<p>Parents play a major role in the choices their children make regarding academics, leisure activities, and college and career preparation. Parent outcome expectations and behaviors are informed by their parenting self-efficacy in a specific subject or task. Parenting self-efficacy is the confidence parents have in their abilities to influence their children’s motivation, environments, and behaviors that could result in positive youth development. Parenting self-efficacy is informed by personal factors and experiences. Parenting self-efficacy can help to describe why or why not a parent engages in certain activities with their child.</p> <p>The purpose of this study was to explore and describe how the motivation of parents of urban middle school students plays a role in their child’s interest in agriculture or STEM-related activities. The convenience sample for this study were parents of urban middle schools in Indianapolis, IN (<i>N </i>= 53) who’s children participated in afterschool programs. Quantitative data were collected using a parenting self-efficacy questionnaire, which included items related to participants’ parenting self-efficacy (PSE) as it pertains to their child’s academics, STEM and agricultural activities; parent outcome expectations (POE) as it pertains to their child’s college and career preparation, and discussing STEM and agriculture activities with their child; and, parents’ perceptions of their child’s post-secondary career and educational options and intended career field. Descriptive statistics including means, standard deviations, frequencies, and percentages were used to analyze the data. Correlations were computed to explore the relationships between the variables.</p> <p>There were four conclusions for this study. First, urban parents were self-efficacious regarding their child’s academic performance and STEM activities, and had positive outcomes expectations regarding their child’s college and career preparation and engaging their child in agriculture and STEM activities. Second, on average urban parents reported participating in four different types of activities with their child, and recreational sports, visiting museums, computer games, and visiting the zoo were most popular. Third, urban parents agreed that their child would most likely pursue an associate or bachelor’s degree in arts, humanities, and social sciences as their post-secondary options. Finally, urban parents’ parenting self-efficacy for academic performance, STEM, and agriculture were positively related to parents’ outcome expectations regarding agricultural activities. Moreover, parenting self-efficacy regarding agricultural activities was positively related to the number of activities parents did with their children. Implications for practice and recommendations for future research were discussed.</p>

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Stem cell pluripotency and self-renewal are two important attributes of human embryonic stem cells which have led to enhanced interest in stem cell research. Understanding the mechanisms that underlie the regulation and maintenance of these properties is imperative to the clinical application of stem cells. Pluripotency and self-renewal are regulated by different genes, transcription factors and other co-factors such as FoxD3 and Klf4. Oct4, Nanog and Sox2 are central to the stem cell regulatory circuitry. They form interactions with co-factors to promote cell proliferation and inhibit differentiation by negatively regulating differentiation markers. However, there are other novel pluripotency associated factors yet to be studied. In this study, bioinformatics and functional analyses were employed to identify a potential pluripotency gene called YY1AP1 from our lab's pre-existing microarray data. YY1AP1, a transcription regulatory gene, showed consistent down-regulation with induced cell differentiation. It was further investigated. First, its co-localization with Oct4 in both hESCs and iPSCs was confirmed by immunofluorescence staining. Knockdown experiments were then performed on this gene to investigate effects of knocking it down on gene expression in hESCs. Knocked-down cells were characterized for markers of pluripotency and differentiation at the transcript level. Results showed a down-regulation of pluripotency genes with no specific promotion of any of the germ layer markers. Gene expression at the protein level in knocked down cells was then assessed for YY1AP1, and its binding partner YY1, and pluripotency markers. Results showed that proteins of YY1AP1, YY1, Oct4, Nanog and CTCF were down regulated while the tumour suppressor gene protein, p53, was up-regulated in YY1AP1 deficient stem cells. Protein to protein interaction studies showed that YY1AP1, YY1, Nanog and CTCF proteins directly interacted with each other. Differentiation of YY1AP1deficient cells into EBs led to an almost complete shutdown of all gene expression, an indication that the cells did not form 'real' EBs. Differentiation of YY1AP1 ablated cells did not support any lineage promotion either. These results suggest a potentially new role for YY1AP1 in proliferation and self-renewal of stem cells through its possible direct binding to CTCF or its indirect binding to CTCF in complex with YY1.

The aim of this thesis is to investigate different functionalization strategy of graphene nanomaterials for graphene-based different biotechnological applications such as graphene-directed stem cell growth and differentiation and graphene-based biosensors. Chemical functionalization of graphene is required in many biological applications; in this thesis we have focused on exploiting the carboxylic groups available on GO molecules and non-covalent functionalization of graphene. GO has been a promising material for stem cell culture due to high specific surface area, ease of functionalization, its ability to support cell proliferation and to not cause cytotoxicity when stem cells are cultured on its substrate. The impact of biochemical functionalization on stem cell differentiation was not widely researched, and many research groups worldwide have been focusing on GO and rGO surfaces only. The approach of this thesis is to fabricate and characterize different graphene-based substrates to investigate the impact of biochemical functionalization of GO in directing adipose stem cell differentiation and to influence the gene expression pathways of Schwann-like differentiated adipose stem cells. The fabrication of graphene based biosensors is still challenging as biological molecules need to be attached to graphene-based sensors to increase both the specificity and the selectivity of the biosensors. In this thesis, two different chemical functionalization approaches were considered. Firstly, the covalent immobilization of membrane proteins embedded on a lipid nanodisc structure on GO was achieved. Secondly, the feasibility of using dip-pen nanolithography as a tool to locally functionalize graphene arrays with phospholipids was demonstrated. Phospholipid interface layer can act as bioactive layer which can be used for the protein insertion of tail-anchoring recombinant proteins as a new route for a non-covalent biological functionalization of graphene array.

Rearrangements involving the mixed lineage leukaemia (MLL) gene are found in 5-10% of human leukaemias and are likely propagated by a deregulated self renewing pool of leukaemia stem cells (LSCs). Targeting of the LSC pool represents a key novel strategy for the treatment of AML. In recent years epigenetic dysfunction has been identified as a key driving factor in a range of solid tumours and haematological malignancies. Evidence for this includes identification of mutations in the genes coding for critical epigenetic modifiers, characterisation of localised regions of abnormal chromatin at oncogene or tumour suppressor genes and the efficacious use of epigenetic-targeted therapies already present in the clinic. The data submitted in this thesis identify the histone demethylase KDM1A as a critical regulator of LSC potential in MLL-AF9 acute myeloid leukaemia (AML). Of all the histone demethylases, we found that only Kdm1a expression correlated positively and significantly with LSC frequency in murine models of human MLL fusion AML. Genetic knockdown or Cre-mediated excision of Kdm1a resulted in loss of LSC potential, reduced expression of LSC maintenance transcriptional programs and induction of macrophage differentiation in MLL-AF9 cells. These effects were phenocopied by chemical inhibition of KDM1A using the monoamine oxidase inhibitor tranylcypromine (TCP), as well as novel TCP analogues which inhibit KDM1A with greater potency and selectivity. These results were seen in murine, human cell line and primary patient cells harbouring MLL rearrangements. Global transcriptome and epigenome analyses revealed a key role for KDM1A in maintaining the histone three lysine four (H3K4) methylation status at highly expressed MLL-AF9-bound genes. In vivo transplantation of Kdm1a knockdown MLL-AF9 cells conferred a significant survival advantage compared with control littermates. Similarly, TCP analogue treatment of mice transplanted with MLL-AF9 cells revealed a reduction in LSC potential of the donor-derived AML cells but little impact on normal recipient haematopoietic stem and progenitor cells (HSPCs). Critically the clonogenic and repopulating potential of normal HSPCS, of both murine and human origin, was spared following either knockdown or chemical inhibition of KDM1A. Taken together, the data presented establish KDM1A as a potential therapeutic target in MLL fusion leukaemia.

Adult stem cells are vital for tissue maintenance. Stem cell over proliferation results in tumour formation, whilst loss of stem cells causes tissue degeneration and a variety of diseases. Stem cell maintenance and proliferation is regulated through somatic structures called niches. The germline stem cell niche in Drosophila ovary has been well defined and it is useful to better understand the interactions between niche and stem cells. Notch signalling is needed for germline stem cell niche creation and maintenance. The aim of this thesis is to better understand both the regulation of Notch signalling during development and its requirement in the adult niche. The first paper, "Reversible regulation of stem cell niche size through dietary control of Notch signalling", revolves around the dynamicity of the niche. The niche is found to respond to diet stimuli and has the ability to be restored. Notch was previously found to be involved in the maintenance of the niche. We found that Notch signalling is altered by diet, and we dissect its different maintenance and recovery roles in the ovary. In the second paper, "ZO-1 controls stem cell niche assembly by acting as an upstream regulator of Deltex-dependent Notch signalling", we show how Notch signalling is finely regulated during niche formation through interplay with the proteins Polychaetoid and Deltex. This paper leads to a better understanding of how the niche is assembled and how Notch signalling is regulated in a context-dependent way. The obtained results from both papers will help understand the dynamics of the model germline stem cell niche, and how Notch signalling is found at the convergence between internal and external stimuli regulating the ovary's response to a changing environment.

Targeted differentiation of human embryonic stem cells (hESCs) towards definitive endoderm (DE) is the first step in generating hepatic or pancreatic cell types with potential for clinical application. Characterisation and efficiency of DE differentiation is improving, however the specific effects of the different exogenous growth factors used, and the changing presence and activity of endogenous factors, are still not well understood. One such endogenous factor, the TGFβ ligand Nodal, is known to drive patterning and differentiation of the primitive streak and DE in the developing mouse embryo. The effect of Nodal signalling during hESC DE differentiation is unknown, and the common use of a related exogenous ligand Activin A may also serve to upregulate rather than simply mimic it. In order to explore this, Activin A differentiation of hESCs in defined culture conditions was analysed. The expression of characteristic mesendoderm and DE markers increased during Activin A treatment, which was significantly enhanced by the inclusion of exogenous Wnt3a. A maintained presence of the pluripotency factor Nanog was observed in most cells expressing markers of DE. The levels of Nodal and its co-receptor Cripto, which were raised during the early stage of Activin A treatment, were also marginally enhanced by Wnt3a, and evidence of Nodal endocytosis further suggested an active signalling presence. RNA interference (RNAi) of Nodal negatively affected both pluripotency maintenance during normal pluripotent culture, and the capacity to differentiate towards DE. Use of a Cripto blocking antibody also inhibited differentiation towards DE. The results strongly suggested the presence of Nodal signalling, as well as possible roles for Nanog, Wnt-related signalling, and Nodal signalling during Activin A-mediated DE differentiation. The results contribute to current understanding of how DE differentiation in hESCs is regulated. They also identify clear targets for further investigation, which would lead to improved characterisation and differentiation of DE from hESCs.

Embryonic stem (ES) cell maintenance and differentiation are dynamic processes controlled by various intrinsic and extrinsic factors. Identifying these factors will enhance the understanding about developmental process and improve the application of stem cells in clinic. Previous studies highlight a shift between non-oxidative and oxidative energy metabolism to play roles during differentiation. Oxidative metabolism is a major source of reactive oxygen species (ROS) which is regulated by a cytoprotective transcription factor, Nuclear factor erythroid 2-related factor 2 (Nrf2). Therefore, this study investigate relationship between metabolism, ROS, and Nrf2 during mouse ES cell differentiation. In vitro models representing early lineage differentiation were used. By measuring metabolic profiles, ROS, and Nrf2 levels from the models, Nrf2 was found related to pluripotency and ROS. However, relationship among metabolism and Nrf2 or ROS could not be detected. Gain- and loss-of-function experiments by pharmacological activator, short hairpin RNA knockdown, and CRISPR-Cas9 genome editing showed that Nrf2 could promote pluripotency and inhibit differentiation, especially during early differentiation toward neural lineage. This study suggested a new player in transcription control that governs pluripotency and differentiation.

In mammalian development, endoderm formation occurs in two phases and the fate of these populations is different. In the blastocyst, inner cell mass (ICM) cells generate the primitive endoderm (PrE), which will give rise to the extra-embryonic parietal (PE) and visceral endoderm (VE). Hematopoietically expressed homeobox (Hhex) protein is initially expressed throughout the PrE and subsequently becomes restricted to the anterior visceral endoderm (AVE), one of two important early embryonic signalling centres in the mouse. During gastrulation a second wave of endoderm differentiation occurs, the definitive endoderm (DE), generating the foregut. Immediately following the induction of DE, regional identity is initially established in the anterior region with the expression of Hhex. One of the earliest specification events in this lineage is the specification of anterior fate by Hhex, this time in a second signalling centre, the anterior definitive endoderm (ADE). The ADE is both important for embryonic patterning, and as the precursor population for differentiating to the foregut and its derivatives the thyroid, liver and pancreas. The literature surrounding these early embryonic patterning events is covered in depth in chapter 1. Embryonic stem cells (ESCs) are normal cell lines derived from the mammalian blastocyst at the time that it is making PrE. A number of laboratories have generated protocols to make endoderm from ESCs and in my thesis I define approaches to distinguish between PrE and DE. I generated a new ESC reporter line utilising a gene normally expressed in both the PrE and later in hepatic endoderm; this reporter contains a GFP in the first exon of the Hnf4α locus. This was combined with a second fluorescent reporter containing DSRed in the Hhex locus. This cell line is described and characterised in chapter 3. As Hnf4α is initially expressed in PrE prior to Hhex, but in the DE following Hhex, I was able to use the temporal expression of this reporter to distinguish the induction of PrE from DE. As Activin and Wnt are known to induce endoderm from ESCs, I was then able to ask what sort of endoderm the combination of these two signals induced. In chapter 4 I found that normal ESCs would readily differentiate to iPrE in the presence of Activin and Wnt3a. While this has not been described previously, my analysis suggests that ESC protocols applying these cytokines directly to ESCs have produced PrE. Given that ESCs are derived from the blastocyst, the generation of iPrE from Wnt3a/Activin treatment fits with developmental paradigms. However, Act/Wnt3a is used routinely on Human ESCs (hESCs) and so I attempted to reconcile these observations. HESCs, while derived from the blastocyst, appear to progress developmentally in vitro, to a stage closer to the epiblast, immediately prior to gastrulation. I therefore assessed the effect of Activin and Wnt3a on mouse stem cell lines derived from the epiblast (Epiblast Stem Cells, EpiSCs), that are grown under similar conditions to hESCs. When Wnt3a/Act is applied to these cells I found that they made DE rather than PrE, which I describe in chapter 4. Taken together my observations suggest that Act/Wnt3a are general endoderm inducers that induce context specific differentiation in vitro. The cell type derived in response to this treatment depends on the developmental stage of the starting stem cell culture. During the course of this work, I also observed that PrE was growing under Activin/Wnt3a treatment. As a number of cell culture systems have been established that reflect PE, but not truly bipotent PrE, I investigated the conditions under which PrE can be expanded. In chapter 5 I characterize a new PrE culture system, in which bipotent extra-embryonic endoderm can be expanded indefinitely in culture. I also explore a bit more precisely the nature of the starting cells that initially become exposed to Activin/Wnt3a treatment. Previous work has extensively characterized the existence of a primed population of PrE in ESC culture and in chapter 6 I explore the existence of a primed DE population in EpiSC culture. Taken together, my thesis is the first demonstration that Activin/Wnt3a can induce different endoderm populations in different embryonic stem cell populations. It underlies the notion that the evolutionary origin of both cell types is the same and that the pathways evolved for extra-embryonic development in mammals just exploit the ancient modes of germ layer specification that evolved with gastrulation.