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A dissertation submitted in fulfilment of the requirements for the degree of Master of Science in Medicine Faculty of Health Sciences University of the Witwatersrand Johannesburg 2015 / Treatment of hormone-dependent breast tumours is typically conducted using hormone-therapy, with NK cell immunotherapy a novel modality. In this study, the effects of hormone-therapy and combined hormone-therapy with immunotherapy in MCF-7 breast cancer cells was investigated. A hormone pre-treatment of 17β-oestradiol and progesterone was performed to simulate the in vivo microenvironment. Subsequently cells were treated with the hormone-therapy drugs Anastrozole or RU486 alone, or with continued hormone simulation. Combined therapy was conducted with in vitro activated NK cells co-cultured with MCF-7 cells undergoing hormone-therapy. Biomarkers ERα, PR and MUC1 were immunolocalised and expression analysed qualitatively, and quantitatively using image analysis software. Hormone pre-treatment reduced biomarker expression, stressing the importance of hormone environment simulation for in vitro experimentation. Hormone-therapy increased cytoplasmic ERα and decreased PR expression. Anastrozole increased MUC1 and RU486 decreased nuclear MUC1. With continued hormone simulation, Anastrozole further decreased all biomarkers whereas RU486 decreased ERα, increased PR expression with variable effects on MUC1 expression. RU486 induced MUC1/PR and MUC1/ERα correlation, which, under continued hormone simulation, was maintained in the nucleus only. Anastrozole induced MUC1/PR correlation in the cytoplasm which was maintained under continued hormone simulation. Hormone-therapy also induced a decrease in apoptosis, with continued hormone simulation abrogating Anastrozole induced apoptosis. While hormone-therapy did not increase proliferation, the associated changes observed in biomarker expression are linked with tumour progression indicating that short-term treatment may be detrimental for overall survival. Combined NK cell immunotherapy resulted in decreased PR, while ERα and MUC1 expression increased in a hormone-dependent manner. Biomarker correlation was evident, albeit reduced with continued hormone simulation. Independently of hormone-therapy and hormone stimulation, immunotherapy reduced apoptosis, contrary to expectation. Proliferation was marginally reduced by immunotherapy. The results indicate that immune cell function is inhibited by interaction with tumour cells, an effect that hormone-therapy cannot abrogate. Furthermore, that this study shows treatment alters both nuclear and cytoplasmic expression of biomarkers, indicates that diagnostic procedures should consider both cellular compartments in tumour progression. It is further shown that qualitative analysis of biomarker expression is not always validated by quantitative analysis, with the latter proposed as a more objective and precise method to be used diagnostically.

by Wong, Cheuk-fai. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 95-99). / List of figures --- p.iv / List of tables --- p.v / Abbreviations --- p.vi / Abstract --- p.vii / Chapter I. --- INTRODUCTION --- p.1 / Chapter 1. --- Inhibin a brief review --- p.1 / Chapter 1.1 --- "Definition and nomenclature, including related substances" --- p.1 / Chapter 1.2 --- Structure --- p.2 / Chapter 1.3 --- Historical background of the inhibin concept --- p.4 / Chapter 1.4 --- Actions of inhibin --- p.9 / Chapter 1.5 --- Control of inhibin production --- p.10 / Chapter 1.6 --- Measurement --- p.11 / Chapter 1.6.1 --- Immunoassay --- p.11 / Chapter 1.6.2 --- Bioassay --- p.13 / Chapter 1.6.2.1 --- In vivo methods --- p.13 / Chapter 1.6.2.2 --- In vitro methods --- p.14 / Chapter 1.7 --- Inhibin in clinical studies --- p.15 / Chapter 2. --- Project design --- p.17 / Chapter 2.1 --- Background --- p.17 / Chapter 2.2 --- Objectives --- p.20 / Chapter II. --- MATERIALS AND METHODS --- p.21 / Chapter 1. --- Materials --- p.21 / Chapter 1.1 --- Tracer preparation and purification --- p.21 / Chapter 1.2 --- Inhibin RIA --- p.21 / Chapter 1.3 --- Other immunoassays --- p.22 / Chapter 2. --- In-house inhibin RIA development --- p.22 / Chapter 2.1 --- The tracer preparation --- p.22 / Chapter 2.2 --- The radioimmunoassay --- p.25 / Chapter 2.3 --- Optimization of assay parameters --- p.26 / Chapter 2.3.1 --- Optimization of serum content and second antibody titre --- p.26 / Chapter 2.3.2 --- Verification of second antibodies' precipitating activity --- p.27 / Chapter 2. --- INHIBIN-EASIA --- p.27 / Chapter 3. --- Progesterone --- p.28 / Chapter 4. --- "Oestradiol, LH, and FSH" --- p.28 / Chapter III. --- RESULTS --- p.30 / Chapter Part I. --- In-house inhibin assay development --- p.30 / Chapter 1. --- Iodination and purification --- p.30 / Chapter 1.1 --- Step 1:Iodination followed by Sephadex column purification --- p.30 / Chapter 1.2 --- Step 2: Red A gel column purification --- p.33 / Chapter 1.3 --- Step 3: Sephadex column purification --- p.33 / Chapter 2. --- Inhibin RIA: Binding and antibody dilution curve experiment --- p.36 / Chapter 3.1 --- Verification of binding activity of the second antibody --- p.38 / Chapter 3.2 --- Optimization of serum content/ second antibody titre --- p.39 / Chapter 4. --- Discussion and conclusion --- p.41 / Chapter Part II. --- Hormone results of women undergoing in vitro fertilization --- p.42 / Chapter 1. --- Presentation of analytical results --- p.42 / Chapter 2. --- Comparison of hormone profiles of patients in the two GnRH agonist regimes --- p.43 / Chapter 2.1 --- Gonadotropins --- p.43 / Chapter 2.2 --- E2 --- p.56 / Chapter 2.3 --- Progesterone --- p.62 / Chapter 2.4 --- Inhibin --- p.68 / Chapter 3. --- Relationship between hormone --- p.74 / Chapter 3.1 --- Relationship between hormone changes --- p.74 / Chapter 3.2 --- Regression analysis --- p.87 / Chapter IV. --- DISCUSSSION --- p.89 / Chapter 1. --- Hormone profiles --- p.89 / Chapter 2. --- Hormone correlation --- p.91 / Chapter V. --- CONCLUSION --- p.94 / Chapter VI. --- REFERENCES --- p.95 / Chapter VIII. --- Appendix 1 Protocol for study on IVF and inhibin --- p.100 / Chapter IX. --- Appendix 2 Protocol for the management of IVF cycles --- p.101 / Chapter X. --- Appendix 3 Patients' results (table) --- p.103 / Chapter XI. --- Appendix 4 Patients ' results (graph) --- p.110

Orientadores: Ricardo Barini, Isabela Nelly Machado / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-11-27T10:51:33Z (GMT). No. of bitstreams: 1 Klimesch_Yvonne_M.pdf: 1424571 bytes, checksum: a70b152ae87de0a973c2d34af4320957 (MD5) Previous issue date: 2014 / Resumo: Introdução: As altas taxas de falha de implantação embrionária vêm sendo uma das principais causas no insucesso dos ciclos de fertilização in vitro. A incompatibilidade imunológica entre o embrião e a decídua materna gera uma ineficiência na produção de anticorpos bloqueadores com um aumento no predomínio de citocinas do tipo Th1 que são prejudiciais ao processo da gestação. A imunoterapia com linfócitos paternos surge como tratamento adjuvante com a função de reverter essa alteração imunológica para um predomínio do tipo Th2 favorecendo assim a tolerância imunológica permitindo a implantação embrionária e posterior desenvolvimento gestacional aumentando as taxas nos ciclos de fertilização assistida. Objetivos: Avaliar o tratamento de imunização com linfócitos paternos, os resultados maternos fetais e perinatais dos casais que tiveram falhas de implantação em ciclos de fertilização in vitro (FIV/ICSI). Materiais e métodos: Através de busca ativa pelos prontuários médicos foram incluídos no estudo 52 casais que apresentavam pelo menos duas falhas nos ciclos de fertilização previamente a imunoterapia. A imunização com linfócitos paternos foi administrada via intradérmica a cada 21 dias, totalizando 3 doses. Após 3 semanas da última dose realizou-se o teste de crossmatch, cujo percentual aceito é superior a 75% para ser considerado positivo e assim indicando a sensibilização da paciente com antígenos de histocompatibilidade do esposo. Resultados: Dos 52 casais incluídos no estudo, 20 casais não conseguiram uma gestação e 32 casais conseguiram uma gestação. No grupo que engravidou, a taxa de gravidez clínica foi de 61,5% (32/52) e a taxa de gravidez viável foi de 57,7% (30/52). Das 32 gestações, 30 evoluíram para parto e duas evoluíram para aborto de primeiro trimestre. A taxa de prematuridade foi de 53%. Conclusão: A imunoterapia com linfócitos paternos pode aumentar as taxas de gestação em mulheres que tiveram falhas prévias nos ciclos de fertilização assistida / Abstract: Introduction: High rates of embryo implantation failures have been a major cause of failure in IVF cycles. The immunological incompatibility between the embryo and the maternal decidua produces inefficiency in the production of blocking antibodies with an increase in the predominance of Th1-type cytokines that are detrimental to the process of gestation. Immunotherapy with paternal lymphocytes appears as adjuvant treatment with immune function reverse this change to a predominance of Th2 thus promoting immune tolerance allowing embryonic development and subsequent gestational increasing implantation rates in assisted fertilization cycles.Objective: To evaluate the treatment of immunization with paternal lymphocytes, fetal and perinatal maternal outcomes of couples who had implantation failure in in vitro fertilization cycles (IVF / ICSI). Material and Methods: Through active search for medical records were included in the study 52 couples with at least two flaws in fertilization cycles prior to immunotherapy. The immunization with paternal lymphocytes was administered intradermic every 21 days, totaling 3 doses. 3 weeks after the last dose was held for the crossmatch test, which is accepted percentage above 75% to be considered positive, thus indicating awareness of patient histocompatibility antigens husband.Results: Of the 52 couples in the study, 20 couples failed gestation and 32 couples achieved a pregnancy. In the group who became pregnant, the clinical pregnancy rate was 61.5% (32/52) and the viable pregnancy rate was 57.7% (30/52). Of the 32 pregnancies, 30 evolved into childbirth and two progressed to first trimester abortion. The prematurity rate was 53%.Conclusions: Immunotherapy with paternal lymphocytes can increase pregnancy rates in women who had previous failures in assisted fertilization cycles / Mestrado / Saúde Materna e Perinatal / Mestra em Ciências da Saúde

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 161-197). Also available in print.

Thesis (M.C.S.)--Regent College, 1999. / Includes abstract and vita. Includes bibliographical references (leaves 180-186).

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A mouse embryo culture medium which would allow for in vitro development from 1-cell stage to blastocyst stage could offer many benefits for human research. Previous researchfrom our lab has demonstrated a mouse embryo culture medium named CZB seems to allow for in vivo-like conditions for development. Compared to other commonly used mouse embryo culture media, CZB medium promotes a higher frequency of 1 cell mouse embryos developing to blastocyst stage (Chatot et 1989). A key difference between CZB and other mouse embryo culture media is that CZB contains the amino acid glutamine metabolism is glutamate dehydrogenase (GDH). In order to determine if CZB cultured embryos follow in vivo-like patterns of gene expression for GDH, a quantitative competitive RT-PCR system was designed. A mutant GDH mRNA template was created which lacked a specific restriction enzyme site and was used as a competitive template in quantitative RT-PCR. This system was used to determine the amount of GDH mRNA present in in vivo grown blastocyst stage mouse embryos. It was determined that the amount of GDH mRNA present in in vivo blastocyst stage embryos was 282 fg/embryo. It is believed this system will also allow for quantitation of GDH mRNA in the earlier preimplantation stages of in vivo grown embryos, as well as the preimplantation stages 2-cell to blastocyst of CZB cultured embryos. / Department of Biology

What determines parenthood? The advent of IVF and the rapid growth of reproductive technologies have challenged the significance historically associated with biological relationships. It is now possible for a child to have many different people in the role of genetic, gestational, nurturing or legal parent and for the formation of many novel types of families. While frequently some or all of these roles are combined, it is now possible for someone to be a ‘parent’ in one sense, without necessarily taking on the obligations and rights associated with parenthood in a moral sense. Despite the expanded options for constructing families and the proliferation of novel arrangements for raising children, the essential feature of what it means to be a ‘real parent’ and to have a child of ‘one’s own’ is often grounded in the transmission of genes. This thesis examines the claim that genes define ‘moral’ parenthood. It investigates whether or not genetic relatedness is morally weighty in determining which individuals incur obligations for and rights over children. My thesis adopts a novel approach to address this question. It combines the analysis of both people’s views as captured through a qualitative study and those found in philosophical literature relating to the moral significance of genetic parenthood. I design and conduct a study to capture more directly the meanings that people attach to passing on their genes, which acts as a starting point for identifying and evaluating possible arguments about the moral relevance of genetic parenthood. I then analyse the principles imbedded in the participants’ views in light of the current philosophical literature.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.