Thesis (DPhil (Philosophy))--University of Stellenbosch, 2007. / Should surplus embryos which are destined to be discarded be protected at all cost, to the extent that they cannot contribute to medical knowledge - knowledge which could benefit society at large? Are embryos people or merely items of property? Different moral theories address these questions in different ways. Deontologists argue that the end never justifies the means and that the right not to be killed is more fundamental than the obligation to save. Utilitarians, on the other hand, argue that certain criteria should be met before moral significance can be contributed to an entity. The question of the moral status of the embryo is, as my discussion will show, one of the most widely discussed issues in the history of bioethics. Extensive literature exists on the topic. This study holds that an Ethics of Responsibility (ER) should by applied when answering the questions posed above as it encourages one to accept responsibility for the choices or decisions made and to defend them accordingly. I have endeavoured to answer the question of the personhood and rights of the embryo within the framework of the Ethics of Responsibility. Although these concepts overlap in many ways they remain central to the debate surrounding the sanctioning or prevention of the use of human embryonic stem cells in research. After identifying the micro-issues surrounding the human embryonic stem cell debate and explaining why both the deontologist and utilitarians fail to provide any adequate answers in this respect, I turn my attention to macro-issues such as safety concerns surrounding the usages and storage of stem cells. Commercialization, power issues, accessibility and the allocation of limited resources are also examined. Living in a society such as South Africa one cannot be blind to the inequalities of our health system. On a macro level I cannot but conclude that stem cell research does not seem to be a viable exercise within the South African context. South Africa faces a health care crisis far greater than the benefits stem cell research currently has to offer. However, the need still exists for a policy to guide future lawmakers who might need to address stem cell research and to guide decisions and actions. This brings me to my final chapter, namely proposing a morally justified policy for South Africa. I propose a policy which respects and values the autonomy of the progenitors’ choices (provided they have not been coerced) and which focuses on the beneficence of the greater society. Furthermore, it is paramount that the goal of any stem cell research should be for therapeutic use ONLY. Before commencing with the extraction of the stem cells, scientists should be obligated first to present convincing evidence that they have tried alternative ways to reach the same result. Once this has been proven, a regulatory body could issue the scientist/team with a license to undertake the specific research with a specific therapy as goal in order to prevent abuse. If they are found guilty of any unethical conduct their licenses should be revoked and an investigation launched.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

No description available.

Source: Masters Abstracts International, Volume: 47-02, page: 0981. Adviser: Luisa Franzini. Includes bibliographical references.

The mammalian zygote and resulting embryo is the starting point of life, and thus must overcome continuous insult from DNA stress and damage while maintaining genome stability and integrity. This thesis examines genome stability in the context of chromosome changes, both in the context of ploidy and whole genome duplications as well as double-strand DNA breakage and chromosome loss. Regarding the ploidy portion of this work, while possible to derive and maintain, mammalian haploid stem cells undergo spontaneous, irreversible diploidization. Here, we investigated the mechanisms driving diploidization using human and mouse embryos, and human embryonic stem cells experimental systems. We demonstrate that diploidization occurs early in development and is often unproductive, with diploidized cells failing to contribute to the developing embryo. Diploidization involves delayed mitotic progression, incomplete alignment of chromosomes, and occurs through mitotic slippage or failed cytokinesis after exit from mitosis without formation of a midbody. Diploidization is associated with DNA damage and aneuploidies, with an upstream component being a decreased nuclear to cytoplasmic ratio. Increasing this ratio in haploid mouse embryos improves developmental outcomes and decreasing this ratio in diploids results in poor outcomes. A sensor of the nuclear to cytoplasmic ratio, CHK1, is required for haploid maintenance as inhibition increases binucleation and diploidization in haploid human embryonic stem cells. Thus, we demonstrate the earliest upstream driver of diploidization as being the nuclear-cytoplasmic ratio in haploid mammalian cells, rather than the actual haploid state. Regarding the double-strand DNA breakage portion of this work, the preferred mechanism by which human embryos repair double-strand breaks was investigated. Utilizing allele-specific CRISPR-Cas9 cleavage, we show that human embryos repair double-strand breaks primarily through non-homologous end joining. In embryos left unrepaired or misrepaired, partial or whole chromosome loss occurs, which can be easily overlooked and misinterpreted with common on-target analyses such as PCR. Off-target Cas-9 activity recapitulated findings on an entirely separate chromosome, confirming the preference of the human embryo for non-homologous end joining and microhomology-mediated end joining, as well as chromosome loss where repair was unsuccessful.

Indiana University-Purdue University Indianapolis (IUPUI) / Ethical considerations of the human embryo have involved heated dispute and seem always to result in the same interminable debate. A history of this debate, however, shows a shift in the language used to distinguish between degrees of moral status – while the debate once focused on the presence or absence of “human life,” now it is more likely to hear whether the qualifications for “personhood” have been met. In other words, any member of the human species may deserve some level of respect, but only the “persons” deserve full moral respect. This leaves open the possibility for a human being who is not actually a person – a “nonperson human being.” As an answer to the question of exactly what kind of respect to give the human embryo, Aristotelian moral philosophy offers a unique perspective, one which is distinctive from the familiar debate. Aristotle’s concept of ergon, or function, is a key to understanding what is essential in any human being, because it reveals the importance of potentiality to our nature as rational beings. A philosophical view of function, combined with the data of modern embryology, makes the case that our proper function is the vital part of who we are as human beings, and that a disruption of human function constitutes a true harm. This thesis contrasts Aristotelian proper human function with the modern understanding of a “nonperson human being,” especially as articulated within the ethical theory of Peter Singer. This understanding of function, revealing the essence of human potential and linked with human development, offers a sort of “common-sense morality” response to modern views on personhood.

Indiana University-Purdue University Indianapolis (IUPUI) / Serotonin (5-hydroxytryptamine, 5-HT) is a monoamine neurotransmitter that is synthesized from the amino acid L-tryptophan and is reported to localize in mitochondria of embryonic stem cells. Even before its role as a neurotransmitter in mature brain was discovered, 5-HT has been shown to play an important role in regulating brain development. However, there is a lack of knowledge about the downstream target genes regulated by serotonin in embryonic stem (ES) cells. Towards this end, our study helps in understanding transcriptional regulatory mechanisms of 5-HT responsive genes in ES cells. By combining the gene expression data with motif prediction algorithms, literature validation and comparison with public domain data, gene targets specific to endogenous or exogenous 5-HT in ES cells were identified. By performing one-way ANOVA, and volcano plots using GeneSpring software, we identified 44 5-HT induced and 29 5-HT suppressed genes, likely to be transcriptionally regulated by 4 & 2 TFs respectively. Motif enrichment analysis on these target genes using MotifScanner revealed that the transcription factor TFAP2A plays a key role in regulating the expression of 5-HT responsive genes. Furthermore, by comparing our dataset with published expression profiles of ES cells, we observed a number of 5-HT responsive target genes showing enrichment in ES cells. Genes such as Nanog, Slc38a5, Hoxb1 and Eif2s1 from this analysis have been observed to be components of ‘stemness’ phenotypes reported in literature. Functional annotation of the 5-HT responsive genes identified gene ontologies such as regulation of translation in response to stress and energy derivation by oxidation, suggesting a regulatory role for 5-HT in mitochondrial functions of ES cells. Additionally, enrichment of other biological process terms such as development of various parts of nervous system, cell adhesion, and apoptosis suggests that 5-HT target genes may play an important role in ES cell differentiation. Our study implemented a new combinatorial approach for identifying gene regulatory mechanisms involved in 5-HT responsive genes and proposed potential mediatory role for serotonin in ES cell differentiation and growth. Thus, this study provides potential 5-HT target genes in ES cells for biological validation.

Tse, Chi Lok. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 116-131). / Abstracts in English and Chinese. / Declaration --- p.i / Abstract --- p.iii / Abstract in Chinese --- p.v / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Figures --- p.X / List of Tables --- p.xii / List of Abbreviations --- p.xiii / Chapter CHAPTER 1 --- General Introduction / Chapter 1.1 --- Regenerative Medicine --- p.1 / Chapter 1.2 --- Embryonic Stem Cells and Reprogramming --- p.3 / Chapter 1.3 --- Transdifferentiation --- p.6 / Chapter 1.4 --- The Cerebellum --- p.7 / Chapter 1.4.1 --- Functions of the cerebellum --- p.7 / Chapter 1.4.2 --- Structure and organization of the cerebellum --- p.8 / Chapter 1.4.3 --- Principle cellular components in the cerebellum --- p.12 / Chapter 1.4.3.1 --- Purkinje cells --- p.12 / Chapter 1.4.3.2 --- Granule cells --- p.12 / Chapter 1.4.3.3 --- Mossy fibres --- p.13 / Chapter 1.4.3.4 --- Climbing fibres --- p.13 / Chapter 1.4.3.5 --- Deep cerebellar nuclei --- p.13 / Chapter 1.4.3.6 --- Other cerebellar neurons --- p.14 / Chapter 1.4.3.7 --- Neuroglia of the cerebellum --- p.16 / Chapter 1.4.4 --- Circuitry of the cerebellum --- p.17 / Chapter 1.5 --- Development of the Cerebellum --- p.21 / Chapter 1.5.1 --- Anatomical changes during cerebellar development --- p.21 / Chapter 1.5.2 --- Molecular control of cerebellar development --- p.25 / Chapter 1.5.2.1 --- Specification of the cerebellar region --- p.25 / Chapter 1.5.2.2 --- Neurogenesis from the ventricular zone --- p.26 / Chapter 1.5.2.3 --- Neurogenesis from rhombic lip --- p.29 / Chapter 1.6 --- Scope of the Thesis --- p.33 / Chapter CHAPTER 2 --- Materials and General Methods / Chapter 2.1 --- Materials for Molecular Biological Work --- p.35 / Chapter 2.1.1 --- Enzymes --- p.35 / Chapter 2.1.2 --- Chemicals and others --- p.35 / Chapter 2.1.3 --- Plasmid vectors and plasmid --- p.36 / Chapter 2.1.4 --- Solutions and media --- p.36 / Chapter 2.2 --- Materials for Tissue/Cell Culture --- p.38 / Chapter 2.2.1 --- Chemicals --- p.38 / Chapter 2.2.2 --- Culture media and solutions --- p.38 / Chapter 2.2.3 --- Culture cells --- p.39 / Chapter 2.3 --- Animals --- p.40 / Chapter 2.4 --- Materials for Immunocytochemistry --- p.40 / Chapter 2.5 --- Oligonucleotide Primers --- p.41 / Chapter 2.6 --- RNA Extraction --- p.44 / Chapter 2.7 --- Generation of cDNA from mRNA --- p.44 / Chapter 2.8 --- Preparation of Recombinant Plasmid DNA --- p.45 / Chapter 2.8.1 --- Small scale preparation of DNA --- p.45 / Chapter 2.8.2 --- QLAGEN plasmid midiprep kit method --- p.46 / Chapter 2.9 --- Preparation of Specific DNA Fragment from Agarose Gel --- p.46 / Chapter 2.10 --- Subcloning of DNA Fragments --- p.47 / Chapter 2.10.1 --- Preparation of cloning vectors --- p.47 / Chapter 2.10.2 --- Subcloning of DNA fragment --- p.48 / Chapter 2.10.3 --- Transformation of DNA into competent cells --- p.48 / Chapter 2.11 --- Preparation of Competent Cells --- p.48 / Chapter CHAPTER 3 --- Generation and Characterization of Induced Neurons / Chapter 3.1 --- Introduction --- p.50 / Chapter 3.2 --- Experimental Procedures --- p.51 / Chapter 3.2.1 --- Construction of expression vector --- p.51 / Chapter 3.2.1.1 --- Preparation of insert DNA --- p.51 / Chapter 3.2.1.2 --- Construction of entry vector --- p.52 / Chapter 3.2.1.3 --- Construction of destination vector --- p.52 / Chapter 3.2.1.4 --- Construction of expression vector --- p.52 / Chapter 3.2.2 --- Generation of induced neural cells --- p.57 / Chapter 3.2.2.1 --- Culture of mouse embryonic fibroblasts (MEF) --- p.57 / Chapter 3.2.2.2 --- Production of expression vector containing retroviruses --- p.57 / Chapter 3.2.2.3 --- Transfection and induction of neural fate of MEF --- p.57 / Chapter 3.2.3 --- Immunocytochemcial analysis --- p.58 / Chapter 3.2.4 --- Efficiency calculation --- p.59 / Chapter 3.3 --- Results --- p.62 / Chapter 3.3.1 --- A screen for cerebellar Purkinje and granule cell fate-inducing factors --- p.62 / Chapter 3.3.2 --- Characterization of the induced neurons --- p.67 / Chapter 3.3.2.1 --- Granule cell induction --- p.67 / Chapter 3.3.2.2 --- Purkinje cell induction --- p.71 / Chapter 3.4 --- Discussion --- p.102 / Chapter 3.4.1 --- Roles of inducing factors in Purkinje cells and granule cells development --- p.102 / Chapter 3.4.2 --- Mechanism of neural transdifferentiation --- p.107 / Chapter CHAPTER 4 --- Future Directions / Chapter 4.1 --- Complete Induction of Purkinje Cell Fate --- p.111 / Chapter 4.2 --- Induced Neurons of Different Subtypes --- p.112 / Chapter 4.3 --- Mechanism of Transdifferentiation --- p.114 / Chapter 4.4 --- Transdifferentiation and Regenerative Medicine --- p.114 / Bibliography --- p.116

Stem and progenitor cells are an attractive cell source for the treatment of degenerative diseases due to their potential to differentiate into multiple cell types and provide large cell yields. Thus far, however, clinical applications have been limited due to inefficient differentiation into desired cell types with sufficient yields for adequate tissue repair and regeneration. The ability to spontaneously aggregate in suspension makes embryonic stem cells (ESCs) amenable to large-scale culture techniques for the production of large yields of differentiating cell spheroids (termed embryoid bodies or EBs); however, the introduction of hydrodynamic conditions may alter differentiation profiles within EBs and should be methodically examined. The work presented here employs a novel, laboratory-scale hydrodynamic culture model to systematically interrogate the effects of ESC culture hydrodynamics on cardiomyocyte differentiation through the modulation of a developmentally-relevant signaling pathway. The fluidic environment was defined using computational fluid dynamic modeling, and the effects of hydrodynamic conditions on EB formation, morphology and structure were assessed. Additionally, EB differentiation was examined through gene and protein expression, and indicated that hydrodynamic conditions modulate differentiation patterns, particularly cardiogenic lineage development. This work illustrates that mixing conditions can modulate common signaling pathways active in ESC differentiation and suggests that differentiation may be regulated via bioprocessing parameters and bioreactor design.

Current therapeutic strategies to stimulate endogenous angiogenic processes within injured tissue areas are typically based on introducing exogenous pro-angiogenic molecules or cell populations. Stem cell transplantation for angiogenic therapy aims to deliver populations of cells that secrete angiogenic factors and/or engraft in the new branching vasculature within the damaged tissue. Utilizing stem or progenitor cells has been shown to induce a rather robust angiogenic response despite minimal repopulation of the host vasculature, suggesting that stem cells may provide paracrine factors that transiently induce endogenous angiogenesis of tissues undergoing regeneration. Early differentiating embryonic stem cell (ESC) aggregates, referred to as embryoid bodies (EBs), can undergo vasculogenic differentiation, and also produce extracellular matrix and growth factors that induce proliferation, differentiation, and tissue morphogenesis. Taken together, the ESC extracellular environment may be an effective means by which to manipulate cell behavior. Thus, the objective of this project was to harness morphogens derived from ESCs undergoing differentiation and analyze their bioactive potential. To examine the expression of extracellular factors within EBs, gene expression arrays in conjunction with a variety of analytical tools were utilized to gain an understanding of the importance of extracellular factors in ESC differentiation. Furthermore, the soluble fraction of secreted factors contained within EB-conditioned media was compared to the matrix-associated factors produced by EBs, which led to the development of novel ESC-derived matrices via mechanical acellularization methods. Acellular embryonic stem cell-derived matrices demonstrated the retention of bioactive factors that impacted aspects of angiogenesis. In conclusion, extracellular factors were modulated in response to the progression of EB differentiation and can further be harnessed via acellularization techniques, in order to deliver bioactive ESC-secreted factors in a cell-free manner.