The Christian Right has been a major contributor to the policy process since the 1980s, helping shape the national agenda by illuminating a number of social issues and influencing elections with strong grassroots campaigns. For political scientists, Christian Right organizations provide a rich source of information for studying interest group activity, electioneering, and general political theory. In particular, their efforts to lobby various policy issues such as prayer in school, education, abortion, and traditional marriage, has caused them to become a distinct coalition of advocacy groups, and the focus of much research by many scholars. However, as we advance into the twenty-first century, new biotechnology-related issues have emerged that challenge Christian Right organizations and their values. The purpose of this dissertation was to investigate the involvement of the Christian Right between 2001 and the beginning of 2009 in legislative debates regarding stem cell research policy, and attempt to distinguish its effective and non-effective lobbying strategies, and the general perception of its influence. This study addressed three research questions. First, to what extent do Christian Right organizations participate in the legislative process regarding stem cell research? Second, what is the perceived influence of its lobbying activities on federal stem cell research legislation? Third, in what ways does the Christian Right engage in lobbying legislators on stem cell research legislation? Within these broad research questions, the following subsequent study objectives were pursued: 1) learn about the reasons for the Christian Right’s influence or lack of influence; 2) understand the goals of its advocacy efforts; 3) learn about its use of outside and inside lobbying strategies; 4) better understand the approach used by Christian Right organizations in lobbying legislators who were undecided about a particular stem cell research legislation; 5) learn about the kind of rhetoric it used; and 6) find out what, if any, forms of coalition building it engaged in as part of its advocacy efforts. In addition, this study examined why legislators voted against the majority of their political party when it came to stem cell research legislation. The epistemological approach for this study was qualitative. Data consisted of verbal responses to semi-structured questions during telephone interviews with representatives from Christian Right organizations, advocacy groups that support the expansion of stem cell research policy, former legislators, and current staff members. In addition to the in-depth interviews, data was also obtained through organizational and government documents. Finally, this dissertation analyzed the Christian Right and its participation in the development of stem cell research legislation through the lens of the advocacy coalition framework. In doing so, the study captures of the essence of the stem cell debate and the role of the Christian Right within it, and offers a new theoretical framework for examining the Christian Right.

Adult stem cells are a small population present within several tissues of an individual, possessing two unique properties: one, the ability to differentiate to give rise to all the cell types of the tissue, and second, the ability to self-renew and make more of their own kind. Owing to these two properties, stem cells underlie the process of organogenesis during development and tissue homeostasis in adult life. In the past decade a small sub-population of cells having phenotypic and functional properties similar to normal stem cells have been identified within several tumors. Only this sub-population of cancer cells seems to have the ability to both initiate and maintain tumors. These cells have been termed as ‘cancer stem cells’ (CSCs) owing to their striking similarities with the normal stem cells of the tissue. It is therefore of fundamental importance to understand normal stem cell biology in order to understand tumorigenesis. The rarity of normal stem cells within adult tissues, the absence of specific cell surface markers to identify and isolate them, and the absence of suitable culture conditions to maintain them has marred our understanding of stem cell behaviour. Recently, growth of mammary cells in serum free suspension cultures resulted in the generation of floating spheroids termed “mammospheres” that were shown to be enriched in stem/progenitor cell population. We established the mammosphere system in our laboratory using mastectomy samples obtained from the Kidwai Memorial Institute of Oncology. In order to understand the composition of the spheres, the stem cell characteristics within them, and the long term self renewal potential of human mammary epithelial stem cells, a detailed phenotypic and functional characterization of the mammospheres was carried out. Phenotypic Characterization: Confocal microscopy of propidium iodide stained mammospheres demonstrated that these spheres are cellular and not hollow structures. Immunostaining revealed that primary mammospheres expressed the epithelial markers like E Cadherin, ESA, CK14, CK18 and CK 19, but failed to express nestin or CD34, indicating their epithelial origin, devoid of contamination from haematopoeitic or neural stem cells. The sizes of mammospheres ranged from 40 to 110 μm, while that of the cells within them ranged from 9-15 μm. Although the sizes of the largest and smallest spheres through subsequent passages remained consistent, the proportion of small spheres increased in later passages. These results indicate the difference in the sphere initiating cells. While a large sphere might be generated by a stem cell, a smaller sphere might be originating from a progenitor. Thus, heterogeneity exists within mammospheres, with respect to size and composition. Unique cell surface markers coupled with flow cytometry serves as useful tools to isolate stem cells. However, no specific marker profile has been reported for normal human breast stem cells. In several tissues, like blood, brain etc, markers of normal stem cells have been successfully used to isolate cancer stem cells within that tissue. Since breast cancer stem cells have already been identified as CD24low/-44high cells, we explored if the same marker profile would hold true to identify normal breast stem cells as well. Two-colour based flow cytometry revealed that only the CD24low/-44high subpopulation of mammospheres could re-generate mammospheres, as well as give rise to all the other cellular fractions. These data demonstrated that normal and cancerous breast stem cells share identical marker profile. Functional Characterization: In addition to cell surface markers, a Hoechst dye based strategy used to isolate stem cells, exploits their unique property to efflux certain lipophilic drugs and small molecules due to the overexpression of ABC family of cell surface transporters. Cells effluxing Hoechst appear as a low fluorescing ‘Side population’ (SP) in a bivariate FACS plot. We detected a small, but distinct SP in human breast cells, which had a CD24low44low profile, and failed to initiate new mammospheres. Thus, the SP cells in mammospheres failed to correspond to the stem cell subpopulation. The hallmark feature of a stem cell is its long term self renewal ability, given that it is the longest lived cell in the body. Long term culture of mammospheres was carried out by passaging the spheres every week. We failed to observe mammosphere formation beyond four passages though there were live, proliferating and undifferentiated cells in fourth passage spheres. These results suggested that either the mammopsheres didn’t contain stem cells to begin with, or their stemness is restricted to four in vitro passages. In order to assess if mammospheres contained stem cells to begin with, we assayed for telomerase activity, since in the adult tissue, only stem cells retain telomerase activity. Telomerase, an enzyme that maintains the length of telomeres through multiple rounds of cell division, is not active in somatic cells. We detected the expression and activity of this enzyme in primary mammospheres, suggesting that the spheres may contain stem cells withinthem Another unique property of a stem cell is its ‘quiescence’, owing to their infrequent divisions. This property is studied by chasing a label (like BrdU or H3-Thymidine), which is taken up by the cells at an earlier time point and retained within the cell after prolonged periods, like weeks or months. In long term culture of mammospheres, using BrdU as the label, 1-2 distinct cells could be detected within late passage spheres which had retained the label, indicating that stem cells may be present within the fourth passage mammospheres as well. Staining for β-Galactosidase activity revealed that almost 70% cells derived from fourth passage spheres were senescent. We speculated that this senescent environment might be one of the inhibitory reasons for further mammosphere formation. Alteration of mammosphere culture conditions for long term maintenance of stem cells. A high level of atmospheric O2 is known to be one of the reasons for inducing senescence in cells. Culturing cells in conventional tissue culture conditions exposes them to high levels of O2 (21%) as against the physiological levels of 1-3% O2. Therefore, to assess the effects of lowered, or physiologically relevant levels of O2 on mammosphere stem cell biology, the mammospheres were cultured in 3% O2. Under this altered condition, a close to 3-fold increase was observed in the number of mammospheres formed coupled with a significant increase in their survival and proliferation. In order to understand the molecular basis of this observation, a microarray based global gene expression profiling was carried out. We observed a significant upregulation of VEGF, a gene responsive to hypoxia; three growth factor related genes, namely adrenomedullin, cMET and osteopontin. Upregulation of β Catenin, the downstream effector of the Wnt signaling pathway was also observed, indicating a possible mechanism for the increase in self renewal seen in 3% O2. We also observed downregulation of the cell cycle inhibitor, Chk1, which in part might explain the observed increase in proliferation. The increase in the number of proliferating cells might be one of the reasons for an increase in the number of spheres, as observed in 3% O2. Even though a significant decrease in the number of senescent cells was detected at 3% O2, mammosphere formation was not seen beyond four passages. It is therefore possible that there are other physico-chemical parameters, comprising the niche of the mammospheres, coupled to the O2 level, which need to be improvised for long term culture of human mammary epithelial stem cells. To summarize, this work reports for the first time that human mammary epithelial stem cells have an identical marker profile as breast cancer stem cells, which is CD24low/-CD44high. It has also been demonstrated for the first time that in long term mammosphere culture, the number of self renewal divisions of human mammary stem cells is restricted to four in vitro passages, at which most of the cells undergo senescence. Altering one of the parameters of the niche, by culturing mammospheres at physiological O2 level failed to prolong the in vitro lifespan of the spheres, although cell survival, proliferation and sphere formation increased, indicating that the niche requirements of human mammary epithelial stem cells for their long term self renewal needs to be further characterized.

Human organoids could facilitate research of complex and currently incurable neuropathologies, such as age-related macular degeneration (AMD) which causes blindness. Here, we establish a human retinal organoid system reproducing several parameters of the human retina, including some within the macula, to model a complex combination of photoreceptor and glial pathologies. We show that combined application of TNF and HBEGF, factors associated with neuropathologies, is sufficient to induce photoreceptor degeneration, glial pathologies, dyslamination, and scar formation: These develop simultaneously and progressively as one complex phenotype. Histologic, transcriptome, live-imaging, and mechanistic studies reveal a previously unknown pathomechanism: Photoreceptor neurodegeneration via cell extrusion. This could be relevant for aging, AMD, and some inherited diseases. Pharmacological inhibitors of the mechanosensor PIEZO1, MAPK, and actomyosin each avert pathogenesis; a PIEZO1 activator induces photoreceptor extrusion. Our model offers mechanistic insights, hypotheses for neuropathologies, and it could be used to develop therapies to prevent vision loss or to regenerate the retina in patients suffering from AMD and other diseases.

Pluripotent stem cells (PSCs) are specialized cells, which have remarkable ability to maintain in an undifferentiated state and are capable of undergoing differentiation to three germ-layer lineage cell types, under differentiation-enabling conditions. PSCs include embryonic stem (ES)-cells, embryonal carcinoma (EC)-cells and embryonic germ (EG)-cells. ES-cells are derived from the inner cell mass (ICM) of day 3.5 blastocysts (mouse). On the other hand, EC- and EG-cells have different source of origin and exhibit some differences in terms of their differentiation abilities and culture requirements. These PSCs act as an ideal in-vitro model system to study early mammalian development and cell differentiation and, they could potentially be used for experimental cell-based therapy for a number of diseases. However, one of the problems encountered is the immune rejection of transplanted cells. For this, immune-matched induced pluripotent stem (iPS)-cells have been derived from somatic cells, by forced expression of a few stemness genes. Although, human PSCs lines are being experimented, their cell-therapeutic potential is still far from being thoroughly tested due to lack of our understanding regarding lineage-specific differentiation, homing and structural-functional integration of differentiated cell types in the host environment. To understand these mechanisms, it is desirable to have fluorescently-marked PSCs and their differentiated cell-types, which could facilitate experimental cell transplantation studies. In this regard, our laboratory has earlier generated enhanced green fluorescent protein (EGFP)-expressing FVB/N transgenic ‘green’ mouse: GU-3 line (Devgan et al., 2003). This transgenic mouse has been an excellent source of intrinsically green fluorescent cell types. Recently, we have derived a ‘GS-2’ ES-cell line from the GU-3 mouse line (Singh et al., 2012). Additionally, we envisaged the need for developing an iPS-cell line from the GU-3 mouse and then use them for studying cell differentiation. Thus, aims of the study described in the thesis are to: (1) develop an experimental system to derive EGFP-expressing fluorescently-marked iPS-cell line from a genetically non-permissive FVB/N mouse strain, characterize the established iPS-cell line and achieve differentiation of various cell types from EGFP-expressing iPS-cell line; (2) to study differentiation phenomenon, in particular to cardiac lineage, using select-cardiogenesis modulators and (3) to assess the gene-expression profiles and signaling system associated with cardiomyocyte differentiation of PSCs. This thesis is divided into four chapters with the 1st chapter being a review of literature followed by three data chapters. In the chapter I of the thesis, a comprehensive up-to¬date review of literature is provided pertaining to PSCs, their classification, derivation strategies especially for reprogramming of somatic cells for iPSC generation, their differentiation potential and characterization, particularly to cardiac lineage. Various molecular regulators involved in cardiac differentiation of PSCs with emphasis on epigenetic regulation involving DNA methylation and signaling pathways involved are described in detail. Subsequently, various approaches used for enhanced cardiac differentiation of PSCs and the therapeutic potential of PSC-derived differentiated cell types to treat disease(s) are discussed. Chapter-II describes the successful establishment of a permanent iPS-cell line (named ‘N9’ iPS-cell line) from the non-permissive FVB/N EGFP-transgenic GU-3 ‘green’ mouse. This chapter provides results pertaining to detailed derivation strategy and characterization of the ‘N9’ iPS-cell line which includes colony morphology, expansion (proliferation) efficiency, alkaline phosphatase staining, pluripotent markers’ expression analysis by qPCR and immunostaining approaches and karyotyping analysis. Further, in order to thoroughly assess the differentiation competence of the ‘N9’ iPS¬cell line, assessment of in-vitro and in-vivo differentiation potential of the ‘N9’ iPS-cell line by embryoid body (EB) formation and teratoma formation in nude mice and its detailed histological analysis showing three germ layer cell types and their derivatives were performed, followed by the generation of chimeric blastocysts by aggregation method. This established N9 iPS-cell line could potentially offer a suitable model system to study cardiac differentiation along with other established PSC lines such as the GS-2 and D3 ES-cell lines and the P19 EC-cell line. Following the establishment of the system to study cardiac differentiation of PSC lines, efforts were made to understand the biology of cardiac differentiation of PSCs (wild¬type and EGFP-transgenic PSC lines and P19 EC-cell line) using small molecules as modulators. Data pertaining to this is described in Chapter-III. The possible involvement of epigenetic regulation of cardiogenesis for example, DNA methylation changes in cardiogenesis-associated genes is studied using 5-aza cytidine as one of the chromatin modifiers. In order to understand the cardiac differentiation phenomenon, as a consequence of using 5-aza cytidine in cell culture, it was important to investigate its ability to induce/mediate cardiac differentiation. This involved an assessment by quantitating the cardiac beating phenotype and correlating this with enhanced cardiac-gene expression profiles. Further, DNA methylation regulation of cardiogenesis¬associated genes is described using various DNA methylation analysis techniques. Moreover, the possible involvement of other signaling members in mediating the cardiac differentiation is also studied using the P19 EC-cells. Results pertaining to the above findings are described in detail in the Chapter-III. Chapter-IV is focused on various efforts made towards investigating the ability of ascorbic acid to enhance cardiac differentiation of mouse ES-cells (GS-2 and D3 lines). Ascorbic acid has been implicated to be influencing cardiogenesis and it is reported to enhance differentiation of various cell types under certain culture conditions. Results pertaining to enhancement of cardiac differentiation of PSCs using ascorbic acid are presented in this chapter. This included assessment by quantitating cardiac beating phenotype and its correlation with enhanced cardiogenesis-associated gene expression profiles. Besides, estimation on the sorted cardiomyocyte population, derived from PSCs was also made using mature-cardiac marker. The possible underlying signaling mechanism involved was also studied in detail, using specific inhibitors for pERK (U0126), integrin signaling (pFAK; PP2) and collagen synthesis (DHP), in order to ascertain their involvement in ascorbic acid-mediated cardiac differentiation of mouse ES-cells. Subsequent to the three data chapters (II-IV), separate sections are provided for ‘Summary and Conclusion’ and for ‘Bibliography’, cited in the thesis. The overall scope of the study has been to understand the basic biology of cardiac differentiation from PSCs (EC-cells, iPS-cells and transgenic and wild-type ES-cells) and to assess, by using certain small molecules, whether PSCs could be coaxed to enhance the differentiation to a particular cell type (cardiac). The data contained in this thesis addresses the above theme.

Tumors exhibit complex organization and contain a variety of cell populations. The realization that the regenerative properties of a tumor may be largely confined to a cell subpopulation (cancer stem cell) is driving a new era of anti-cancer research. Cancer stem cells from Glioblastoma Multiforme tumors express markers that are also expressed in non-cancerous neural stem cells, including nestin and Sox2. We previously showed that the transcription factor Hes3 is a marker of neural stem cells, and that its expression is inhibited by JAK activity. Here we show that Hes3 is also expressed in cultures from glioblastoma multiforme which express neural stem cell markers, can differentiate into neurons and glia, and can recapitulate the tumor of origin when transplanted into immunocompromised mice. Similar to observations in neural stem cells, JAK inhibits Hes3 expression. Hes3 RNA interference reduces the number of cultured glioblastoma cells suggesting a novel therapeutic strategy.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-19T13:49:54Z No. of bitstreams: 1 Gabrielle Viana Martins Gonçalves Geração...2015.pdf: 6199357 bytes, checksum: 605427028fc638e77cf5015ba759917c (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-02-19T13:50:09Z (GMT) No. of bitstreams: 1 Gabrielle Viana Martins Gonçalves Geração...2015.pdf: 6199357 bytes, checksum: 605427028fc638e77cf5015ba759917c (MD5) / Made available in DSpace on 2016-02-19T13:50:09Z (GMT). No. of bitstreams: 1 Gabrielle Viana Martins Gonçalves Geração...2015.pdf: 6199357 bytes, checksum: 605427028fc638e77cf5015ba759917c (MD5) Previous issue date: 2015-01 / Fundação Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / As células-tronco mesenquimais (CTM) constituem uma ferramenta promissora para o campo de terapia celular. Além de seu potencial de diferenciação em diferentes tipos celulares, as CTM apresentam a habilidade de secretar moléculas bioativas e, assim, exercer múltiplos efeitos biológicos, tais como indução da regeneração de tecidos lesionados, redução de fibrose e modulação do sistema imune. A superexpressão dos fatores de crescimento G-CSF e IGF-1, conhecidos por seus efeitos sobre os processos de imunomodulação, sobrevivência celular e reparo tecidual, pode ampliar as ações terapêuticas das CTM. O objetivo deste trabalho consiste em gerar e caracterizar linhagens de CTM de camundongo superexpressando hGCSF ou hIGF-1. Um sistema lentiviral de segunda geração foi utilizado para modificação de CTM para expressão ectópica dos genes de interesse. As sequências codificantes de hG-CSF e hIGF-1 foram amplificadas por PCR e subclonadas em um vetor lentiviral de transferência, contendo um promotor constitutivo. As partículas lentivirais foram produzidas a partir da cotransfecção de células da linhagem HEK293FT com os vetores constituintes do sistema lentiviral. Em seguida, as CTM obtidas da medula óssea de camundongos transgênicos para proteína fluorescente verde (GFP) foram transduzidas com partículas lentivirais infectantes contendo hG-CSF ou hIGF-1. A expressão gênica de hG-CSF ou hIGF-1 pelas linhagens geradas foi quantificada por qRTPCR, e a produção da proteína por ELISA. As linhagens foram caracterizadas por imunofenotipagem e avaliadas quanto ao seu potencial de diferenciação celular. Foram geradas duas linhagens de CTM superexpressando hG-CSF e três linhagens superexpressando hIGF-1. Todas demonstraram por qRTPCR, estar efetivamente expressando os genes de interesse. Foi possível detectar e quantificar a síntese proteica de G-CSF e IGF-1. Todas as linhagens geradas foram capazes de se diferenciar em osteócitos, condrócitos e adipócitos, demonstrando a manutenção de seu fenótipo estromal. Neste contexto, este trabalho resultou em ferramentas funcionais para a avaliação dos efeitos terapêuticos de IGF-1 e G-CSF combinados à CTM, em modelos de lesões animais, em comparação com CTM não-modificadas geneticamente. Além disso, estas ferramentas poderão ser empregadas em estudos de pesquisa básica, para melhor compreensão dos efeitos de hIGF-1 e hG-CSF sobre a biologia das CTM. / Mesenchymal stem cells (MSCs) are a promising tool for the cell therapy field. In addition to their potential for differentiation into different cell types, MSCs have the ability to secrete bioactive molecules and thus exert multiple biological effects such as induction of the injured tissue regeneration, fibrosis reduction and modulation of the immune system. The overexpression of the growth factors G-CSF and IGF-1, known for their effects on immune modulation processes, cell survival and tissue repair, can result in a magnification of MSCs' therapeutic actions. The objective of this work is to generate and characterize mouse MSCs lines overexpressing hG-CSF or hIGF-1. A second generation lentiviral system was used to modify MSCs derived from mice for the ectopic expression of the genes of interest. The coding sequences of hG-CSF and hIGF-1 were amplified by PCR and subcloned into a lentiviral transfer vector containing a constitutive promoter. The lentiviral particles were produced from the co-transfection of HEK293FT lineage cells with the lentiviral vectors. Subsequently, MSCs obtained from the bone marrow of transgenic mice for green fluorescent protein (GFP) were transduced with infectious lentiviral particles containing hG-CSF or hIGF-1. The gene expression of hG-CSF or hIGF-1 by the generated cell lines was quantified by qRTPCR, and the protein production by ELISA. The lineages were characterized by immunophenotyping and evaluated for their potential of cellular differentiation. Two lines of MSCs overexpressing hG-CSF and three lines overexpressing hIGF-1 were generated. All the cell lines demonstrated to be effectively expressing the genes of interest by qRTPCR. It was possible to detect and quantify the protein synthesis of G-CSF and IGF-1. Moreover, all the generated lines were capable of differentiating into osteocytes, chondrocytes and adipocytes, indicating the conservation of their stromal phenotype even after genetic modification. In this context, this study resulted in functional tools for evaluating the IGF-1 and G-CSF therapeutic effects when combined with MSCs, to be tested in experimental animal models in comparison to non-genetically modified MSCs. Furthermore, these tools may be employed for basic research studies, for a better understanding of the effects of hIGF-1 and hG-CSF on MSCs' biology

The aim of this dissertation is to examine and analyse the judicial framework with regard to embryonic stem cell research and cloning in South Africa. The examination is conducted within the framework of the South African and United Kingdom's legal systems. Focus is placed on aspects of medical law, human rights law as envisaged in the Constitution of the Republic of South Africa, and the law of persons. The specific focus of this dissertation is to examine the intense debate on the moral and legal status of the embryo and fetus in South Africa. A comparative study is undertaken, with the United Kingdom as a background against which recommendations for the South African framework are made. The study firstly provides a clinical overview of stem cell research and cloning. Secondly, the concept of life, in particular human life; the protection of the embryo and fetus under the constitutional guarantee of the right to life, among other constitutionally protected rights, are examined. In this context, the most important finding is that although the fetus is not a bearer of constitutional rights the state has a constitutional duty to protect fetal life in terms of an objective value system. Thereby, the state is permitted to regulate abortion, fetal tissue research, and embryo research to protect fetal life. In particular, the aim of this dissertation is to present a critical summary of the major debates and policy responses relating to embryonic stem cell research and cloning techniques, drawing attention to some of the challenges posed by conflicting moral values in an era of global scientific endeavour, and to provide an analysis of the key ethical and regulatory implications for stem cell therapy. The most important findings are that current South African legislation remains fragmented and ineffective in the manner in which embryonic stem cell research and cloning are regulated. This finding leads to a summary of recommendations, which attempts to provide specific remedies in order to adapt the current regulatory framework in South Africa. / Dissertation (LLM (Public Law))--University of Pretoria, 2007. / Public Law / LLM / unrestricted

Tumors exhibit complex organization and contain a variety of cell populations. The realization that the regenerative properties of a tumor may be largely confined to a cell subpopulation (cancer stem cell) is driving a new era of anti-cancer research. Cancer stem cells from Glioblastoma Multiforme tumors express markers that are also expressed in non-cancerous neural stem cells, including nestin and Sox2. We previously showed that the transcription factor Hes3 is a marker of neural stem cells, and that its expression is inhibited by JAK activity. Here we show that Hes3 is also expressed in cultures from glioblastoma multiforme which express neural stem cell markers, can differentiate into neurons and glia, and can recapitulate the tumor of origin when transplanted into immunocompromised mice. Similar to observations in neural stem cells, JAK inhibits Hes3 expression. Hes3 RNA interference reduces the number of cultured glioblastoma cells suggesting a novel therapeutic strategy.

No description available.