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Limbal stem cells maintain the corneal epithelium through a process of clonal growth and ordered migration. In X-inactivation mosaic female mice, that express LacZ from one of their X-chromosomes, random clumps of LacZ-positive cells are seen in the cornea at 3-6 weeks of life. This pattern resolves between 6-10 weeks forming radial stripes thought to represent chords of clonally related, inwardly migrating cells. By measuring the number and width of stripes and correcting for the effects of different proportions of LacZ-positive cells, an estimate of the number of coherent stem cell clones maintaining the tissue can be derived. Analysis at 5 ages demonstrated that the estimated number of coherent stem cell clones is reduced from ~100 at 15 weeks to ~50 at 39 weeks and is then stable at least until 52 weeks. An automated method was developed using image analysis software to analyse these striping patterns. This method produced results that did not differ significantly from the above. The dosage of the transcription factor Pax6 is crucial for normal eye development. In Pax6 heterozygous animals the estimated number of coherent stem cell clones is reduced to ~50 at 15 weeks with no further reduction up to 30 weeks. Mice hemizygous for the PAX77 transgene over-express human PAX6. In PAX77 hemizygous X-inactivation mosaics, estimated clone number was similarly reduced to ~50 with no further decline. Mice heterozygous for both Gli3 and Pax6 have a distinct striping phenotype, highlighted by an increase in coherent clones. When the corneal epithelium is injured the surrounding epithelial cells migrate along the corneal stroma to cover the wound. X-gal staining of healed, centrally wounded X-inactivation eyes reveals that striping patterns are reconstituted during wound healing in ex-vivo culture. In GFP mosaics the healing process can be imaged using time-lapse confocal microscopy. This technique demonstrated that clones remain contiguous throughout their migration. Healing of peripheral wounds was observed to form de-novo whorling patterns, revealing that basal cells in the epithelium can migrate both away from and towards the limbal region.

Pluripotency is defined as the ability of a cell to give rise to all the cell types of the adult organism. In vivo this property is possessed transiently by the cells of the epiblast in the developing embryo but it can be maintained indefinitely by deriving embryonic stem (ES) cells. How the pluripotent state is established in the cells of the early embryo and how it is ‘captured’ and maintained in the form of ES cells is a fascinating question for biology with practical implications. It is hoped that ES cells will be of use in biomedical research and cell replacement therapy. Our understanding of their biology and our ability to manipulate the cells in vitro will be of great importance if these hopes are to be realised. The starting point for the work presented in this thesis was the development of a novel culture condition for the derivation and maintenance of mouse ES cells (Q-L. Ying and J. Nichols). The media is formed by the addition of three small molecule inhibitors to a previously described serum-free media, N2B27, and is termed 3i (three inhibitors).The inhibitors are SU5402, PD184352 and CHIRON99021, and they inhibit the FGF receptor, mitogen activated protein/extracellular signalregulated kinase (ERK) kinase (MEK), and glycogen synthase kinase 3 (GSK3) respectively. I attempt to further our understanding of pluripotency and self-renewal in ES cells by genetic and biochemical examination of ES cells cultured in 3i. Analysis of intracellular signalling pathways together with descriptions of genetic mutants for the targets of the inhibitors validates the mode of action and the specificity of the three inhibitors. Self-renewal of mouse ES cells is considered dependent on activation of STAT3 through provision of the cytokine leukaemia inhibitory factor (LIF). I demonstrate unequivocally that this pathway is not required for self-renewal in 3i by characterising Stat3-null ES cells. Further experiments reveal that preventing activation of ERK downstream of the growth factor FGF4, produced by the ES cells themselves, is key to preventing differentiation. Pleiotropic effects of GSK3 inhibition are observed and candidate GSK3 targets with known or predicted effects on self-renewal are investigated as potential downstream effectors. I propose that activation of canonical Wnt signalling, together with a global derepression of biosynthetic capacity, mediate the pro-self-renewal effects of GSK3 inhibition. The description of culture conditions that function independently of signalling pathways previously thought essential for self-renewal provides fresh insight into the nature of ES cell self-renewal and the relationship of ES cells to the pluripotent cells of the developing embryo. There are practical implications for ES cell biology as there is reason to hope that the new conditions will translate more readily to other mammalian species to facilitate the derivation of ES cells and will provide an optimal platform for differentiation of ES cells into somatic cell types of interest.

La Universidad Peruana de Ciencias Aplicadas (UPC), a través de su Facultad de Educación, organizó el Congreso Internacional de Educadores UPC 2016 - “Innovación en la Educación”. Este importante evento que se realizó los días 2, 3 y 4 de febrero en el Centro Convenciones del Colegio Médico del Perú y en el Campus Villa de la UPC, contó con la participación de un distinguido grupo de 9 expositores internacionales provenientes de países como Estados Unidos, España, Australia, Canadá y Alemania, y 20 destacados especialistas nacionales quienes compartieron su conocimiento a través de Conferencias Magistrales y Paneles de expertos. http://bit.ly/1T6hIiN / Conferencia presentada en el Congreso Internacional de Educadores UPC 2016 – Innovación en la Educación por el ponente internacional Bernardo León De la Barra de Australia quien es académico e investigador en la Escuela de Ingeniería y Tecnologías de la Información y Comunicación de la Universidad de Tasmania, Australia.

Histones are proteins found in the nuclei of eukaryotic cells where they are complexed to DNA in chromatin. Rephcation-dependent histones are expressed only during S-phase. Regulation of expression of replication-dependent histone genes requires a highly conserved hairpin RNA element in the 3' untranslated region of histone mRNAs. Replication-dependent histone mRNAs are not polyadenylated; their 3' end is formed by an endonucleolytic cleavage event, 3' of a hairpin element, which is recognised by the Hairpin Binding Protein, HBP (also known as Stem-Loop Binding Protein, SLBP). This protein-RNA interaction is important for the endonucleolytic cleavage that generates the mature mRNA 3' end. The 3' hairpin, and presumably HBP, are also required for nucleocytoplasmic transport, translation and stability of histone mRNAs. It is therefore important to understand this interaction. The hairpin is highly conserved and I have demonstrated that residues in the hairpin loop are important for binding the HBP. This complimented structural studies that showed that the same residues are involved in stacking interactions in the RNA loop. In cell culture, expression of replication-dependent histone genes is S phase specific as is the expresion of HBP. Here I demonstrated that in Caenorhabditis elegans the HBP promoter is active in dividing cells during embryonic and postembryonic development. Depletion of HBP by RNAi leads to an embryonic lethal phenotype associated with defects in chromosome condensation. Postembryonic depletion of HBP results in defects in cell fate during late larval development, specifically in vulval development. A similar phenotype was obtained when histone H3 and H2A were depleted by RNAi suggesting that the phenotype of the hbp (RNAi) worms was due to a lack of histone proteins. I have confirmed this by showing that histone proteins are indeed reduced in hbp (RNAi) worms. I have also shown that depletion of HBP leads to a change in expression of a number of other proteins and specifically an up-regulation of a histone H3 like protein with an apparent molecular mass of 34 kDa. I have evidence that suggests that this protein is the centromer specific protein, CENP-A. As this protein was up-regulated when RNAi was used to deplete histones proteins, this suggests that there could be a compensatory mechanism that helps the animal to deal with the shortage of histone proteins.

Migraine is a highly prevalent headache disorder imposing a significant burden of disability on human health worldwide. The headache is believed to arise from activation of trigeminal pain pathways, with CNS regions also playing an integral role in attack initiation and progression. Recent genetic associations have been made, but there is a need to convert these into relevant experimental models to study underlying disease mechanisms. Herein, I detail functional analysis of two deleterious variants in the genes KCNK18 and SLC12A3, that segregate with migraine with aura in one large pedigree. Gene function has been studied in a range of cell models, from heterologous expression systems and primary neuronal cultures, to Induced Pluripotent Stem (iPS) cell-derived nociceptors. In this context, the protein products of KCNK18 and SLC12A3 have been shown to modulate parameters of neuronal excitability, including baseline membrane properties and firing patterns. Migraine attacks are not wholly attributable to perturbations in peripheral pathways. I have shown that these genes are also expressed within the CNS in a small number of discreet regions, suggesting a possible role in central processing. Utilizing recently defined genetic variants and physiological cell- based models, will provide a platform for mechanistic insights into migraine pathogenesis and allow for the development of drug screening assays for new migraine therapies.

Murine embryonic stem cells (ESCs) are self-renewing, pluripotent cells able to differentiate into cells of all three germ layers. Pluripotency and self-renewal are maintained primarily by the core transcriptional factors Nanog, Oct4 and Sox2, but require the cooperation of other factors and coregulators and an efficient telomere maintenance mechanism. In mammals, telomere maintenance is achieved via a telomerase reverse transcriptase (Tert) that acts together with an RNA component (Terc). Maintenance of functional telomeres is essential to allow ESC proliferation, nevertheless if and how it is involved in the achievement and preservation of cell differentiation is still unknown. Here, we used Tert deficient mouse ESCs to elucidate the role of telomere length in differentiation. We found that Tert-/- ESCs with critically short telomeres are delayed, but still capable, to achieve differentiation after leukemia inhibitory factor (LIF) withdrawal and all-trans retinoic acid (ATRA) treatment, but failed to maintain it after LIF re-introduction to the growth medium. Telomere shortening effect on differentiation was accompanied by pluripotency gene dysregulation (e.g. Nanog overexpression), DNA hypomethylation and epigenetic disorders. This phenotype of metastable differentiation could be rescued by telomere lengthening via re-introduction of Tert, depletion of Nanog via short hairpin RNA, or via enforced expression of the de novo DNA methyltransferase 3b. These results reveal an unanticipated role of telomeres in the epigenetic regulation of gene expression and cell fate determination during physiological or pathological processes.

The thymus is the principal site of T-cell development and maturation. Failure to develop a functional thymus leads to severe immunodeficiency, while partially incorrect function of the organ can lead to a variety of autoimmune diseases as well as higher risk for infections and cancer. The thymus is organized into cortical and medullary regions, which are functionally distinct. The diverse array of thymic epithelial cells (TEC) are the key components of the thymic stroma, both the cortical and medullary TEC subsets are responsible for the establishment of a self-tolerant and self-restricted T-cell repertoire. The thymus is most active in young individuals, and undergoes a progressive naturally occurring involution from birth, which accelerates after puberty. Thymic involution is characterized by loss of thymus organization and function, including an overall reduction in the amount of functional thymic tissue. This results in decreased production of new naïve T-cells, and contributes to the diminished capacity of the aged immune system to adequately respond to new antigenic challenge. Involution of the thymus, both natural and in response to different therapies such as chemotherapy, raises interest in developing cell based treatment methods that will allow the restoration of the thymic architecture and so elevate immune reconstitution in vivo. The cellular mechanisms by which the postnatal thymus is maintained during homeostasis and involution are currently unknown. The earliest thymic progenitors in the thymus express Plet1; it has been established that from E12.5 to E15.5 these cells when purified are able to generate all thymic epithelial cell types and initiate thymus organogenesis. However, at least the latter capacity is reported to be lost from E18.5. A number of papers published provide evidence for the existence of both bipotent and unipotent TEC progenitors in the adult thymus. However the identity of these cells remains unknown, nor has the relationship between the mature and immature postnatal TEC compartments been established. The aim of my research was to investigate the cellular mechanism(s) that maintain the postnatal thymus. Specifically, I aimed to determine whether the thymus is maintained by a stem cell mechanism or by division of terminally differentiated thymic epithelial cells, and whether or not postnatal thymic epithelial stem/progenitor cells express functionally relevant levels of the transcription factor Foxn1. To address these aims, I used two approaches: in vivo genetically heritable lineage tracing and a novel grafting assay to assess the contribution of different lineages of TEC. This thesis describes the characterization of a novel mouse strain, the Foxn1CreERt2 line, which was predicted to allow conditional inducible manipulation of gene expression in TEC. I show that this deletor strain, while thymic epithelial cellspecific, could induce cre-mediated recombination in only in a low proportion of TEC and thus could not be used to address the initial aim of this work as described above. However, lineage tracing experiments using this line have provided evidence for a persistent cortical thymic epithelial progenitor/stem cell type, that was capable of rapid expansion within the cortical compartment over time. In parallel with characterisation of the Foxn1CreERt2 strain, I investigated the potential of various defined epithelial populations to contribute to the thymic environment in an assay of TEC potency. Using this technique I have established the potential of defined TEC subpopulations isolated from postnatal mice to generate cortical and medullary TEC. Among the populations analysed I have identified a minor TEC subset that can robustly contribute to both cortical and medullary TEC that coexpress Ly51 and Plet1. I have further shown, using a limiting dilution approach, that this population contains a postnatal common thymic epithelial stem/progenitor cells, present at a frequency of between 87.5 and 92.5 within this population. I have also produced evidence of a unipotent cortical progenitor population that is capable of long term expansion in vivo.

This thesis demonstrates a pulse-chase assay that describes the turnover of the .r J normal human oesophageal epithelium and Barretr~- metaplasia. to be approximately 11 days. This assay also identified the existence of label-retaining cells after 67 days within the basal layer of the human oesophagus and showed that these cells were epithelial in origin, capable of division yet are not frequently dividing. Furthermore, label-retaining cells were identified within the base of Barrett's glands and were also epithelial and primarily undifferentiated. This thesis suggests that these cells may represent a population of stem cells within the human oesophagus. Using mtDNA mutations as markers of clonal expansion, this thesis demonstrates the presence of a stem cell niche within the normal human oesophagus. Furthermore, clonal patches covering large areas of squamous epithelium were observed. In Barrett's oesophagus, partially-mutated glands were observed indicating that glands are maintained by multiple stem cells. Wholly-mutated Barrett's glands contain all the expected differentiated cell lineages; demonstrating multilineage differentiation from single stem cells. Patches of clonally-related Barrett's glands were also observed; indicating that glands can divide by fission. In one patient the squamous epithelium and the underlying glandular tissue were shown to be derived from a common progenitor cell. TNFa was shown to induce migration of Barrett's cells in vitro suggesting that the inflammatory environment contributes to the expansion of Barrett's lesion. Furthermore, data presented here shows that NSAIDs can act as TNFa inhibitors in the human oesophagus, by decreasing epithelial cell membrane TNFa levels in vivo. This suggests that anti-TNFa therapy may prevent further growth of Barrett's lesions. III

Hematopoietic Stem Cells (HSCs) are rare, self-renewing, and multipotent cells that sustain lifelong production of blood and immune cells. Much of our understanding of hematopoiesis, including the process of divergence and commitment into specific lineages during differentiation, is derived from the analysis of static composition of HSC and progenitor compartments as well as the measurement of their potential using transplantation-based studies. As such, the dynamics of endogenous HSCs, including the kinetics of their differentiation and their interactions with the bone marrow (BM) niche in real-time is poorly understood. The current study aims to characterize HSCs in their native, unperturbed environment by using inducible lineage tracing in combination with high-dimensional flow cytometry and single cell transcriptomics. Our findings provide an unbiased kinetic roadmap of early steps of hematopoietic differentiation and reveal fundamental differences in the sequence of lineage emergence from HSCs. We found a rapid and preferential emergence of megakaryocytic lineage followed by erythroid and myeloid lineages, whereas a substantial delay in lymphopoiesis at steady state. We also used intravital microscopy to visualize endogenous HSCs in the BM of live animals and discovered them to undergo short-range directional movements with extensive morphological changes. Furthermore, our findings revealed profound changes in HSC behavior following treatment with drugs that are used to induce their mobilization into peripheral blood. Overall, the present study offers novel insights into the fundamental features of endogenous HSC differentiation and their in-vivo dynamics during steady state.