The cancer stem cell (CSC) theory proposes that a minor population in tumor cells with specific features, such as self-renewal and reproducible tumor phenotype could contribute to tumor relapse and chemotherapy resistance. Several studies have convincingly documented the existence of ovarian CSC, but questions related to the biologic behavior and specific biomarkers of ovarian CSC remain to be clarified. In the present study, we firstly established a tumor cell line with capability of regenerating tumors through serial transplantation of ovarian tumor tissue in non-obese/severe combined immunodeficient (SCID) mice. After separation of CD133+ cells with magnetic beads, we compared the phenotype and biologic behavior of CD133+ versus CD133-cells. It was found that the CD133+ cells were much more potent to produce colonies in semi-solid agar culture than CD133-cells. The proportion of the cells in G0/1 cell cycle is much higher in CD133+ cells than in CD133-cells. Furthermore, in vivo experiments demonstrated that the CD133+ cells were capable of repeatedly regenerate tumors in NOD/SCID mice, while the CD133-cells were not. Compared with CD133-cells, the CD133+ cells expressed much higher levels of the stem cell markers Oct4, Sox2, Nanog and Mcl-1. Clinically, among a total of 290 ovarian epithelial cancers, increased level of CD133 expression was positively correlated with a high cancer stage and had a worse 5-year survival rate. Taken together, the results suggest that the CD133+ cells from human ovarian cancer have the characteristics of CSC, which may contribute to ovarian cancer relapse and anti-apoptotic activity. The method of ovarian CSC enrichment we established provides a feasible and practical way of ovarian cancer research in a molecular level. In addition, CD133 may be used as a prognostic marker for ovarian epithelial cancer, which may have a role for future therapeutic effect.

Master of Arts / Department of Economics / Florence Neymotin / Florence Neymotin / This report provides review of the existing literature on the role model effect in higher education and identifies the methodological and conceptual issues that have complicated the research program before discussing how research on similar areas may provide insight into the relationship between female students and female faculty members. By examining the related literature on the determinants of college major, peer effects, and critical mass theory, the existing studies of role model effect can be interpreted as support for a more specified theory of the role model effect in higher education that highlights the importance of the gender composition of the course or field of study.

>Magister Scientiae - MSc / According to the literature, women are still unrepresented in the field of information technology. Although many women study computer science at first year level, the dropout rate for women—before they graduate—is high, and if they graduate many decide neither to continue with their studies nor to work in the field of information technology. Research has shown that women who have been exposed to computing at some point during their school career or early in their lives, tend to be more open to choosing a career in computer science and are in many cases successful at it. The aim of this research is to understand what factors influence women to choose a career within computer science and to consider whether gaming would encourage young women to enter the field. Design Science Research was the methodology employed. It was chosen since an artefact was developed. For each of the five steps of this methodology a different method was chosen to address the research question. Surveys were administered to first year computer science students and both IT high school teachers and computer science honours students were interviewed. Non-computer science students were involved in the participatory design process.

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This thesis deals with the issue of stem cell research and its legal aspects. The issue raises many ethical questions, especially due to its closed connection to the topics of embryonic research and right to life. In the first introductory chapter the thesis explains the topic of stem cells from biological point of view and defines and classifies stem cells by their origin and differential potential. In the second chapter the thesis briefly summarizes the history of stem cell research and deals with the ethical questions raised by closed connection of the topics of embryonic stem cell research and in vitro fertilization to the right to life. The second chapter also notes some recent possibilities and medical methods connected with stem cells, such as gene therapy and diagnostics and new eugenics. In the following chapters the thesis concentrates on the legal aspects of the topic on both national and international level. Specifically, the third chapter puts the issue of stem cells research to the contexts of the two most important conventions - the Convention for the Protection of Human Rights and Fundamental Freedoms and the Convention on Human Rights and Biomedicine. The fourth chapter concentrates on legislation of Czech Republic regulating the legal status of embryo and its protection in the...

O envelhecimento é um processo gradual e intrínseco que ocorre devido a mudanças fisiológicas e fenotípicas com o avanço da idade e que acarreta na diminuição da capacidade de manter a homeostase e reparo tecidual. A perda do controle homeostático e o possível envolvimento de células-tronco e progenitores, provavelmente, é uma das causas das fisiopatologias do sistema hematopoético que acompanham o envelhecimento. O declínio na competência do sistema imune adaptativo, o aumento de doenças mielóides, leucemias e o desenvolvimento de anemias são algumas mudanças significantes e decorrentes do processo de envelhecimento. Durante a transição ontológica, a habilidade de células-tronco hematopoéticas originarem células progenitoras diminui progressivamente, sugerindo perda da capacidade de autorrenovação e diferenciação das células-tronco com o avanço da idade. O microambiente medular se divide em duas áreas distintas: nicho endosteal e nicho vascular, conhecidos por controlar a homeostase das células-tronco hematopoéticas; e é composto por uma mistura heterogênea de células, dentre elas as células-tronco mesenquimais que expressam moléculas que controlam algumas funções das células-tronco hematopoéticas. De acordo com estas observações, este trabalho investiga o papel do envelhecimento das células-tronco mesenquimais no processo de autorrenovação, multipotência e diferenciação das células-tronco hematopoéticas. Neste trabalho, avaliamos a percentagem de células-tronco hematopoéticas Lin-CD34+ e subpopulações em co-cultura com células-tronco mesenquimais derivadas de medula óssea de diferentes idades, bem como sua capacidade de autorrenovação, diferenciação, secreção da quimiocina CXCL-12 e a expressão do receptor CXCR-4. Nossos resultados mostraram diferenças significativas nos parâmetros fenotípicos e funcionais das células-tronco hematopoéticas co-cultivadas com células-tronco mesenquimais de doadores idosos. Estes dados sugerem que o envelhecimento das células-tronco mesenquimais podem influenciar na homeostase do microambiente medular / Certainly, aging is one of the best identified features of the human biology, and is also the least understood. This is largely attributed to the fact that aging is gradual and fundamentally complex, due to all modifications in the physiological and phenotypic aspects occurred during the age advancing. One of the most striking features of aging is the decreased ability to maintain homeostasis and tissue repair. Consistent with those findings, many of the pathophysiological conditions affecting aging, such as anemia, dysplasia, leukemia and anemia suggest an imbalance between cell losses and the ability to self-renew or differentiation. The decline in homeostatic maintenance and regenerative potential of tissues during aging has been associated with changes in stem cells. Increasing evidences point to the stem cells as major accountable for the aging pathophysiology in several tissues. Thus, studies in mammals comprise a careful evaluation of mechanisms connected to stem cells. The increasing age is accompanied by many pathophysiological changes in the hematopoietic system wherein the etiology suggests loss of homeostatic control and a possible involvement of stem and progenitor cells. The clinically relevant changes are related to adaptive immune system diminished competence, the increase of myeloid diseases including leukemia and the onset of anemia in the elderly. The hematopoietic stem cell microenvironment is located in the bone marrow and is divided in two domains: the endosteal niche near to the bone surface and vascular niche associated with the sinusoidal endothelium; the niche consist of several heterogeneous cells types, among them, the mesenchymal stem cells. The mesenchymal stem cells express molecules that control hematopoietic stem cells functions. Therefore, this study investigates the role of mesenchymal stem cells aging in the self-renewal, multipotency and differentiation of hematopoietic stem cells. This study evaluated the percentage of hematopoietic stem cell Lin-CD34+ and subpopulations in co-culture with mesenchymal stem cell bone marrow-derived from donors with different ages, their ability of self-renewal, differentiation, secretion of chemokine CXCL-12 and expression of the CXCR-4 receptor. Our results suggest that the mesenchymal stem cells aging can affect the bone marrow niche homeostasis

Hematopoietic stem cells (HSCs) take on the extraordinary role of sustaining life-long production of blood cells. Despite their indisputable therapeutic potential, HSC biology is poorly understood, and the field remains limited by the inability to maintain, expand, or generate HSCs in vitro. The aim of this study was to elucidate a particular gap in our understanding of the organellar cell biology of HSCs, specifically the role and function of the mitochondria. Several signaling pathways and biological processes converge onto the mitochondria, yet these organelles were found to be largely dispensable in HSCs on the basis of their predominantly glycolytic metabolism and reports of low mitochondrial content. Our studies show that MitoTracker Green (MTG), a frequently used fluorescent dye to measure mitochondrial mass in hematopoietic populations, is effluxed by HSCs resulting in their systematic and deceptive enrichment in the subset of cells with the lowest MTG fluorescence. Using dye-independent methods we discovered that HSCs have elevated mitochondrial content despite their reliance on glycolysis for ATP production. Moreover, mechanisms of mitochondrial quality control and clearance by autophagy appear to be comparatively lower in HSCs than in any other hematopoietic population we analyzed, suggesting HSCs maintain their mitochondria over time. To investigate the function of mitochondria in HSCs we generated mice with disruption of mitofusins (MFN) 1 and 2. These proteins are key mediators of mitochondrial fusion, a process that in coordination with mitochondrial fission regulates mitochondrial size, number, and function. Mice with deletion of Mfn1 and Mfn2 (DKO) die perinatally, are pale in appearance and their HSCs show complete loss of regenerative capacity. Several processes linked to dysfunctional mitochondrial fusion and known to be tightly regulated in HSCs are altered in these mutants, including mitochondrial morphology, mitochondrial mass, proliferation, and altered metabolism. Interestingly, one allele of Mfn1 is sufficient to rescue the hematopoietic function and lethality of DKO mice, while one allele of Mfn2 only rescues myeloid reconstitution. Taken together, our findings highlight the importance and complexity of mitochondrial function and dynamics in HSCs and have contributed to the recently increased appreciation of a vital role for mitochondria in HSCs.

Lau Fung Yi. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 107-130). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

The first transplantable hematopoietic stem cells (HSC) arise in the aorta-gonad mesonephros region (AGM) during early stages of embryo development. Specifically, ventral aspect of embryonic dorsal aorta (DA) contains HSC that upon transplantation into irradiated recipients can reconstitute all lineages of the haematopoietic system [Medvinsky et al. 1993; Muller and Medvinsky, 1994; Medvinsky and Dzierzak, 1996; Cumano et al., 1996; Tavian et al., 1996; Peault and Tavian, 2003; Taoudi and Medvinsky, 2007; Ivanovs et al., 2011, 2014]. The ventral aspect of DA bears so-called intra-aortic cell clusters (IAC), the appearance of which coincides with the emergence of HSC [Babovic and Eaves, 2014; Bhatia, 2007; Boisset et al., 2010, 2011; Bollerot et al., 2005; de Bruijin et al., 2002; Bertrand et al., 2010]. According to recent reports, HSC are a heterogeneous population of cells [Dykstra et al., 2007; Seita and Weissman, 2010; Muller-Sieburg et al., 2012]. It is unclear whether all HSC precursors originate from the same location, for example, DA lining, IAC or sub-aortic tissues; or HSC precursors migrate into DA lining from other parts of the embryo [Tavian et al., 1999; Yoder et al., 1997; Oberlin et al., 2002; Peault and Tavian, 2003; Dzierzak, 2003; Samokhvalov et al., 2007; Medvinsky et al., 2011]. To elucidate ontogeny of early HSC precursors (pro-HSC), two approaches were applied in this PhD project. First, we mapped potential pro-HSC in pre-circulation mouse embryos (embryonic day 6-8.5, E6-E8.5). We defined potential pro-HSC as cells co-expressing the transcription factor Runx1, endothelial markers (VE-Cad or CD31) and/or haematopoietic markers (CD45, CD41) [Oberlin et al., 2002; de Bruijn and Dzierzak, 2012; Liakhovitskaia et al., 2009, 2014]. In E6-E8 mouse embryo, prospective pro-HSC were found to be located in chorionic plate, yolk sac and in allantoic core domain. In early somitic mouse embryo (E8-8.5) cells with pro-HSC phenotype (Runx1+CD31+CD41+) were found to be in cell clusters in forming vessel of confluence and in nascent dorsal aortae lining. Pro-HSC are not directly transplantable [Cumano et al., 1996., 2001; Godin et al., 1993; 1995; Batta et al., 2016; Matsuoka et al., 2001; Nishikawa et al., 1998]. Therefore, cells and tissues containing prospective pro-HSC were initially matured using several in-vitro culture systems. According to our results, E8 mouse embryo pro-HSC are only preserved in explant cultures, but not in co-aggregate cultures with stroma cells. After culture, cells were transplanted into sub-lethally irradiated recipients. Six weeks after transplantation 19 out of 82 transplanted recipients had donor derived blood cells' chimerism at the level of 0.1-0.3%. Forty six percent of these grafts were derived from rostral part of the embryo tissues (head, heart, upper somites). Only one out of 82 recipients had donor cells contribution above 1% (1.2 %). This recipient was engrafted with cells derived from the E8 mouse embryo head and heart region. Recipients having blood chimerism at the range of 0.1-0.3% had mainly lymphoid donor derived cells in their peripheral blood. The only recipient showing the high donor cells contribution (1.2%) had contribution mainly to myeloid lineage. Recorded low levels of blood chimersims are in line with those reported by Rybtsov et al. (2014) for early E9 mouse embryos. Donor derived cells formed clearly distinguishable populations on cytometry plots. This population of cells were absent from control engraftment experiments with carrier cells only. Previously, lymphoid potential was detected in paraaortic spnanchnopleura (P-Sp) of E8.5-9 mouse embryos, but not in E8 mouse embryos (0-5 somites, pre-circulation) and later in yolk sac [Cumano et al., 1996; Nishikawa et al., 1998; Fraser et al., 2002; Yokota et al., 2006]. However, prior works used different criteria to establish recipient reconstitution. Therefore, it is possible that recipients repopulated with E8 derived cells at the level of 0.1% were not considered as repopulated and hence, presence of lymphoid lineage precursors was overlooked in early somitic mouse embryos. The only recipient showing substantial myeloid cells contribution (73% Mac1+Gr1+ cells of donor derived cells) received engrafted cells from an older (6-13 sp) embryo and therefore potentially has yolk sac derived myeloid cells. Yolk sac cell contribution to myeloid lineage, specifically to the brain microglia was reported in prior works [Samokhvalov et al., 2007]. Our data show that early E8 AGM cells do not expand in in vitro conditions. While in AGM, cells from E9 mouse embryo expand in culture [Rybtsov et al., 2014]. We have analysed Runx1 expression pattern and dorsal aorta morphology at the time when E9 HSC precursors acquire ability to expand in in vitro culture. Runx1 expression becomes clearly polarised at the time point (22-26 sp), when paired dorsal aortae fusion is initiated. We envision that intimate connection between DA fusion events and induction of pro-HSC maturation exists. According to prior reports, Bmp, Shh and VEGF signalling regulate DA fusion [Garriock et al., 2010]. Thereofore, to enhance in vitro HSC maturation system, DA fusion triggers (for example, Bmp4) might be added to culture. Since, pro-HSC maturation methods established to date are not efficient to expand and differentiate E8 pro-HSC into potent HSC, another approach had to be implemented to study HSC ontogeny. The second approach we utilized was to trace the origin of HSC in chicken embryo, starting from the very beginning of cell fate specification, i.e. from gastrulation stages. Chick embryo haematopoiesis is similar in both human and mouse: precursors of HSC arise in the embryo proper in AGM, and IAC are formed in DA ventral aspect [Dieterlen-Lièvre, 1975; Dieterlen-Lièvre and Martin, 1981; Dieterlen-Lièvre and Jaffredo, 2009; Jaffredo et al., 2000; Le Douarin and Dieterlen-Lièvre, 2013]. In contrast to mammals, chick embryo develops ex vivo, making direct labelling and cell tracing possible. We aimed to identify cells giving rise to regions of DA that produce IAC. Therefore, segments of primitive streak (PS) were labelled with lipophilic dyes or by substituting segments of host PS with PS sections derived from transgenic (GFP+) stage matched chicken embryos. Our results show that in an 18-25h chicken embryo (Hamburger and Hamilton developmental stage 4-6, HH4-6) cells giving rise to DA ingress through the wide region of PS (35-60% of its length) [Hamburger and Hamilton, 1951]. We identified that the section of DA producing HSC is formed by cells ingressing through PS in region of 40-55% of its length at 18-25h of chick embryo development. Regardless of the embryo development stage (HH4-6), in chimeras grafted at 40-55% of PS length, GFP+ cells contributed to DA and to the IAC. Within GFP+ labelled areas, we observed clusters consisting entirely of GFP+ and clusters having a mixture of GFP+ and GFP- cells. Entirely GFP+ clusters were found in the stretch of DA that had the entire aortic endothelial lining labelled. Clusters formed on the mosaic (GFP+/GFP-) aortic endothelium also had mosaic nature. According to our data, multiple descendants of PS contribute to the same stretch of dorsal aorta. This explains mosaicity of dorsal aorta lining and IAC labelling. Since we encountered clusters with mixture of GFP+ and GFP- cells, we conclude that IAC are not clonal formations. Mosaicity of IAC also does not exclude a scenario when cells migrate in and out of a cluster. Further tracing experiments are required to establish HSC nature of cells within a cluster.

"The effectiveness of exogenous cellular therapies has been limited by the ability to efficiently and locally deliver cells to a region of interest. We have developed biological sutures, formed from fibrin microthreads, to overcome these delivery issues and demonstrated increased cell engraftment compared to the current gold standard. However, the cell seeding efficiency onto the sutures is low and during implantation cells are subjected to shear forces as the sutures are pulled through the tissue. As a result, cells go unused after seeding and an uneven distribution of cells from the entry point to exit of the suture. By adding cell attachment and adhesion promoters and increasing culture time we proposed to overcome these issues. We have developed a shear loading method to evaluate the changes in cellular adhesion. Either poly-l-lysine or vitronectin was used to coat sutures. Uncoated control and coated sutures were then seeded with 100,000 human mesenchymal stem cells (hMSCs) for 24hrs or control sutures were seeded for 48hrs. An in vitro shear stress model was created by spinning seeded sutures with a centrifuge. Cell number per unit length prior to and post spinning were compared. To compare the effect of modifications on cell morphology cells were qualitatively assessed and nuclear alignment was evaluated as a robust measurement for overall cellular angle. Control sutures were found to have 6,821±739cells/cm prior to spinning, while sutures modified with poly-l-lysine resulted in 4,226±1,003cells/cm and vitronectin had 19,604±1,829cells/cm (p<0.05 vs. control and poly-l-lysine). 48hrs seeding resulted in a cell number to 4,417±2,266 cells/cm. Spinning resulted in relative decreases in cell number for control and coated sutures. Cells remained attached after sutures were spun after increased incubation time. Cells aligned along the long axis of individual microthreads; the alignment on control sutures was significantly different from all modifications. There was no difference in alignment between modifications, although they were significantly different compared to cells grown on topographically flat tissue culture plastic. These results demonstrated increased cell seeding efficiency and cell number for vitronectin coated biological sutures and increased cell adhesion following increased incubation time. The combination of these two modifications may lead to increased quantity and more evenly distributed cells delivered to diseased tissues by increasing initial cell number, increasing cell engraftment, and increased resistance to shear."