Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 89-109 and 224-228).

Thesis (PhD)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Although the progestins medroxyprogesterone acetate (MPA) and norethisterone acetate (NET-A) are widely used in reproductive therapy, the steroid receptors and their target genes involved in the actions of MPA and NET-A are not well understood. Surprisingly, it had not yet been investigated whether doses of MPA and NET-A used for contraception and HRT cause significant side effects through various target genes via the glucocorticoid receptor (GR). In this thesis results of in vitro studies showed that, MPA, like dexamethasone (dex) and prog, significantly repressed tumour necrosis factor (TN F)-stimulated IL-6 protein production, and IL-6 and IL-8 promoter reporter constructs at the transcriptional level in L929sA cells, via interference with nuclear factor KB (NFKB) and activator protein-1 (AP-1) transcription factors. Like dex and prog, MPA did not affect NFKB DNA-binding activity. Furthermore, unlike dex and prog, MPA did not inhibit mitogen-activated protein kinase (MAPK) activity. The antagonistic effects of the GR and progesterone receptor (PR) antagonist, RU486, as well as the MPAinduced nuclear translocation of the GR, strongly suggest that the actions of MPA in these cells are mediated at least in part via the GR. Although the mechanism was not investigated as extensively as for MPA, NET-A was shown to repress IL-8 promoter reporter activity very weakly relative to dex, MPA and prog in Hek293 cells stably transfected with the rat GR. Furthermore, NET-A, like MPA, dex and prog did not interfere with the DNA-binding activity of NFKB. Significant transactivation of a GRE-driven promoter reporter construct by MPA and dex in L929sA via endogenous GR and COS-1 cells via expressed rat GR, and by MPA, dex and prog in Hek293 cells via expressed rat GR was also observed. In contrast, NET-A, unlike MPA, dex and prog showed no transactivation in Hek293 cells. MPA, NET-A and prog were shown to compete with dex for binding to the endogenous human GR in human lung carcinoma A549 cells. Similarly, MPA and NET-A were shown to compete with dex for binding to expressed rat GR in COS-1 cells. MPA displayed a higher relative binding affinity than NET-A for the GR in both systems, and a higher relative binding affinity than prog in A549 cells. Equilibrium dissociation constants (Ki values) for MPA (Ki = 10.8 ± 1.1 nM), NET-A (Ki = 270 ± 1.3 nM) and prog (Ki = 215 ± 1.1 nM) towards the human GR in A549 cells were also established. Furthermore, dose-response curves showed that MPA displays significantly greater GC agonist potency and efficacy than NET-A and prog for both transactivation of a synthetic GRE-reporter construct and transrepression of a synthetic IL-8 reporter construct via expressed rat GR in Hek293 cells, as NET-A showed no transactivation and very weak partial agonist activity for transrepression. Based on these observations, MPA behaves as a GR agonist whereas NET-A is proposed to be a weak antagonist. These results show that MPA and NET-A are not alike and not the same as prog in their mechanism of action via the GR, which may have serious health implications in vivo. Such insights may provide women and their clinicians with more information to facilitate the selection of contraception or reproductive therapy regimes with fewer side effects. / AFRIKAANSE OPSOMMING: Alhoewel MPA en NET-A algemeen gebruik word in hormoontherapie, is dit nie duidelik watter steroïedreseptore en teikengene betrokke is by die werking van MPA en NET-A nie. Verrassend is dat geen studie nog gedoen is om te bepaal of die dosisse van MPA en NET-A wat gebruik word in voorbehoeding en hormoonvervangingsterapie (HVT), newe-effekte veroorsaak deur die glukokortikoïedreseptor (GR) en verskeie teikengene nie. In hierdie tesis is in L929sA selle aangetoon dat MPA, net soos deksametasoon (dex) en prog, TNF-gestimuleerde IL-6 produksie onderdruk, en dat IL-6 en IL-8 promoter-rapporteerderkonstrukte op transkripsionele vlak onderdruk word deur middel van inmenging met NF-KB en AP-1 transkripsie-faktore. Net soos dex en prog het MPA nie die DNA-bindingsaktiwiteit van NF-KB beïnvloed nie. Anders as dex en prog het MPA egter nie MAPK aktiwiteit onderdruk nie. Die antagonistiese effekte van RU486, asook die MPA-geïnduseerde translokasie van die GR na die selkern, dui sterk daarop dat die effekte van MPA in hierdie selle ten minste gedeeltelik deur die GR geskied. Alhoewel die meganisme vir NET -A nie so breedvoerig bestudeer is as dié van MPA nie, is tog aangetoon dat, in Hek293 selle wat stabiel getransfekteer is met die rot GR, die onderdrukking van die IL-8 promoter deur NET-A baie swakker is as met dex, prog en MPA. Verder is daar ook gevind dat NET-A, net soos MPA, dex en prog, nie kon inmeng met die DNA-bindingsaktiwiteit van NF-KB nie. Beduidende transaktivering van 'n GRE-bevattende promoterrapporteerderkonstruk deur MPA en dex in L929sA en COS-1 selle, en deur MPA, dex en prog in Hek293 selle, is ook gevind. Daarteenoor het NET-A, anders as MPA, dex en prog, geen transaktivering in Hek293 selle getoon nie. Verder moes die relatiewe bindingsaffiniteit (ewewigs-dissosiasiekonstantes) van MPA, NET-A en prog vir die GR, asook die relatiewe sterkte en effektiwiteit vir transaktivering en transonderdrukking van verskeie teikengene deur die GR, ook bepaal word. Daar is gevind dat MPA, NET-A en prog meeding met dex vir binding aan die endogene GR in mens longkarsinoom A549 selle. Soortgelyk hieraan is ook gevind dat MPA en NET-A meeding met dex vir binding aan rot GR wat in COS-1 selle uitgedruk is. MPA het in beide sisteme 'n hoër relatiewe bindingsaffiniteit vir die GR getoon as NET-A, asook 'n hoër relatiewe bindingsaffiniteit as prog in A549 selle. Ewewigs-dissosiasiekonstantes (Ki waardes) vir MPA (Ki = 10.8 ± 1.1 nM), NET- A (Ki = 270 ± 1.3 nM) en prog (Ki = 215 ± 1.1 nM) vir die mens GR in A549 selle is ook bereken. Dosisrespons-grafieke het ook aangedui dat MPA 'n beduidend beter GC sterkte en effektiwiteit as NET-A en prog het, vir beide transaktivering van 'n sintetiese GRE-rapporteerderkonstruk en transonderdrukking van 'n sintetiese IL-8 rapporteerderkonstruk via rot GR wat uitgedruk is in Hek293 selle. Dit kon afgelei word aangesien NET-A geen transaktivering en slegs baie swak gedeeltelike agonisaktiwiteit vir transonderdrukking getoon het. Op grond van hierdie waarnemings tree MPA op as 'n GR agonis, terwyl dit lyk asof NET-A 'n swak antagonis is. Hierdie resultate dui aan dat MPA en NET-A nie dieselfde is nie, en ook nie dieselfde meganisme van werking deur die GR het as prog nie. Dit kan ernstige gesondheidsimplikasies inhou in vivo. Hierdie insigte kan dus meer inligting aan vroue en kliniese personeel verskaf om sodoende die keuse van voorbehoeding of voortplantingsterapie met minder newe-effekte te vergemaklik.

Thesis (MScMed)--University of Stellenbosch, 2005. / Bibliography. Also available via the Internet.

Thesis(M.P.H.M. (Primary Health Care Management))--Mahidol University, 2004.

Orientador: Adriana Vitorino Rossi / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-09T10:06:34Z (GMT). No. of bitstreams: 1 Pinheiro_TaniaAparecidaLopes_M.pdf: 1079209 bytes, checksum: 7315b8210dfb843979f6a17ba083ed67 (MD5) Previous issue date: 2006 / Resumo: Acetato de medroxiprogesterona (AMP) é um derivado da progesterona que vem sendo usado para contracepção por cerca de 30 milhões de mulheres e também vem aplicado em tratamentos de câncer. Como em alguns casos, AMP gera efeitos colaterais desfavoráveis, sua dosagem deve ser bem controlada. Neste trabalho, foi desenvolvido um método espectrofotométrico para quantificação de AMP em fármacos pela reação com Mo(VI) em meio fortemente ácido. Testes preliminares como avaliação do ponto de fusão e spot test com ácido sulfúrico foram introduzidos para identificar o AMP de outros compostos, como derivados de cortisona que também podem reagir com Mo(VI). A reação foi otimizada a partir da avaliação de algumas variáveis do sistema reacional: tempo e temperatura de aquecimento, solvente e concentração de ácido. Os resultados obtidos indicaram que as melhores condições para o sistema reacional são: solução 9 mol L de H2SO4, 70 minutos de aquecimento a 90 °C e uso de AMP em acetonitrila. Nestas condições, foram obtidas curvas analíticas com a absorbância em 406 nm do produto formado cuja concentração é diretamente proporcional à concentração de AMP na faixa de 5,87x10 a 10,00x10 mg mL, com coeficiente de correlação linear = 0,9972 e limite de detecção = 1,77x10 mg mL. A precisão foi avaliada por intra (repetibilidade) e inter (precisão intermediária) testes, tendo sido obtidos desvios máximos de + 1,9%. Quando comparado com um método de quantificação de AMP em medicamentos comerciais por cromatografia líquida de alta eficiência, os resultados com o método proposto não apresentaram diferenças estatisticamente significativas com 95 % de confiança, indicando bom desempenho do método baseado na reação colorimétrica, que tem favoráveis características de simplicidade instrumental / Abstract: Acetate of medroxiprogesterona (AMP) is one derivative of the progesterone, that have been used used as contraceptive method for about 30 million women. And in treatments of cancer. In some cases, AMP cause favorable collateral effect, therefore its dosage must be well controlled. In this work, a spectrophotometric method for quantification of AMP in pharmaceutical preparation using the reaction with Mo(VI) in strong acid was developed. Preliminary tests as evaluation of the fusing point and spot test with H2SO4 had been introduced in order to identify the AMP from other compounds, as corticoids, that ca also react with Mo(VI). The reaction was optimized from the evaluation of some reaction variable: time and temperature of heating, AMP solvent, acid concentration. The optimized conditions are 9 mol L H2SO4; heating for 70 minutes at 90 °C and acetonirile solvent for AMP. In these conditions, analytical curves of absorbance at 406 nm of the product formed whose concentration is directly proportional to the initial concentration of AMP were obtained from 5,87x10 a 10,00x10 mg mL , with linear coefficient = 0,9972 and detection limit = 1,77x10 mg mL. The precision was evaluated by intra (repeatability) and inter (intermediate precision) tests, having been achieved maximum relative deviation = + 1,9 %. When compared with a HPLC method of quantification of AMP in commercial medicines, the results achieved with this method did give statistical significant differences at 95 % confidence level, indicating good performance of the colorimetric method, which has the advantage of instrumental simplicity / Mestrado / Quimica Analitica / Mestre em Química

Orientador: Luis Guillermo Bahamondes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T05:05:19Z (GMT). No. of bitstreams: 1 Viola_AlexandredeSouza_D.pdf: 11179010 bytes, checksum: b640fe59edc62c7e4656a85dd2252e89 (MD5) Previous issue date: 2011 / Resumo: Objetivos: Avaliar a densidade mineral óssea (DMO) em usuárias por pelo menos um ano e ex-usuárias na pós-menopausa do contraceptivo injetável com acetato de medroxiprogesterona de depósito (AMP-D). Sujeitos e métodos: Foram realizados dois estudos, ambos de corte transversal com usuárias atuais em idade reprodutiva de AMP-D e outro com mulheres na pós-menopausa que usaram AMP-D até a menopausa. Cada usuária foi comparada com uma mulher nunca usuária de AMP-D, da mesma idade (± 1) e mesmo índice de massa corporal (kg/m2) (± 1). Nelas, foram medidas a DMO nas porções distal e ultradistal do rádio do antebraço não dominante pela técnica de DXA (double X-ray absoptiometry). No primeiro estudo foram incorporadas 232 mulheres usuárias do AMP-D e igual número que usavam dispositivo intrauterino (DIU) TCu380A como grupo de controle. As mulheres foram divididas em 5 grupos (1-5) de acordo com o tempo de uso do AMPD: 1-3; 4-6; 7-9; 10-12 e 13-15 anos, respectivamente. No segundo estudo foram incorporadas 79 mulheres com até 5 anos desde a menopausa, sendo 24 ex-usuárias de AMP-D até a menopausa e 55 ex-usuárias de DIU TCu380A também até a menopausa e que tinham avaliado previamente a DMO aos 1-2 e 3 anos após a menopausa. Resultados: No primeiro estudo, a média da idade (± EPM) das usuárias de AMP-D e DIU foi 38,3±0,5 e 38,1±0,57 anos e a média do IMC (índice de massa corporal) (kg/m2) (± EPM) foi de 26,4±0,3 e 26,3±0,3 (kg/m2) para cada grupo, respectivamente. As usuárias de ambos os grupos com pouco tempo de uso dos métodos eram mais jovens que aquelas que os usavam por longo período de tempo, e ambos os grupos apresentavam menor IMC nos primeiros 5 anos de uso. Paridade, cor, prática de atividade física manualmente foram significativamente maiores no grupo das usuárias de DIU (p<0,053, p<0,040 e p<0,012), e o ato de lavar roupas, nas usuárias de AMP-D (p<0,025). Os outros dados sociodemográficos, antropométricos e obstétricos não apresentaram diferenças significantes. As médias da DMO nas regiões distal e ultradistal do rádio foram similares entre as usuárias de AMP-D e DIU, exceto nas usuárias de AMP-D com mais de 13 anos de uso, e que apresentaram DMO significativamente menor (p<0,041 e 0,042, respectivamente). Houve relação direta nos dois grupos entre a DMO e o IMC (kg/m2) e relação inversa entre DMO e idade, nas regiões distal e ultradistal do rádio. No segundo estudo, o tempo de uso de ambos contraceptivos foi de 9,4 ± 3,8 e 14,7 ± 6,2 anos para o AMP-D e DIU, respectivamente. A DMO na região distal nos anos 1; 2-3 e 5-6 após a menopausa foi de 0,426 e 0,445; 0,421 e 0,434 e 0,437 e 0,427 nas usuárias de AMP-D e DIU, respectivamente. Na região ultradistal os valores foram: 0,350 e 0,380; 0,360 e 0,367 e 0,388 e 0,373, respectivamente, todos sem significância estatística. Conclusão: Não foi detectado efeito deletério na DMO do antebraço nas usuárias de AMP-D, com exceção daquelas que o usaram por mais de 13 anos, ou ex-usuárias de AMP-D que estavam na pós-menopausa, quando comparadas às usuárias de DIU. Também foi observada uma relação direta entre DMO e IMC (kg/m2) e indireta entre DMO e idade / Abstract: Objectives: Assess bone mineral density (BMD) in users for at least a year and ex-users in injectable contraceptive postmenopausal with medroxyprogesterone acetate depot (DMPA). Subjects and methods: Two studies have been conducted, both transversal cutting with current users of reproductive age of DMPA and another with postmenopausal women who have used DMPA until menopause. Each user was compared with a woman never using DMPA, the same age (± 1) and the same body mass index (2kg/m) (± 1). In them, were measures the DMO in distal portion and ultra-distal of non-dominant forearm radio technique by DXA (double X-ray absoptiometry). In the first study were incorporated into 232 women users of DMPA and an equal number who used intrauterine device (IUD) TCu380A as control group. Women were divided into 5 groups (1-5) in accordance with the usage time of DMPA: 1 -3; 4-6; 7-9; 10-12 and 13-15 years, respectively. In the second study were incorporated into 79 women with up to 5 years since menopause, being 24 ex-users of DMPA until menopause and 55 ex-users IUD TCu380A also until menopause and who had previously evaluated the DMO to 1-2 and 3 years after menopause. Results: in the first study, the average age (± EPM) of users of DMPA and IUD was 38.1 and 38.3 ± 0.5 ± 0.57 years and average BMI (body mass index) (kg/m2) (± EPM) was 0.3 ± ± 26.3 and 26.4 0.3 (kg/m2) for each group, respectively The users of both groups with little time using the methods were younger than those who used over a long period of time, and both groups had lower BMI in the first 5 years of use. Parity, color, physical activity practice manually were significantly higher in the Group of IUD users (p <0.053, p< 0.040 and p<0.012), and the Act of washing clothes, in the users of DMPA (p < 0.025). The other demographic and anthropometric data partners did not show significant differences obstetric. The average of the DMO in the distal region of the ultra-distal and radio were similar between the users of DMPA and IUD users, except for DMPA with more than 13 years of use with DMO had significantly lower (p < 0.041 and 0.042, respectively). There was no direct relation in two groups between the DMO and the BMI (kg/m2) and inverse relationship between DMO and age, in the region and distal radio ultra-distal. In second study, the time using both contraceptives was 9.4 ± 3.8 and 14.7 ± 6.2 years for DMPA and IUD, respectively. The DMO in distal region in the years 1; 2-3 and 5-6 after menopause was 0.426 and 0.445; 0.434 and 0.421 and 0.437 and requirements in 0.427 DMPA and IUD, respectively. In the ultra-distal region the figures were: 0.350 and 0.380; 0.360 and 0.367 and 0.388 0.373, respectively, and all without statistical significance. Conclusion: No deleterious effects on the DMO requirements in forearm DMPA, with the exception of those who have used for over 13 years, or DMPA ex-users were in post-menopausal women, compared the IUD users. Also we see a direct relationship between the DMO and BMI (kg/m2) and indirectly between DMO and age / Doutorado / Fisiopatologia Ginecológica / Doutor em Ciências da Saúde

Background: HSV-2 was estimated to infect 491 million people worldwide, with women disproportionately affected by HSV-2. Understanding factors that influence susceptibility to HSV-2 in women is important in preventing infections. Through various studies, the progestin-based contraceptive DMPA exhibited immunosuppressive effects, and has shown increased susceptibility to HIV and HSV-2. Studies comparing DMPA to other contraceptives like NET suggest that NET may be safer. In vivo NET effects have not been characterized thoroughly to better understand the effect of NET on susceptibility to HSV-2. Therefore, this study aimed to compare the effects of NET and DMPA in mouse models that affect susceptibility to HSV-2. We hypothesized that NET treated mice will have decreased susceptibility to HSV-2 compared to DMPA but elevated compared to normal mice. Method of study: Ovariectomized mice were treated with DMPA (2mg) and NET (2 mg injections, 2.5 mg pellets or 5 mg pellets) for 10 days and intravaginally immunized with HSV-2 TK-, then intravaginally challenged with WT HSV-2 ~4-7 weeks later. Primary intravaginal WT HSV-2 challenges were conducted in ovariectomized and normal mice after 10 days of DMPA and NET treatment. Viral titers, pathology and survival were examined. Mucus production in the vagina was investigated through immunohistology. Effects of hormones on immune cells were explored in the lymph nodes, spleens, and vaginal tracts through flow cytometry. Results: Increased mucus was consistently observed in the vaginal tracts of mice after treatment with NET 2.5 mg and 5 mg treated mice, but not with DMPA Therefore, NET treated mice displayed reduced viral shedding and delayed pathology compared to DMPA treated mice. No significant changes occurred in immune cells analyzed post DMPA and NET treatment, although there were trends of increased T cells in progestin treated mice. However, more experiments need to be conducted to confirm observed trends. Conclusion: NET treatment in mice results in mucus production in the vaginal tract, a potential mechanism impeding intravaginal HSV-2 infection and could be applied to other STIs. This provides insight into protective effects of NET compared to DMPA allowing women to make informed decisions regarding hormonal contraceptives. / Thesis / Master of Science in Medical Sciences (MSMS)

Thesis (PhD)--Stellenbosch University, 2012. / Bibliography / ENGLISH ABSTRACT: Most individuals latently infected with Mycobacterium tuberculosis (Mtb) contain the infection by a balance of effector and regulatory immune responses. However, this balance can be influenced by steroid hormones such as glucocorticoids (GCs), which are known to increase the risk of reactivation of TB. The contraceptive medroxyprogesterone acetate (MPA), which also possesses selective glucocorticoid activity, is widely used in developing countries with approximately 60% of women on contraceptives using MPA in our study cohort. Therefore, our aim was to investigate the effect of this hormone on protective immune responses to BCG in HIV negative household contacts of active TB patients. When PBMCs of TB household contacts were stimulated with BCG in the presence of 10 μM MPA; this hormone displayed both glucocorticoid as well as progestogenic properties. Similarly to cortisol, MPA suppressed antigen specific expression of a range of cytokines including IL-1α, IL-1ra, IL- 17, TNFα, IL-5 and IFNγ. Dose response curves showed that MPA can also alter expression of some cytokines at lower contraceptive doses (in the nano molar range). To assess whether this effect of MPA in vitro also occurs in women using this hormone as contraceptive the PBMCs of MPA users and controls were stimulated with BCG and the levels of up to 29 different cytokines measured by luminex analysis. PBMCs of MPA users produced significantly lower levels of cytokines involved in immune responses against Mtb such as IL-12p40, IL-1α, IL-10, IL-13 and G-CSF, which corresponds with lower numbers of circulating monocytes observed in these women. These findings warrant further investigation and clinical trials should investigate the risk of progression from latent to active TB disease in women using this contraceptive. These trials, however, require a large number of participants and are prohibitively expensive; therefore it was decided to setup an Mtb/MPA mouse model to determine the effect of MPA on the disease outcome. BALB/c and C57BL/6 mice were injected with a weekly dose of one mg MPA or PBS and infected with 30 colony forming units of Mtb H37Rv one week after commencing the hormonal treatment. Both strains were included to establish which strain best represents the human model. Three and eight weeks post infection the MPA treated C57BL/6 mice had a significantly higher bacterial load in their lungs compared to untreated mice, whereas no difference was found in the bacterial loads of the BALB/c mice. MPA treated C57BL/6 mice had significantly lower serum levels of IL-10 and G-CSF and MPA treated BALB/c mice lower serum levels of IFNγ, when compared to untreated mice. Furthermore, cells isolated from the MLNs of MPA treated C57BL/6 mice, produced significantly less TNFα, significantly more IP-10 and less IL-10 in response to PPD, while MLN cells of MPA treated BALB/c mice produced significantly less IFNγ, IL-2, IL-17, GM-CSF and MCP-1. Data of the C57BL/6 mouse strain correlated with our human data and can it therefore be said that the C57BL/6 mouse strain, together with the serum concentration of MPA used in these experiments, is a good model to determine the effect of MPA in the context of a low dose Mtb infection. To conclude MPA use could therefore alter susceptibility to TB, TB disease severity as well as change the efficacy of new BCG-based vaccines, especially prime-boost vaccine strategies which may be administered to adult of adolescent women in the future. / AFRIKAANSE OPSOMMING: Die meeste mense wat latent met Mycobacterium tuberculosis (Mtb) geïnfekteer is, hou die infeksie onder beheer deur ʼn balans te handhaaf tussen effektor en regulatoriese immuunresponse. Hierdie balans kan egter beïnvloed word deur steroïedhormone soos glukokortikoïede (GCs), wat bewys is om die risiko van die heraktivering van TB te verhoog. Die voorbehoedmiddel medroksiprogesteroon-asetaat (MPA), wat ook selektiewe glukokortikoïed-aktiwiteit toon, word wyd gebruik in ontwikkelende lande en omtrent 60% van die vrouens in ons studie-bevolking wat voorbehoedmiddels gebruik, gebruik MPA. Om dié rede wou ons die effek van hierdie hormoon op die beskermende immuun-response teenoor M.bovis Bacilli Calmette-Guérin (BCG) in HIV negatiewe huishoudelike kontakte (HHKe) van pasiënte met aktiewe TB ondersoek. Ons het gevind dat wanneer perifere bloed mononukleêre selle (PBMSe) met BCG gestimuleer word in die teenwoordigheid van 10 μM MPA, hierdie hormoon beide glukokortikoïede en progesterogeniese eienskappe toon. Soos kortisol het MPA die antigeenspesifieke-uitdrukking van ʼn reeks sitokiene, insluitend IL-1α, IL-1ra, IL-17, TNFα, IL-5 en IFNγ, onderdruk. Respons kurwes wat verskillende konsentrasies van hormoon insluit, het getoon dat MPA ook by laer (nano-molare) dosisse die uitdrukking van sommige sitokiene kon verander. Om te bepaal of hierdie in vitro effek van MPA ook in vrouens wat MPA as voorbehoedmiddel gebruik voorkom, het ons PBMSe van MPA-gebruikers and kontroles met BCG gestimuleer en die vlakke van tot 29 verskillende sitokiene met behulp van Luminexanalise gemeet. PBMSe van MPA-gebruikers produseer beduidende laer vlakke van IL-12p40, IL-1α, IL- 10, IL-13 en G-CSF, wat elk in imuunafweerreaksies teen Mtb betrokke is. Die afname in dié sitokiene het gepaard gegaan met laer hoeveelhede sirkulerende monosiete. Ons resultate regverdig verdere ondersoeke en kliniese proewe behoort die risiko van progressie vanaf latente tot aktiewe TB in vrouens wat hierdie voorbehoedmiddel gebruik te bepaal. Sulke proewe vereis egter groot getalle deelnemers en is skrikwekkend duur, om die rede het ons besluit om ʼn Mtb/MPA muis-model op te stel om sodoende die algehele effek van MPA op die uitkoms van die siekte te bepaal. BALB/c en C57BL/6 muise is met ʼn weeklikse dosis van een mg MPA of sout oplossing ingespuit en een week na die aanvang van die hormoon behandeling met 30 kolonie-vormende eenhede Mtb H37Rv geïnfekteer. Beide muis tipes was ingesluit om sodoende te bepaal watter tipe die mens data die beste verteenwoordig. Drie en agt weke na die infeksie het die MPA-behandelde C57BL/6 muise ‘n beduidende hoër bakteriële lading in hul longe gehad as die onbehandelde muise, maar was daar geen verskil in die bakteriële ladings in die longe van die BALB/c muise nie. MPA-behandelde C57BL/6 muise het beduidende laer serumvlakke van IL-10 en G-CSF gehad, terwyl MPA-behandelde BALB/c muise laer serumvlakke van IFNγ gehad het. Verder het ons gevind dat die geisoleerde limfosiete van MPA-behandelde C57BL/6 muise beduidend minder TNFα, beduidend meer IP-10 en minder IL-10 geproduseer het na stimulasie met PPD, terwyl die limfosiete van MPA-behandelde BALB/c muise beduidend minder IFNγ, IL-2, IL-17, GM-CSF en MCP-1 geproduseer het. Data van die C57BL/6 muise stem ooreen met die van ons mens studie en ons kan dus vermeld dat die C57BL/6 muise, tesame met die spesifieke serumkonsentrasie van MPA wat gebruik is, ʼn goeie model is om die effek van MPA in die konteks van ʼn lae-dosis Mtb-infeksie te bestudeer. MPA gebruik kan dus die vatbaarheid vir TB, asook die erns van die siekte verander en kan ook die effektiwiteit van nuwe BCG-gebaseerde entstowwe, veral prima-hupstoot enstowwe, wat moontlik in die nabye toekoms vir volwasse en adolessente vroue toegedien kan word, verander.

Thesis (PhD (Biochemistry))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Medroxyprogesterone acetate (MPA) and norethisterone (NET) and its derivatives (norethisterone enanthate (NET-EN); norethisterone acetate (NETA)), are used by millions of women as contraceptives and in hormone replacement therapy (HRT). Although both progestins are widely used, very little is known about their mechanism of action at the molecular level. In this thesis, the differential regulation of gene expression and molecular mechanism of action via different steroid receptors by these synthetic progestons, as compared to progesterone (Prog) was investigated in human cell lines. In the first part of the study, the effect of Prog, MPA and NET-A on the expression of endogenous cytokine genes was investigated in two epithelial cell lines of the human female genital tract, Ect1/E6E7 (an ectocervical cell line) and Vk2/E6E7 (a vaginal cell line). Quantitative realtime RT-PCR (QPCR) showed ligand-specific and cell-specific regulation of the interleukin (IL)-6, IL-8 and RANTES (Regulated-upon-Activation, Normal T cell Expressed and Secreted) genes with Prog, MPA and NET-A. Moreover, the repression of the TNF -induced RANTES gene by MPA in the Ect1/E6E7 cell line was found to be mediated by the androgen receptor (AR). The second part of the study focused on elucidating the androgenic activities of these two progestins, in comparison to Prog. Competitive binding in whole cells revealed that Prog, MPA and NET-A have a similar binding affinity for the hAR as the natural androgen dihydrotestosterone (DHT). Both transactivation and transrepression transcriptional assays demonstrate that, unlike Prog, MPA and NET-A are efficacious AR agonists, with activities comparable to DHT. Using a mammalian two-hydrid assay, it was shown that MPA and NET-A exert their androgenic actions by different mechanisms. NET-A, like DHT and other well-characterised androgens, induces the ligand-dependent interaction between the NH2- and COOH-terminal domains (N/C-interaction) of the AR independent of promoter-context, while MPA does this in a promoterdependent manner. In the third part of this study, competitive binding revealed that MPA and NET-A have a similar binding affinity to each other, but about a 100-fold lower affinity than Prog for the human mineralocorticoid receptor (hMR), while RU486 has an even lower affinity for the hMR. Promoter-reporter assays showed that MPA, NET-A and RU486 are all antagonists of the hMR, but unlike Prog, they have weak antagonistic activity. However, on the endogenous MR-regulated Orm-1 (a-glycolytic protein or orosomucoid-1) gene expressed in a rat cardiomyocyte cell line, NET-A and RU486, but not MPA, has similar antagonistic activity as Prog. This study is the first to show that, NET-A and RU486, but not MPA, can dissociate between transrepression and transactivation via the hMR. Taken together, these results show that natural Prog and the synthetic progestins, MPA and NET-A display differential promoter-, cell- and receptor-specific effects on gene expression. Furthermore they may have important implications for cervicovaginal immune function, cardiovascular and other physiological functions. / AFRIKAANSE OPSOMMING: Medroksieprogesteroon asetaat (MPA), noretisteroon (NET) en derivate daarvan (noretisteroon enantaat (NET-EN); noretisteroon asetaat (NET-A), word deur miljoene vroue gebruik as voorbehoedmiddels en vir hormoon vervangingsterapie (HVT). Tenspyte daarvan dat beide hierdie progestiene algemeen gebruik word, is min bekend oor hulle meganisme van werking op molekulêre vlak. In hierdie proefskrif word die differensiële regulering van geenuitdrukking asook die molekulêre meganisme van werking deur middel van steroïedreseptore van beide hierdie sintetiese progestiene, ondersoek, en vergelyk met progesteroon (Prog), in menslike sellyne. In die eerste deel van die studie is die effek van Prog, MPA en NET-A op die uitdrukking van endogene sitokinien gene ondersoek in twee epiteel sellyne van die menslike vroulike geslagskanaal, Ect1/E6E7 (‘n ektoservikale sellyn) en Vk2/E6E7 (‘n vaginale sellyn). Kwantitatiewe intydse RT-PKR het ligand-spesifieke en selspesifieke regulering van interleukien (IL)-6, IL-8 en RANTES (Regulering-na- Aktivering, Normale T-sel Uitgedrukte en Afgeskei) gene getoon met Prog, MPA en NET-A. Verder is gevind dat die onderdrukking van die TNF- - geïnduseerde RANTES geen deur MPA in die Ect1/E6E7 sellyn bemiddel word deur die androgeen reseptor (AR). Die tweede deel van die studie het gefokus op die toeligting van die androgeniese aktiwiteit van die twee progestiene in vergelyking met Prog. Kompeterende binding in volselle het getoon dat Prog, MPA en NET-A ‘n soortelyke bindings affiniteit vir die menslike AR as die natuurlike androgeen dehidrotestosteroon (DHT) vir die menslike AR het. Beide transaktiverings en transonderdrukkings transkripsionele analieses toon dat, anders as Prog, MPA en NET-A effektiewe AR agoniste is met aktiwiteite wat vergelykbaar is met die van DHT. Deur die gebruik van ‘n soogdier twee-hibried toets, kon gewys word dat MPA en NET-A hul androgeniese effekte uitoefen deur verskillende meganismes. NET-A, soos DHT en ander goed gekarakteriseerde androgene, induseer die ligand-afhanklike interaksie tussen die NH2- en COOH-terminale domeine (N/C-interaksie) van die AR, onafhanklik van die promoter-konteks. MPA, aan die ander kant, doen dit op ‘n promoter-afhanklike manier. In die derde deel van die studie het kompeterende binding getoon dat MPA en NETA soortelyke relatiewe bindings affiniteite vir die menslike mineralokortikoïed reseptor (hMR) het, maar dat hierdie affiniteit ongeveer 100-voud laer is as die van Prog en dat die affiniteit van RU486 vir hMR selfs nog laer is. Promoter-rapporteerder toetse het getoon dat MPA, NET-A en RU486 almal antagoniste van die hMR is, maar anders as Prog, is hierdie ‘n swak antagonistiese aktiwiteit. Nietemin, op die endogene MR-gereguleerde Orm-1 ( -glikolitiese proteïen of orosomukoïed-1) geen, uitgedruk in ‘n rot kardiomiosiet sellyn, het NET-A en RU486, maar nie MPA nie, ‘n soortgelyke antagonistiese aktiwiteit as Prog. Hierdie studie is die eerste om te wys dat NET-A en RU486, maar nie MPA nie, kan onderskei tussen transrepressie en transaktivering deur middel van die hMR. Samevattend toon die resultate dat natuurlike Prog en die sintetiese progestiene, MPA en NET-A, ‘n differentiële promoter-, sel- en reseptor-spesifieke effek op geenuitdrukking het. Verder mag die resultate belangrike implikasies vir servikovaginale immuunfunksie, asook kardiovaskulêre en ander fisiologiese funksies, inhou.

Objetivo: Avaliar os efeitos dos estrogênios conjugados eqüinos (ECE), isolados ou associados ao acetato de medroxiprogesterona (AMP) sobre os glicosaminoglicanos do colo e do corno do útero de ratas. Métodos: 40 ratas adultas, após 30 dias de ooforectomia foram distribuídas em quatro grupos: GI - controle (veículo); GII - ECE (50 g/Kg, por dia); GIII - AMP (0,2 mg/Kg, por dia) e GIV - ECE (50 g/Kg, por dia) + AMP (0,2 mg/Kg, por dia). As substâncias foram administradas por gavagem por 28 dias consecutivos, sendo que ao final, após anestesia, o colo e o corpo do útero foram retirados e mergulhados em acetona para detecção e quantificação dos glicosaminoglicanos. Os glicosaminoglicanos sulfatados foram submetidos a eletroforese em gel de agarose e o ácido hialurônico a ensaio fluorimétrico (ELISA-like). Os resultados foram analisados pelo teste de t de Student e de ANOVA, complementado pelo teste de Tukey-Kramer (p<=0,05). Resultados: Foi detectada, em todos os grupos, maior concentração de glicosaminoglicanos sulfatados tanto no colo como nos cornos uterinos, em especial do dermatam sulfato. Já a concentração de ácido hialurônico foi maior no corno do que no colo. Com relação ao dermatam sulfato, os estrogênios promoveram incremento, tanto no colo quanto no corpo, sendo que a medroxiprogesterona bloqueou esta ação nos cornos uterinos. Os estrogênios e a medroxiprogesterona, por sua vez, apresentaram ação positiva nos níveis de heparam sulfato no colo do útero, já no corno este efeito foi atribuído à medroxiprogesterona. Conclusões: O perfil e a quantificação dos glicosaminoglicanos nas duas porções do útero de ratas são diferentes. Os glicosaminoglicanos sulfatados, em especial o dermatam sulfato, mostrou-se em maior concentração tanto no colo quanto no corno uterino. Os estrogênios e a medroxiprogesterona têm ação positiva nos glicosaminoglicanos sulfatados do colo, enquanto a medroxiprogesterona apresenta efeito antagônico no corno. O ácido hialurônico apareceu em maior concentração no corno do que no colo uterino. / Objective: To evaluate the effects of conjugated equine estrogens (CEE) alone or associated to medroxyprogesterone acetate (MPg) on glycosaminoglycans (GAGs) in the cervix and horns of the rat uterus. Methods: Thirty days after ovariectomy, 40 adult rats were randomly divided into four groups: GI, control (treated with drug vehicle); GII, CEE (50 ?g/Kg per day); GIII, MPg (0.2 mg/Kg per day), and GIV, CEE + MPg (doses as in GII and GIII). Drugs and vehicle were given by gavage during 28 days. After this the animals were euthanized, the uterine cervix and body were removed, fixed in acetone and further processed for GAGs quantification. Agarose gel electrophoresis was used for sulphated GAGs analyses; the hyaluronic acid was assayed with an ELISA-like method. Statistical analysis was done by the Student\'s t test and the Tukey-Kramer test. Significant differences were set at p<=0.05. Results: In all groups sulphated GAGs concentration (especially dermatan sulphate) was higher than that of nonsulphated GAGs, both in the cervix and in the uterine horns. Hyaluronic acid concentration in uterine horns was higher than in the cervix. With regard to dermatan sulphate, CEE exerted trophic effects both in horns and cervices, whereas MPg blocked this action in the uterine horn. The cervical concentration of heparan sulphate was rised by CEE and by MPg; in the uterine horns the same rise was observed, and this effect was attributed to MPg. Conclusions: The qualitative and quantitative profiles of GAGs in the horns and cervix of rat uterus are distinct. Sulphated GAGs (especially dermatan sulphate) are more concentrated than the non-sulphated ones in both uterine regions. With regard to sulphated GAGs, estrogens and medroxyprogesterone have trophic actions on sulphated GAGs at the cervix, whereas at the uterine horn MPg shows an antagonistic action. The concentration of hyaluronic acid in the uterine horns was higher than in the cervix.