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Thesis (Ph. D.)--University of Wisconsin--Madison, 1968. / Vita. Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

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A Gonadotrofina Coriônica Eqüina (eCG) é produzida pela égua prenhe e tem ação folículo estimulante e luteinizante em animais domésticos não eqüídeos. Um pool formado por plasma de 4 éguas prenhes, com média de 69 dias de gestação, foi purificado em coluna cromatográfica com resina de afinidade Blue Sepharose FF (BS). As frações que adsorveram à resina BS foram purificadas em coluna cromatográfica com resina de afinidade Concanavalina A 4B (ConA). As frações que não adsorveram à resina BS também foram purificadas em coluna cromatográfica com resina de afinidade ConA. O mesmo pool de palsma foi diafiltrado, em cartucho de hemodiálise. O diafiltrado foi aplicado em coluna cromatográfica com resina de afinidade ConA. Atividade biológica (UI/mL) do plasma, do diafiltrado e das frações purificadas foram quantificadas por ensaio biológico com ratas impúbres. As atividades biológicas encontradas no plasma e no plasma diafiltrado foram de 3,63 e 5,14UI/mL, respectivamente. A atividade biológica encontrada nas frações que adsorveram à BS foi de 3,50UI/mL. Não foi encontrarda atividade biológica nas frações que não adsorveram à BS. A atividade biológica contida nas frações que adsorveram à BS e que também adsorveram a ConA foi de 3,65UI/mL. O rendimento do processo cromatográfico onde o plasma foi adsorvido pela BS e pela ConA, foi de 69,52%. Não foi encontrada atividade biológica nas frações obtidas da aplicação do plasma diafiltrado em coluna de ConA. O processo cromatográfico com uso de BS seguido de ConA mostou-se eficaz em purificar a eCG do plasma de éguas prenhes. / The equine Chorionic Gonadotrophin (eCG) is produced by the pregnant mare and has follicle-stimulant and luteinizing actions on non-equine domestic animals. A pool formed by the plasma of 4 pregnant mares (with mean gestation of 69 days) was purified in chromatographic column with Blue-Sepharose FF affinity resin (BS resin). Fractions adsorbed by BS resin were then purified in chromatographic column with Concavalin A 4B affinity resin (ConA resin). The fractions not adsorbed by the BS resin were also purified in chromatographic column with ConA resin. The same plasma pool was dialyzed in hemodialysis cartridge. The dialyzed was applied in chromatographic column with ConA resin. Biological activities (in IU/mL) of the plasma, of the dialyzed and of the purified fractions were quantified in a biological assay with female rats that did not reach puberty. The biological activities found in the plasma and dialyzed were of 3.63 and 5.14 IU/mL, respectively. Fractions that were adsorbed by BS had a biological activity of 3.50 IU/mL. No biological activity was found in fractions that were not adsorbed by BS. Biological activity found in fractions adsorbed by both BS and ConA was of 3.65 IU/mL. When plasma was both adsorbed by BS and ConA, the chromatographic process yield had results of 69.52%. No biological activity was found in the fractions obtained from the administration of dialyzed plasma in ConA column. The BS - followed by ConA -chromatographic process showed efficacy in purifying the eCG from the plasma of pregnant mares.

A Gonadotrofina Coriônica Eqüina (eCG) é produzida pela égua prenhe e tem ação folículo estimulante e luteinizante em animais domésticos não eqüídeos. Um pool formado por plasma de 4 éguas prenhes, com média de 69 dias de gestação, foi purificado em coluna cromatográfica com resina de afinidade Blue Sepharose FF (BS). As frações que adsorveram à resina BS foram purificadas em coluna cromatográfica com resina de afinidade Concanavalina A 4B (ConA). As frações que não adsorveram à resina BS também foram purificadas em coluna cromatográfica com resina de afinidade ConA. O mesmo pool de palsma foi diafiltrado, em cartucho de hemodiálise. O diafiltrado foi aplicado em coluna cromatográfica com resina de afinidade ConA. Atividade biológica (UI/mL) do plasma, do diafiltrado e das frações purificadas foram quantificadas por ensaio biológico com ratas impúbres. As atividades biológicas encontradas no plasma e no plasma diafiltrado foram de 3,63 e 5,14UI/mL, respectivamente. A atividade biológica encontrada nas frações que adsorveram à BS foi de 3,50UI/mL. Não foi encontrarda atividade biológica nas frações que não adsorveram à BS. A atividade biológica contida nas frações que adsorveram à BS e que também adsorveram a ConA foi de 3,65UI/mL. O rendimento do processo cromatográfico onde o plasma foi adsorvido pela BS e pela ConA, foi de 69,52%. Não foi encontrada atividade biológica nas frações obtidas da aplicação do plasma diafiltrado em coluna de ConA. O processo cromatográfico com uso de BS seguido de ConA mostou-se eficaz em purificar a eCG do plasma de éguas prenhes. / The equine Chorionic Gonadotrophin (eCG) is produced by the pregnant mare and has follicle-stimulant and luteinizing actions on non-equine domestic animals. A pool formed by the plasma of 4 pregnant mares (with mean gestation of 69 days) was purified in chromatographic column with Blue-Sepharose FF affinity resin (BS resin). Fractions adsorbed by BS resin were then purified in chromatographic column with Concavalin A 4B affinity resin (ConA resin). The fractions not adsorbed by the BS resin were also purified in chromatographic column with ConA resin. The same plasma pool was dialyzed in hemodialysis cartridge. The dialyzed was applied in chromatographic column with ConA resin. Biological activities (in IU/mL) of the plasma, of the dialyzed and of the purified fractions were quantified in a biological assay with female rats that did not reach puberty. The biological activities found in the plasma and dialyzed were of 3.63 and 5.14 IU/mL, respectively. Fractions that were adsorbed by BS had a biological activity of 3.50 IU/mL. No biological activity was found in fractions that were not adsorbed by BS. Biological activity found in fractions adsorbed by both BS and ConA was of 3.65 IU/mL. When plasma was both adsorbed by BS and ConA, the chromatographic process yield had results of 69.52%. No biological activity was found in the fractions obtained from the administration of dialyzed plasma in ConA column. The BS - followed by ConA -chromatographic process showed efficacy in purifying the eCG from the plasma of pregnant mares.

Gonadotrophin-releasing hormone (GnRH) can inhibit proliferation of multiple reproductive tissue cancer cell lines through direct interaction with GnRH receptors (GnRHR) on tumour cells. GnRH analogues may therefore have a role in treating some cancers. The signalling pathways associated with these inhibitory effects are poorly defined, and characterising them may help to understand therapeutic sensitivity. To elucidate these pathways, transcriptomic and proteomic approaches were used to compare the effects of the GnRH agonist Triptorelin in responsive GnRHR-transfected HEK293 cells (SCL60) and unresponsive (HEK293) cells both in vitro for up to 24h and in vivo for up to 7 days. Gene expression profiling demonstrated that SCL60 gene expression was temporally regulated with Triptorelin treatment, with expression of some genes increased at one time point but decreased at another. Early and mid-phase gene expression changes comprised mainly transcription factors and late changes included the hormonal signalling component CGA. Pathway analysis implicated mitogen-activated protein kinase and cell cycle pathways, supporting the detection of G2/M arrest. Signalling effects within SCL60 xenografts, 4 and 7 days following Triptorelin treatment, were investigated using a phosphoproteomic antibody array. Changes included cell cycle and apoptosis regulators, as well as cell surface receptors and NFκB signalling pathway members. Reverse-phase protein arrays and western blotting also showed that pAkt was decreased and pNFκB-p65 was increased after Triptorelin treatment in vitro. An NFκB inhibitor enhanced the anti-proliferative effect of Triptorelin in SCL60 cells in vitro, suggesting that NFκB acts as a survival factor in the response to GnRHR stimulation. A range of GnRHR expression was observed in breast cancer tumours by immunohistochemistry, and on average GnRHR expression was significantly higher in the Triple Negative Phenotype (TNP) subgroup and in grade 3 tumours. A GnRHR-transfected breast cancer cell line, MCF7-h14, was developed. Despite this expressing a similar level of GnRHR to responsive SCL60 cells, MCF7-h14 cells were not inhibited by GnRHR activation, indicating that a high level of GnRHR is insufficient for the antiproliferative effects of Triptorelin.

Abstract The effectiveness of intrauterine insemination (IUI) combined with controlled ovarian hyperstimulation (COH) in the treatment of subfertility was investigated in the present study. For this purpose the prognostic factors associated with success of clomiphene citrate (CC)/human menopausal gonadotrophin (HMG)/IUI were identified in 811 treatment cycles. Furthermore, a long gonadotrophin-releasing hormone agonist (GnRHa)/HMG stimulation protocol was compared with a standard CC/HMG protocol. In addition, the usefulness of alternative insemination techniques including fallopian tube sperm perfusion (FSP) and intrafollicular insemination (IFI) was investigated. Finally, the obstetric and perinatal outcome of pregnancies after COH/IUI was examined and compared with those of matched spontaneous and in vitro fertilization(IVF) pregnancies. Female age, duration of infertility, aetiology of infertility, number of large preovulatory follicles and number of the treatment cycle were predictive as regards pregnancy after CC/HMG/IUI. The highest pregnancy rate (PR) was obtained in women of < 40 years of age with infertility duration ≤ 6 years, who did not suffer from endometriosis. A multifollicular ovarian response to CC/HMG resulted in better treatment success than a monofollicular response, indicating the necessity of COH combined with IUI. A significantly higher PR was achieved in the first treatment cycles compared with the others, and 97% of the pregnancies were obtained in the first four treatment cycles. The PR per cycle did not differ significantly between a long GnRHa/HMG and a standard CC/HMG protocol, but the average medication expense of GnRHa/HMG stimulation was four times the cost of CC/HMG stimulation. Therefore, the routine use of a long GnRHa/HMG protocol in IUI treatment remains questionable. The FSP procedure was easy to perform by using a paediatric Foley catheter. The success rate in couples with either FSP or standard IUI did not differ significantly, although there was a trend towards a lower PR in the FSP group. The FSP technique should not replace the simpler and less time-consuming IUI technique in routine use. The IFI technique was also simple to perform and convenient for patients. However, only one normal singleton intrauterine pregnancy resulted in 50 IFI-treated women, indicating that IFI is inefficacious for treating subfertility. The IUI parturients differed from average Finnish parturients in respect to higher maternal age, more frequent primiparity and a higher incidence of multiple pregnancies. The use of antenatal care services was significantly lower in IUI singleton pregnancies compared with IVF singletons, although there were no more complications in IVF pregnancies. The hospitalization and Caesarean section rates were generally high in all pregnancies. The mean birthweight of IUI singletons was significantly lower than that in spontaneous pregnancies, but comparable to that in IVF pregnancies. However, the incidence of preterm birth, low birth weight and other variables describing the outcome of infants were similar in IUI, IVF and spontaneous pregnancies. In summary, the IUI procedure itself does not seem to affect adversely the obstetric and perinatal outcome of pregnancy, and patient characteristics and multiplicity may be more important in this respect.

Este estudo avaliou os efeitos da administração de diferentes doses de eCG em dois momentos distintos no protocolo de sincronização da ovulação para inovulação em tempo fixo. No dia 0, as receptoras foram tratadas com dispositivo intravaginal contendo progesterona (DIP) e 2,0 mg de Benzoato de Estradiol (BE) associados a 50 mg de progesterona (P4) IM. A partir desse momento, os animais foram divididos homogeneamente para receberem 0,15 mg de d-cloprostenol (PGF2&#945;) e 400, 500 ou 600 UI de eCG no dia 5 (G-400d5, n=101; G-500d5, n=98; G-600d5, n=100, respectivamente) ou no dia 8 (G-400d8, n=100; G-500d8, n=99; G-600d8, n=96, respectivamente; fatorial 3x2). O DIP foi retirado no dia 8 e foi administrado 1 mg de BE no dia 9. No dia 17 os animais foram submetidos a ultra-sonografia ovariana e os que apresentaram mais que um corpo lúteo (CL) ou CL único maior que 18 mm de diâmetro receberam um embrião produzido in vitro. Em um grupo de animais, colheu-se amostras de sangue no dia 17 para determinação da concentração plamática de P4 (CPP4). Os animais tratados com eCG no dia 5 apresentaram maior taxa de aproveitamento [87,0 (260/299) vs. 81,7% (241/295), P<0,05], tendência de maior taxa de concepção [51,8 (132/255) vs. 45,0% (108/295), P=0,07], maior taxa de prenhez [44,1 (132/299) vs. 36,6% (108/295), P<0,05], maior número de CL (1,74 ± 0,09 vs. 1,13 ± 0,03, P<0,05) e maior proporção de receptoras com CL único de maior tamanho [CL 22: 65,7 (111/169) vs. 43,1% (94/218), P<0,05] que os tratados no dia 8. Não se observou efeito de dose sobre a taxa de aproveitamento [400: 82,1 (165/201) vs. 500: 83,8 (165/197) vs. 600: 87,2% (171/196), P<0,05], de concepção [400: 51,8 (85/164) vs. 500: 44,1 (71/161) vs. 600: 49,4% (84/170), P<0,05], de prenhez [400: 42,3 (85/201) vs. 500: 36,0 (71/197) vs. 600: 42,9% (84/196), P<0,05] e sobre a quantidade de receptoras com CL único de maior tamanho [400: 46,8 (65/139) vs. 500: 54,9 (73/133) vs. 600: 57,4 (66/115), P<0,05]. Os animais tratados com 600 UI de eCG apresentaram maior número de CL que os que receberam 400 ou 500 UI (400: 1,17 ± 0,03 vs. 500: 1,33 ± 0,06 vs. 600: 1,82 ± 0,12, P<0,05). No grupo que se determinou a CPP4 verificou-se que os animais que receberam eCG no dia 5 apresentaram maiores CPP4 que os do dia 8 (4,68 ± 0,43 vs. 2,73 ± 0,17 ng/ml, P<0,05). As novilhas tratadas com 600 UI de eCG apresentaram maiores CPP que as tratados com 400 ou 500 UI (400: 2,96 ± 0,22 vs. 500: 3,45 ± 0,38 vs. 600: 4,69 ± 0,55, P<0,05). Receptoras com mais de 1 CL apresentaram tendência de menores perdas gestacionais entre 30 e 60 dias de gestação que as com CL único [10,2 (4/49) vs. 17,5% (33/189), P=0,0547]. Os resultados são indicativos de maior eficiência do protocolo de sincronização da ovulação para transferência de embriões em tempo fixo com o uso de eCG no dia 5. A dose de eCG não influenciou na eficiência do tratamento / The aim of this study was to compare the effects of different doses of eCG administrated at two moments in a fixed-time embryo transfer protocol. On day 0, the heifers received a progesterone releasing vaginal insert (PRVI) and an injection of 2.0 mg Estradiol Benzoate (EB) associated with 50 mg progesterone (P4) i.m. The animals were randomly assigned to six treatment groups in a tree by two factorial design to receive 0.15 mg d-cloprostenol (PGF2&#945;) i.m. and 400, 500 or 600 IU eCG on day 5 (G-400d5, n=101; G-500d5, n=98; G-600d5, n=100, respectively) or on day 8 (G-400d8, n=100; G-500d8, n=99; G-600d8, n=96, respectively). On day 8, the PRVI was removed and an injection of 1 mg EB was administered on day 9. On Day 17, all heifers were examined by ultrasonography to determine the number of CL and those with more than one CL or a single CL with a diameter ?18 mm received an in vitro produced embryo. A subset of heifers were bled on day 17 for plasma P4 determination. The animals treated with eCG on day 5 presented higher proportion of heifers selected/treated [87.0 (260/299) vs. 81.7% (241/295), P<0.05], tendency to higher proportion of heifers pregnant/transferred [51.8 (132/255) vs. 45.0% (108/295), P<0.1], higher proportion of heifers pregnant/treated [44.1 (132/299) vs. 36.6% (108/295), P<0.05], higher CL number (1.74 ± 0.09 vs. 1.13 ± 0.03, P<0.05) and higher proportion of recipients with larger single CL [CL 22: 65.7 (111/169) vs. 43.1% (94/218), P<0.05] than those treated with eCG on day 8. Dose effects were not observed in the proportion of heifers selected/treated [400: 82.1 (165/201) vs. 500: 83.8 (165/197) vs. 600: 87.2% (171/196), P<0.05], pregnant/transferred [400: 51.8 (85/164) vs. 500: 44.1 (71/161) vs. 600: 49.4% (84/170), P<0.05], pregnant/treated [400: 42.3 (85/201) vs. 500: 36.0 (71/197) vs. 600: 42.9% (84/196), P<0.05] and proportion of recipients with larger single CL [400: 46.8 (65/139) vs. 500: 54.9 (73/133) vs. 600: 57.4 (66/115), P<0.05]. The animals that received 600 IU eCG presented higher CL number than those received 400 and 500 IU (400: 1.17 ± 0.03 vs. 500: 1.33 ± 0.06 vs. 600: 1.82 ± 0.12, P<0.05). In a subset of heifers that was performed a plasma P4 determination, the group that received eCG on day 5 presented higher plasmatic P4 concentration than those received eCG on day 8 (4.68 ± 0.43 vs. 2.73 ± 0.17 ng/ml, P<0.05). The use of 600 IU eCG presented higher plasmatic P4 concentration than 500 and 600 IU (400: 2.96 ± 0.22 vs. 500: 3.45 ± 0.38 vs. 600: 4.69 ± 0.55, P<0.05). Recipients with more than 1 CL tended to present lower pregnancy losses between 30 and 60 days than those with a single CL [10.2 (4/49) vs. 17.5% (33/189), P=0.0547]. The results suggest higher efficiency in the synchronization of ovulation protocol for fixed-time embryo transfer with eCG administration on day 5. The dose of eCG did not affect the efficiency of treatment