogen levels might previously have been underestimated. Systemic estrogen suppression by treatment with AIs, and possibly also by local inhibition of vaginal aromatase, results in reduced cell adhesion, proliferation, differentiation, and weak aquaporin 3 protein staining. Low proliferation and poor differentiation leads to fewer and less differentiated superficial cells affecting epithelial function and possibly also causing vaginal symptoms. Aquaporin 3 with a possible role in vaginal dryness, cell proliferation, and differentiation should be further explored for the development of non-hormonal treatment options for vaginal symptoms.

Diss. (sammanfattning) Stockholm : Karol. inst., 2007. / Härtill 4 uppsatser.