published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Thesis (M. Med. Sc.)--University of Hong Kong, 2004. / Also available in print.

Background: In the last two decades the discovery of the Toll-Like Receptor (TLR) family in humans revealed the importance of innate immunity in providing host defence against invading pathogens. Moreover, TLRs have a vital role in mediating the interactions between the reproductive and immune systems. Similar to TLRs, (NOD)-like receptors (NLRs); retinoic acid-inducible gene-1, RIG-1-like receptors (RLRs) are also important pattern recognition receptors in the female reproductive system. Successful implantation requires effective reciprocal interactions between receptive endometrium and competent embryo. The endometrial innate immunity during implantation has been intensively investigated. However, little is known about the expression of innate immunity during the early stages of human embryo development. Objective: To investigate the expression of innate immunity molecules during the early stages of human embryo development and to determine the functions of TLRs in blastocysts. Material and methods: The expression of TLRs, a panel of downstream signalling molecules, NLRs, RLRs, inflammatory cytokines, chemokines and the hyaluronic acid system were investigated in the following developmental stages: oocyte, 4- cell, blastomeres, 8- cell, blastocyst, inner cell mass and trophectoderm using Affymetrix Human Genome U133 Plus 2.0 arrays. Microarray data were validated by Q-PCR. TLR function in human blastocysts was investigated by treating day five blastocysts with TLR3 or TLR5 specific ligands; Poly (I:C) and flagellin respectively, for 24 hours. The culture media was analysed for elevated cytokine and chemokine levels using cytometric bead array. Results: TLRs, NLRs, RLRs, TLR downstream signalling molecules, cytokines and chemokines involved during implantation event and the hyaluronic system were all found to be positively expressed in the early stages of human embryo development. The expression levels of the above molecules were generally moderate to low (CT 24-34) and varied across the embryonic developmental stages. Stimulation of TLR3 and TLR5 in day 5 blastocysts produced cytokines and chemokines. In addition, there were alterations in gene expression patterns in the Poly (I:C) and flagellin treated blastocysts in comparison to the untreated blastocysts. Conclusion: The varied expression levels of the investigated molecules involved in early embryonic developmental suggests a potential role for these molecules in early pregnancy loss and implantation failure. Specifically, the relationship between the level of TLR expression, function and embryo quality is worth investigating in the future as a potential marker for embryo competence.

This thesis analyses the nature, rationale, and implementation of United Kingdom and Australian regulation of research involving human embryos and cloning using legal materials, other documents and qualitative interviews with researchers, practitioners and regulators. It considers how law-makers have decided upon what to regulate and where to draw the line between permissible and prohibited activities, and the type of regulatory design strategies and enforcement approaches adopted in each jurisdiction (the ‘how to regulate’ question). It is argued that both jurisdictions have effectively decided upon permissible and prohibited activities as a result of thorough public consultation, research, reviews and the parliamentary process, and have appropriately balanced competing rationales for regulation. However, the type of regulation used in relation to those who are licensed to research in this area is unsuitable due to an over-emphasis on deterrence and the authoritarian approach taken by the regulatory bureaucracies. The central thesis is that a responsive regulatory system for licence-holders should be adopted. It is proposed that such a system would maintain the top level ‘command and control’ design strategies and deterrence approaches present in the current regulatory systems for breaches of legislation by non-licence holders and serious breaches by licence holders. However, greater use of co-regulatory design strategies and cooperative, educative and persuasive enforcement approaches should be used for regulating licensed research activities.

Infertility is a widespread problem around the world, although the available treatment options constantly improve through research and methodological development. A common treatment option mainly for biologically caused infertility is in-vitro fertilization, where in a menstrual cycle multiple oocytes are stimulated to mature and are then fertilized in a laboratory. This often results in multiple good quality embryos which can be cryopreserved through vitrification and used in a later cycle to increase the success chance of the treatment. The purpose of this pilot study was to evaluate vitrification and warming kits from Kitazato and Irvine Scientific regarding results and procedures. Human cleavage stage embryos (n=76) were thawed and cultured to the blastocyst stage. The blastocysts were scored according to Gardner and cryopreserved with vitrification and warming kits from either Kitazato (n=20) or Irvine Scientific (n=20). The warmed blastocysts were controlled after 2 and 4 hours for re-expansion and freeze injuries. The data was analysed with Fisher’s exact test and considered statistically significant if two-tailed p-value <0.05. The results showed no significant difference between the kits after 2 respectively 4 hours regarding re-expansion (p=0.432; p=0.492) or freeze injury (p=1.000; p=0.476). A significant difference was observed between group AB (with higher Gardner-score) and group C (with lower Gardner-score) in the degree of freeze injury (p=0.048; p=0.034), regardless of vitrification kit used. The details of the procedures differed somewhat between the kits, both having pros and cons, although overall procedures were equivalent. Further evaluation is needed before a change in method can be conducted.

La cryopréservation, dans le domaine de la reproduction médicalement assistée, constitue depuis de nombreuses années une branche suscitant beaucoup d’intérêts et d’espoirs. En effet, de nombreuses équipes de recherche se sont attelées à mettre au point et à améliorer des protocoles permettant de conserver les gamètes, les embryons et les tissus reproducteurs.<p>Malgré le fait que la cryopréservation soit une technique très attractive, elle peut avoir des effets délétères sur les cellules. Les protocoles expérimentaux visent donc à minimiser ces effets afin d’augmenter la survie et la compétence cellulaire après décongélation.<p><p>Les deux méthodes les plus utilisées, la congélation lente et la vitrification, présentent chacune des avantages et des inconvénients. En effet, la première ne permet pas d’éliminer la cristallisation intracellulaire. Quant à la seconde, elle empêche la formation de cristaux de glace mais pourrait provoquer une toxicité due à la forte concentration des cryoprotecteurs. <p><p>Cette thèse de doctorat propose plusieurs objectifs :<p><p>•\ / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Univ., Diss.--Regensburg, 2005. / Literaturverz. S. [XXXV] - LXXVII.

Malgré le succès de l’Aide Médicale à la Procréation (AMP), des données récentes suggèrent un lien entre le recours à l’AMP et des pathologies liées à des erreurs épigénétiques. Afin d’évaluer l’impact des procédés in vitro de l’AMP sur la méthylation de l’ADN, nous avons analysé : 1) le profil de méthylation de 2 locus soumis à empreinte, H19DMR et KvDMR1, dans des ovocytes recueillis immatures, vitrifiés, puis mûris in vitro après réchauffement. Nous avons montré pour la première fois que le procédé vitrification/MIV pouvait constituer une alternative prometteuse pour préserver le potentiel procréatif des femmes avant un traitement anticancéreux, puisqu’il n’altérait pas l’empreinte parentale. 2) le profil de méthylation de H19DMR dans des embryons humains en retard de développement ainsi que dans les gamètes des géniteurs. Nous avons mis en évidence des anomalies de l’empreinte dans ces embryons, sans lien apparent avec les indications d’ICSI mais liées à des altérations survenues soit au cours de l’ovogenèse, soit au cours du développement précoce. 3) le profil de méthylation des promoteurs d’OCT4 et NANOG, gènes essentiels à l’expression de la pluripotence, dans des embryons bloqués après culture prolongée. Nous avons montré que le promoteur d’OCT4 était significativement hyperméthylé dans les embryons bloqués, en particulier dans la population de couples présentant uniquement une infertilité masculine, alors que le promoteur de NANOG était hypométhylé / Despite the success of Assisted Reproductive Technologies (ART), recent data suggest a link between the use of ART and diseases related to epigenetic errors. To evaluate the impact of ART on DNA methylation, we analyzed: 1) the methylation profile of 2 imprinted loci, H19DMR and KvDMR1, in immature oocytes which were vitrified and matured in vitro after warming. We have shown for the first time that vitrification/IVM process did not alter the imprinting. Then, this new technology could be a promising alternative to preserve the procreative potential of women before cancer treatment. 2) the methylation profile of H19DMR in human embryos with developmental anomalies and in the gametes of the parents. We have shown that imprinting errors in these embryos have no apparent link with ICSI indications, but were related to alterations in H19DMR occurring either during oogenesis or early development. 3) the methylation profile of the promoters of OCT4 and NANOG, essential genes for the expression of pluripotency in arrested embryos after prolonged culture. We have shown that the promoter of OCT4 was highly methylated in arrested embryos particularly in the population of couples with male infertility only, whereas the NANOG promoter was hypomethylated

Thesis (PhD)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Problem statement For utilitarians, human beings have intrinsic moral significance based on only two acquired characteristics: sentience, or the ability to suffer, and psychological personhood. Sentience is the entrance-requirement for moral significance, but does not justify a "right to life" claim; at most a "right" not to suffer. Personhood, described as some sort of self-conscious awareness with a concept of the future, may justify a "right to life" claim. However, since personhood is absent in prenatal beings, and only develops some time after birth, the implication is that such beings have little moral significance and may, for instance, be killed "at will". The moral problem that I address in this dissertation is to investigate, assess and evaluate the utilitarian position on the moral status or value of prenatal life. Methodology and results I firstly, on the basis of an extensive literature study, make a detailed analysis of the utilitarian position with reference to a number of themes that I have identified in their argument. This is followed by a critical philosophical evaluation of the utilitarian position, based on six particular arguments: • Utilitarianism is philosophically incoherent. It over-simplifies the moral argument in claiming that consequences are all that matter morally. Its underlying moral theory is at odds with moral claims contained in contemporary notions of human rights and individual justice. It ignores the moral significance of special obligations to special groups. • Utilitarianism potentially has unacceptable consequences. It IS inherently discriminatory and may lead to legitimate "slippery slope" fears. • Utilitarianism clashes with our fundamental moral intuitions on the value of prenatal life. These intuitions are cherished in most world religions. • Contrary to the utilitarian position, speciesism is inevitable to the human condition, especially argued from a position of existential phenomenology. Self- constitution, simultaneous constitution of the world as we know it, and the very possibility of morality are possible only within a particular notion of speciesism. • The potentiality of pre-persons to develop into persons cannot be as convincingly ignored as is done by the utilitarian. • There is a basic and underlying need and intuition to protect vulnerable human beings, of which pre-persons are exemplars. These notions clash with utilitarian theory. As an alternative, I introduce, set out and evaluate a two-phased position on the moral significance of pre-personal human life, a position of respectfulness of prenatal and pre-personal human life based upon its humanity, potentiality and separation-viability. This leads, firstly, to the conclusion of a graded, sliding scale conception of human prepersonal moral significance in line with the level of development and with the actuation of potentiality. Secondly, it leads to the conclusion that the advent of separationsurvivability (viability) is a morally significant cut-off point beyond which the human fetus may "normally" have a justifiable right to the continuation of its life. In as far as the application of my argument is concerned, I develop a "moderate" position with reference to the abortion debate. Whilst I recognize that all human prenatal beings of which it can be argued that they have a reasonable chance to develop their intrinsic potentiality, i.e., to become full-fledged persons, should have the opportunity to do so, I also recognize that neither this position, nor the complexities of life make it possible to hold "absolute" positions on the justifiability of abortion. I explore this extremely problematic notion in the text. That having been said, the advent of separation-survivability may imply a "moral cut-off point", beyond which termination is only rarely justified. I argue that I find no moral hindrance to wellmotivated research on human pre-embryos and stem cells. / AFRIKAANSE OPSOMMING: Probleemstelling Utilitariste huldig sterk omlynde standpunte oor die waarde van lewe. Hulle redeneer dat menslike (inderwaarheid, alle lewende) wesens slegs op grond van twee eienskappe intrinsieke morele waarde kan verwerf: sentiëntisme, d.i. die vermoë om lyding te ervaar, en persoonstatus. Sentiëntisme is 'n bepalende vereiste vir morele status, maar regverdig nie 'n "reg op lewe"-aanspraak nie. Persoonsyn, verstaan as 'n vorm van selfbewustheid tesame met 'n bewuste belang by die voortsetting van eie bestaan, mag wel so 'n aanspraak regverdig. Voorgeboortelike (en "voorpersoonlike") wesens is egter nie persone nie; hulle word eers (aansienlik) ná geboorte volwaardige persone. Die implikasie is dat sulke wesens weinig morele status het, en byvoorbeeld, na willekeur gedood mag word. Die morele probleem wat ek in hierdie dissertasie aanspreek is om die utilitaristiese beskouing ten opsigte van die morale status of waarde van voorgeboortelike lewe krities-filosofies te ondersoek en te evalueer. Metodologie en gevolgtrekkings Eerstens maak ek na aanleiding van 'n gedetaileerde literatuurstudie 'n in-diepte analise van van die utilitaristiese posisie aan die hand van 'n aantal temas wat ek in hul argument geïdentifiseer het. Daarna volg 'n krities-filosofiese evaluasie van die utilitaristiese posisie, aan die hand van ses argumente: • Utilitarisme is filosofies onsamehangend. Dit oorvereenvoudig die morele argument deur voor te gee dat gevolge al is wat moreel saakmaak. Die onderliggende utilitaristiese teorie bots met die morele eise vervat in kontemporêre sienings van menseregte en geregtigheid. Dit negeer die morele belangrikheid van spesiale verpligtinge teenoor spesiale belangegroepe. • Utilitarisme het potensieelonaanvaarbare gevolge. Dit IS inherent diskriminerend en kan lei tot onkeerbare glybaan ("slippery slope")-argumente. Utilitarisme bots met ons fundamentele morele intuïsies betreffende die waarde van voorgeboortelike lewe. Hierdie intuïsies word onder meer ondersteun deur die meeste hoofstroom godsdienste. • Spesiësisme is, in kontras met die utilitaristiese beskouing, onafwendbaar vir ons selfverstaan as mense, soos aangetoon kan word met 'n beroep op die eksistensiële fenomenologie. Self-konstituering, gelyktydige konstituering van die wêreld van die mens, en selfs die moontlikheid van moraliteit is slegs moontlik vanuit' n bepaalde spesiësistiese beskouing. • Die potensialiteit van "pre-persone" om tot volwaardige persone te ontwikkel kan nie, soos die utilitaris doen, sonder meer geïgnoreer word nie. • Daar is 'n basiese en onderliggende morele eis om swak en weerlose menslike wesens te beskerm. Hierdie idees bots lynreg met utilitaristiese teorie. As 'n alternatief tot die utilitaristiese beskouing, ontwikkel ek 'n twee-fase posisie betreffende die morele waarde van voorgeboortelike menslike lewe. Ek noem hierdie posisie agting vir voorgeboortelike en voor-persoonlike menslike lewe gebaseer op die menslikheid, potensialiteit en oorleefbaarheid van prenatale mense. Dit lei, eerstens, tot die gevolgtrekking van 'n gegradeerde glyskaal konsepsie van voor-persoonlike menslike morele waarde, min of meer parallel aan die vlak van ontwikkeling en die ontwikkeling van potensialiteit. Tweedens lei dit tot die gevolgtrekking dat die ontwikkeling van lewensvatbaarheid 'n moreel-beduidende afsnypunt is waarna die menslike fetus "normaalweg" aanspraak kan maak op 'n reg dat sy lewe voortgesit moet word. In soverre dit die toepassing van my argument betref, ontwikkel ek 'n "gematigde" posisie vis-á-vis aborsie. Ek redeneer dat alle menslike voorgeboortelike wesens wat 'n redelike kans het dat hul intrinsieke potensialiteit verder sal ontwikkel, die geleentheid daartoe gegun behoort te word. Ek aanvaar ook dat nog hierdie beskouing, nog die kompleksiteit van die menslike bestaan "absolute" posisies moreel regverdig. Die problematiek en inherente spanning tussen hierdie oënskynlik-opponerende posisies word in die teks bespreek. Nogtans beskou ek die ontwikkeling van lewensvatbaarheid as 'n moreel insiggewende afsnypunt waarna terminasie net in buitengewone omstandighede moreel regverdigbaar is.

Made available in DSpace on 2016-04-26T20:25:26Z (GMT). No. of bitstreams: 1 Fernanda Garcia Escane.pdf: 10577423 bytes, checksum: d657a52e1d8f512f4aea237929afa9c6 (MD5) Previous issue date: 2007-05-08 / This study intends to analyze the biosafety law allowing genetic engineering with human biological material, more precisely human embryos generated in laboratories. Chapter I discusses the concepto of life according to science, law and ethics in view of the understanding prevailing in science and law. Chapter II approaches the right to life as seen by the Brazilian Federal Constitution and the principles on which it lies. There is no other right more valuable or important than that to life, all the more so because without it any other fails to deserve protection or even to stand by itself, dependent as they are on the right to life. In chapter III the definition of embryo and unborn child are introduced and a lengthy discussion about personality rights is tackled. Focus is laid on the protection due to the life of embryos and the menace represented by the continuous manipulation of scientists. Chapter IV analyses the biosafety law authorizing embryos to become object of research and therapy and the disregard of scientific parameters determining the inception of life at the exact moment of conception. Finally, chapter V considers the civil liability of those dealing with embryos, given that law must protect life to the fullest, whichever stage it might be, clear from any sort of prejudice may look, because life is not relative: it either exists or does not / Este trabalho tem por objetivo analisar a Lei de Biossegurança que permite a engenharia genética com material biológico humano, mais precisamente, com os embriões humanos, criados em laboratório. No capítulo I, o objetivo é delinear o conceito de vida, pautado pela ciência, pelo direito e pela ética, considerando o entendimento que prevalece na ciência e no direito. No capítulo II, define-se o direito à vida em face da Constituição Federal e o os princípios que o norteiam, dado que não existe nenhum outro bem mais valioso ou importante do que o direito à vida. Sem ela, por força de lógica, desaparece o respaldo a qualquer outro, visto que nenhum deles sobrevive sem a vida, existindo tão-somente por decorrência dela. No capítulo III, definem-se embrião e nascituro e abordam-se os direitos da personalidade, destacando o direito à vida, que deve ser protegida em sua amplitude, independentemente da maneira pela qual se origina, haja vista a possibilidade atual de manipulação do homem. No capítulo IV, analisa-se a Lei de Biossegurança, que autoriza sejam os embriões objeto de pesquisa e terapia, desconsiderando os parâmetros científicos que demonstram iniciar-se a vida no exato momento da concepção. Por fim, o capítulo V destina-se a observar a responsabilidade civil de todos os que lidam com o embrião, já que compete ao direito proteger integralmente a vida, qualquer seja seu estágio, desvinculando-se de todo preconceito, ainda que insignificante na aparência. A vida não é relativa: ou existe ou não