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Human embryonic stem cells (hESCs) derived from the inner cell mass of the blastocyst, are pluripotent, capable of indefinite self-renewal and have the capacity to differentiate into all cells of the three germ layers. Thus, hESCs hold great potential for a wide range of applications such as regenerative medicine and drug development. However, improved propagation and use of hESCs in medical applications requires a better knowledge of the underlying mechanisms that regulate hESC pluripotency. Hypoxia (5% oxygen) has been shown to be beneficial for maintaining pluripotent stem cells. Hypoxia inducible factors are crucial for this process, being stabilised under low oxygen conditions and promoting the expression of several pluripotency associated genes. However, microRNAs (miRNAs) as multi-purpose gene regulators might also have an important role in regulating these processes. Currently, little is known about specific miRNAs that may regulate pluripotency in hESCs in response to changes in environmental oxygen. Thus, this thesis aims to determine the involvement of miRNAs in the hypoxic regulation of hESCs. Using miRNA arrays, differentially expressed miRNAs were found in hESCs cultured at 5% oxygen compared to those maintained at atmospheric, 20% oxygen. Validation of the arrays using RT-qPCR showed that hESCs cultured at 5% oxygen displayed increased levels of the hypoxamir miR-210 and decreased levels of miR-122-5p and miR-223-3p. Using bioinformatic analysis miR-122-5p and miR-223-3p were found to target the NANOG 3’UTR. Using synthetic pre-miRs to transiently up-regulate either miR-122-5p or miR-223-3p in hESCs cultured at 5% oxygen significantly reduced NANOG protein expression. DualLuciferase-Reporter Assays and site-directed mutagenesis confirmed the location of a novel binding-site for miR-223-3p in the NANOG 3’UTR. The data presented in this thesis show that miRNA-223-3p directly regulates NANOG expression and that miRNAs have a physiological role in regulating the response of hESCs to environmental oxygen.

Cells use phagocytosis and macropinocytosis to internalise bulk material, which in phagotrophic organisms supplies the nutrients necessary for growth. Wildtype Dictyostelium amoebae feed on bacteria, but for decades laboratory work has relied on axenic mutants that can also grow on liquid media. We used forward genetics to identify the causative gene underlying this phenotype. This gene encodes the RasGAP Neurofibromin (NF1). Loss of NF1 enables axenic growth by increasing fluid uptake. Mutants form outsized macropinosomes which are promoted by greater Ras and PI3K activity at sites of endocytosis. Relatedly, NF1 mutants can ingest larger-than-normal particles using phagocytosis. An NF1 reporter is recruited to nascent macropinosomes, suggesting that NF1 limits their size by locally inhibiting Ras signalling. Our results link NF1 with macropinocytosis and phagocytosis for the first time, and we propose that NF1 evolved in early phagotrophs to spatially modulate Ras activity, thereby constraining and shaping their feeding structures. DOI: http://dx.doi.org/10.7554/eLife.04940.001, eLife digest Dictyostelium amoebae are microbes that feed on bacteria living in the soil. They are unusual in that the amoebae can survive and grow in a single-celled form, but when food is scarce, many individual cells can gather together to form a simple multicellular organism. To feed on bacteria, the amoebae use a process called phagocytosis, which starts with the membrane that surrounds the cell growing outwards to completely surround the bacteria. This leads to the bacteria entering the amoeba within a membrane compartment called a vesicle, where they are broken down into small molecules by enzymes. The cells can also take up fluids and dissolved molecules using a similar process called macropinocytosis. With its short and relatively simple lifestyle, Dictyostelium is often used in research to study phagocytosis, cell movement and other processes that are also found in larger organisms. For example, some immune cells in animals use phagocytosis to capture and destroy invading microbes. Most studies using Dictyostelium as a model have used amoebae with genetic mutations that allow them to be grown in liquid cultures in the laboratory without needing to feed on bacteria. The mutations allow the ‘mutant’ amoebae to take up more liquid and dissolved nutrients by macropinocytosis, but it is not known where in the genome these mutations are. Here, Bloomfield et al. used genome sequencing to reveal that these mutations alter a gene that encodes a protein called Neurofibromin. The experiments show that the loss of Neurofibromin increases the amount of fluid taken up by the amoebae through macropinocytosis, and also enables the amoebae to take up larger-than-normal particles during phagocytosis. The experiments suggest that Neurofibromin controls both phagocytosis and macropinocytosis by inhibiting the activity of another protein called Ras. Neurofibromin is found in animals and many other organisms so Bloomfield et al. propose that it is an ancient protein that evolved in early single-celled organisms to control the size and shape of their feeding structures. In humans, mutations in the gene that encodes the Neurofibromin protein can lead to the development of a severe disorder—called Neurofibromatosis type 1—in which tumours form in the nervous system. Given that tumour cells can use phagocytosis and macropinocytosis to gain nutrients as they grow, understanding how this protein works in the Dictyostelium amoebae may help to inform future efforts to develop treatments for this human disease. DOI: http://dx.doi.org/10.7554/eLife.04940.002