The governance of cell fate decisions during development is a fundamental biological problem. An important aspect of this is how cells exit a multipotent state and choose their fates in a correct manner and proportion. To tackle an aspect of this problem, I have focused on 2 multipotent models: one infinite self-renewal pluripotency in an artificial environment, and the other, bipotent progenitors in the context of the mouse embryo. The first model aimed to explore the effects of chromatin-associated factors on the ability of pluripotent mouse Embryonic Stem Cells (ESCs) to self-renew, via monitoring gene expression heterogeneity of key genes. The second model focused on Neural Mesodermal Progenitors (NMPs), a bipotent cell population found in the Caudal Lateral Epiblast (CLE) of mammalian embryos, which contributes to the spinal cord and paraxial mesoderm. The aim here was to derive NMPs in vitro which exhibit similar gene expression patterns and function like their mouse embryo counterpart and study their renewal and differentiation in detail. The first multipotent model explores the effects of chromatin remodelling on cell fate decisions, specifically investigating the consequences of inhibiting the histone acetyltransferase Kat2a on the ESCs fate. I found first, that the effect of Kat2a inhibition depends on the pluripotent state of the cells; cells in a ground state exhibit a resistance to Kat2a inhibition and maintain their pluripotency, whereas cells in a naïve state experience destabilization of their pluripotency gene regulatory network and shift towards differentiation. Second, that Kat2a inhibition in the naïve state results in a decline in the gene expression noise strength contributed by the promoter activation operation, which suggests that when ESCs become lineage-primed their transcriptional noise is constrained. In the bipotent model, the NMPs are identified as cells coexpressing Sox2 and T/Brachyury, a criterion used to derive NMP-like cells from ESCs in vitro. Comparison between the different NMPs protocols stresses that Epiblast Stem Cells (EpiSCs) are an effective source for deriving a multipotent population resembling the embryo Caudal Epiblast (CE), that generates NMPs. Furthermore, self-organization of this CE-like population, resulted in axially organized aggregates. Exploiting the mouse embryo CLE as a reference shows that EpiSCs derived NMPs, monolayers and aggregates, consist of a high proportion of cells with the embryo's NMP signature. Importantly, studying this system in vitro sheds light on the sequence of events which lead to NMP emergence in vivo. On this basis, I conclude that understanding the initial state of cells at a crossroads is important to reveal the limitations it imposes on the cells fate exploration, hence makes it possible to mimic more precisely the fate decision process in vitro.

Leung, Sze Lee Cecilia. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 144-153). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese (摘要） --- p.iii / Acknowledgements --- p.v / Table of Content --- p.vi / Abbreviations --- p.xv / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Stem cells --- p.1 / Chapter 1.1.1 --- Adult stem cells --- p.2 / Chapter 1.1.2 --- Embryonic stem cells --- p.2 / Chapter 1.1.3 --- Pros and cons of embryonic and adult stem cells --- p.5 / Chapter 1.1.4 --- Human embryonic stem cells (hESCs) --- p.6 / Chapter 1.1.5 --- Mouse embryonic stem cells (mESCs) --- p.7 / Chapter 1.1.6 --- Characteristics of ESC-derived cardiomyocytes --- p.7 / Chapter 1.2 --- Cardiovascular Diseases (CVD) --- p.9 / Chapter 1.2.1 --- Causes and statistics of CVD --- p.9 / Chapter 1.2.2 --- Current treatment for CVD --- p.10 / Chapter 1.2.3 --- Current hurdles of putting hESC-CMs into clinical use --- p.11 / Chapter 1.3 --- Myosin light chain2v --- p.13 / Chapter 1.4 --- Genetic-engineering of hESCs & their cardiac derivatives by lentiviral-mediate gene transfer --- p.14 / Chapter 1.5 --- Cytokines secretion during myocardial infarction --- p.15 / Chapter 1.6 --- Aims of the Project --- p.19 / Chapter 1.7 --- Significance of the Project --- p.19 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Subcloning --- p.20 / Chapter 2.1.1 --- Amplification of MLC-2v --- p.20 / Chapter 2.1.2 --- Purification of DNA product --- p.21 / Chapter 2.1.3 --- Restriction enzyme digestion --- p.21 / Chapter 2.1.4 --- Ligation of MLC-2v promoter with DuetO 11 vector --- p.22 / Chapter 2.1.5 --- Transformation of ligation product into competent cells --- p.22 / Chapter 2.1.6 --- PCR confirmation of successful ligation --- p.23 / Chapter 2.1.7 --- Small-scale preparation of bacterial plasmid DNA --- p.23 / Chapter 2.1.8 --- Restriction enzyme digestions to reconfirm positive clones --- p.24 / Chapter 2.1.9 --- DNA sequencing of the cloned plasmid DNA --- p.25 / Chapter 2.1.10 --- Large-scale preparation of target recombinant expression vector --- p.25 / Chapter 2.2 --- Mouse Embryonic Fibroblast (MEF) Culture --- p.26 / Chapter 2.2.1 --- Derivation of MEF --- p.26 / Chapter 2.2.2 --- Mouse embryonic fibroblast cells culture --- p.27 / Chapter 2.2.3 --- Irradiation of mouse embryonic fibroblast --- p.28 / Chapter 2.3 --- HESC culture --- p.29 / Chapter 2.3.1 --- Thawing and Plating hESCs --- p.29 / Chapter 2.3.2 --- Splitting hESCs --- p.30 / Chapter 2.3.3 --- "Culture maintainence, selection and colony removal" --- p.31 / Chapter a) --- Distinguish differentiated and undifferentiated cells and colonies / Chapter b) --- "Remove differentiated cells by ""Picking to Remove""" / Chapter c) --- "Remove undifferentiated cells by ""Picking to Keep""" / Chapter 2.3.4 --- Freezing hESCs --- p.31 / Chapter 2.3.5 --- Differentiation of hESCs --- p.32 / Chapter 2.3.6 --- "HESC culture on feeder free system, mTeSR TM1" --- p.34 / Chapter a) --- Preparation of mTeSRTMl / Chapter b) --- Preparation of BD MatrigelTM hESC-qualified Matrix aliquots / Chapter c) --- Coating plates with BD MatrigelTM hESC-qualified Matrix / Chapter d) --- Human Embryonic stem cells culture in mTeSRTMl / Chapter 2.4 --- ES Cell Characterization (Chemicon Cat# SCR001) --- p.36 / Chapter 2.4.1 --- Alkaline Phosphatase Staining --- p.36 / Chapter 2.4.2 --- Immunofluorescence staining --- p.37 / Chapter 2.5 --- MESC culture --- p.38 / Chapter 2.5.1 --- Thawing and Plating mESCs --- p.38 / Chapter 2.5.2 --- Splitting mESCs --- p.38 / Chapter 2.5.3 --- Differentiation of mESCs --- p.39 / Chapter 2.5.4 --- To study the effects of cytokines on mESC differentiation --- p.40 / Chapter 2.6 --- Lentivirus (LV) Packaging --- p.41 / Chapter 2.6.1 --- Transfection of lentiviral vectors into HEK293FT cells --- p.41 / Chapter 2.6.2 --- LV titering --- p.42 / Chapter 2.7 --- MultipleTransduction --- p.43 / Chapter 2.8 --- Selection of transduced cells by hygromycin --- p.43 / Chapter 2.8.1 --- Determination of hygromycin selection dosage --- p.43 / Chapter 2.8.2 --- Selection of stable clones --- p.44 / Chapter 2.9 --- Isolation of green fluorescent cardiomyocytes derived from differentiated hESCs --- p.45 / Chapter 2.9.1 --- Collagenase digestion of embryoid bodies into single cells --- p.45 / Chapter 2.9.2 --- FACS --- p.46 / Chapter 2.10 --- Gene expression study / Chapter 2.10.1 --- Primer design --- p.46 / Chapter 2.10.2 --- RNA extraction --- p.46 / Chapter 2.10.3 --- DNase Treatment --- p.47 / Chapter 2.10.4 --- Synthesis of Double-stranded cDNA from Total RNA --- p.47 / Chapter 2.10.5 --- Quantitative real-time PCR --- p.48 / Chapter 2.10.6 --- Quantification of mRNA expression --- p.49 / Chapter 2.11 --- Protein Expression study --- p.49 / Chapter 2.11.1 --- Crude protein extraction --- p.49 / Chapter 2.11.2 --- Quantitation of protein samples --- p.50 / Chapter 2.11.3 --- SDS-PAGE --- p.50 / Chapter 2.11.4 --- Western Blot --- p.51 / Chapter 2.11.5 --- Western blot luminal detection --- p.52 / Chapter 2.11.6 --- Quantification of protein expression --- p.52 / Chapter CHAPTER 3 --- PURIFICATION OF CARDIOMYOCYTES DERIVED FROM DIFFERENTIATED HESCs / Chapter 3.1 --- Subcloning --- p.57 / Chapter 3.1.1 --- Linearization of DuetO11 and excision of UBC promoter --- p.58 / Chapter 3.1.2 --- PCR cloning of MLC-2V --- p.59 / Chapter 3.1.3 --- Ligation of MLC-2v promoter to linearized DuetO11 --- p.60 / Chapter 3.1.3.1 --- Colony PCR to screen for positive clones --- p.61 / Chapter 3.1.3.2 --- Restriction digestion to confirm the success of ligation --- p.61 / Chapter 3.2 --- Lentivirus (LV) packaging --- p.62 / Chapter 3.2.1 --- Transfection --- p.63 / Chapter 3.2.2 --- LV titering --- p.64 / Chapter 3.3 --- HESC culture --- p.66 / Chapter 3.4 --- Multi-transduction of hESCs with LVs --- p.67 / Chapter 3.5 --- Differentiation after transduction --- p.69 / Chapter 3.6 --- Antibiotic selection --- p.71 / Chapter 3.6.1 --- Characterization of hESCs on feeder free system --- p.72 / Chapter 3.6.1.1 --- Alkaline Phosphatase (AP) staining --- p.72 / Chapter 3.6.1.2 --- Immunostaining with pluripotency marker --- p.73 / Chapter 3.6.2 --- Determination of hygromycin dosage by MTT assay --- p.74 / Chapter 3.6.3 --- HESCs after selection in feeder free system --- p.75 / Chapter 3.7 --- Differentiation of hESCs after selection --- p.76 / Chapter 3.8 --- FACS --- p.77 / Chapter 3.9 --- QPCR of cells after FACS --- p.80 / Chapter 3.9.1 --- Gene expression of Nkx2.5 --- p.81 / Chapter 3.9.2 --- Gene expression of c-Tnl --- p.82 / Chapter 3.9.3 --- Gene expression of c-TnT --- p.83 / Chapter 3.9.3 --- Gene expression of MLC-2v --- p.84 / Chapter CHAPTER 4 --- THE STUDY OF CYTOKINES' EFFECT ON MESC DIFFERENTIATION / Chapter 4.1 --- mESC culture --- p.85 / Chapter 4.2 --- The effect of cytokines on the differentiation of mESCs --- p.86 / Chapter 4.2.1 --- Beating curves of mESCs treated with different concentrations of cytokines at differentiation day 2 to 6 before attachment --- p.88 / Chapter 4.2.2 --- qPCR to determine the cytokines' effect on the differentiation of mESCs --- p.94 / Chapter 4.2.2.1 --- The effect of IL-1α on the expression of cardiac specific genes --- p.95 / Chapter 4.2.2.2 --- The effect of IL-1β on the expression of cardiac specific genes --- p.98 / Chapter 4.2.2.3 --- The effect of IL-6 on the expression of cardiac specific genes --- p.101 / Chapter 4.2.2.4 --- The effect of IL-10 on the expression of cardiac specific genes --- p.104 / Chapter 4.2.2.5 --- The effect of IL-18 on the expression of cardiac specific genes --- p.107 / Chapter 4.2.2.6 --- The effect of TNF-α on the expression of cardiac specific genes --- p.110 / Chapter 4.2.3 --- Western blot analysis of the cytokines' effect on the differentiation of mESCs --- p.113 / Chapter 4.2.3.1 --- The effect of IL-lα on the abundance of cardiac specific proteins --- p.114 / Chapter 4.2.3.2 --- The effect of IL-1β on the abundance of cardiac specific proteins --- p.116 / Chapter 4.2.3.3 --- The effect of IL-6 on the abundance of cardiac specific proteins --- p.118 / Chapter 4.2.3.4 --- The effect of IL-10 on the abundance of cardiac specific proteins --- p.120 / Chapter 4.2.3.5 --- The effect of IL-18 on the abundance of cardiac specific proteins --- p.122 / Chapter 4.2.3.6 --- The effect of TNF-α on the abundance of cardiac specific proteins --- p.124 / Chapter CHAPTER 5 --- DISCUSSION / Chapter 5.1 --- Purification of cardiomyocytes derived from differentiated hESCs --- p.127 / Chapter 5.2 --- Study on the effect of cytokines on mESC differentiation --- p.135 / Chapter 5.3 --- Conclusion --- p.142 / REFERENCES --- p.144

Ng, Wing Chi Linda. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 120-142). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgement --- p.iv / Publications --- p.v / Abbreviations --- p.vi / Table of Contents --- p.viii / List of Tables --- p.xiii / List of Figures --- p.xiv / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Stem Cells --- p.1 / Chapter 1.1.1 --- Definition of Stem Cells --- p.1 / Chapter 1.1.2 --- Different Origins of Stem Cells --- p.2 / Chapter 1.1.3 --- Challenges and Importance of Stem Cell Research --- p.5 / Chapter 1.2 --- Adult Mesenchymal Stem Cells --- p.7 / Chapter 1.2.1 --- Characteristics of Adult Mesenchymal Stem Cells --- p.7 / Chapter 1.2.2 --- Adipose Tissue as an Alternate Source of MSCs --- p.8 / Chapter 1.2.3 --- Adipose Tissue Versus Bone Marrow as a Source of MSCs --- p.10 / Chapter 1.3 --- Adipose Tissue-derived Stem Cells (ATSCs) --- p.11 / Chapter 1.3.1 --- Cell Surface Marker Characteristic of ATSCs --- p.11 / Chapter 1.3.2 --- Global Gene Expression Profile of ATSCs --- p.14 / Chapter 1.3.3 --- Immunomodulatory Effect of ATSCs --- p.15 / Chapter 1.3.4 --- Proliferation Capacity of ATSCs --- p.17 / Chapter 1.3.5 --- Multilineage Differentiation of ATSCs --- p.18 / Chapter 1.3.5.1 --- Differentiation Capability of ATSCs : Adipogenesis --- p.18 / Chapter 1.3.5.2 --- Osteogenesis --- p.19 / Chapter 1.3.5.3 --- Skeletal and Smooth Muscle Myogenesis --- p.21 / Chapter 1.3.5.4 --- Cardiomyogenesis --- p.23 / Chapter 1.3.5.5 --- Chondrogenesis --- p.24 / Chapter 1.3.5.6 --- Neurogenesis --- p.27 / Chapter 1.4 --- Signaling Pathways in Stem Cells --- p.31 / Chapter 1.4.1 --- Wnt Signaling --- p.31 / Chapter 1.4.2 --- Notch Signaling --- p.33 / Chapter 1.4.3 --- Signaling Pathway of the TGF-β Superfamily --- p.34 / Chapter 1.5 --- Pathways Controlling Chondrogenesis --- p.36 / Chapter 1.6 --- MicroRNA --- p.39 / Chapter 1.6.1 --- MicroRNA - A Novel Gene Regulator --- p.39 / Chapter 1.6.2 --- Biogenesis of MicroRNAs --- p.40 / Chapter 1.6.3 --- Post-transcriptional Repression by MicroRNAs --- p.43 / Chapter 1.6.4 --- Role of MicroRNAs in Development --- p.45 / Chapter 1.6.5 --- MicroRNAs in Stem Cell Differentiation --- p.46 / Chapter 1.6.5.1 --- MicroRNA Expression Profile in ESCs --- p.46 / Chapter 1.6.5.2 --- Lineage Differentiation --- p.47 / Chapter 1.7 --- Project Aims --- p.52 / Chapter 1.8 --- Significance of Study --- p.53 / Chapter Chapter 2 --- Materials and Methods --- p.54 / Chapter 2.1 --- Sample Collection --- p.54 / Chapter 2.2 --- Isolation and Culture of ATSCs --- p.54 / Chapter 2.3 --- Measurement of Cell Growth --- p.55 / Chapter 2.4 --- Effect of Estrogen Treatment on ATSC Proliferation --- p.55 / Chapter 2.5 --- Multilineage Differentiation of ATSCs --- p.55 / Chapter 2.5.1 --- Chondrogenic Differentiation --- p.56 / Chapter 2.5.2 --- Neural Differentiation --- p.56 / Chapter 2.6 --- Immunocytochemical Analysis of Surface Markers and Lineage Specific Markers --- p.57 / Chapter 2.7 --- Alcian Blue Staining --- p.58 / Chapter 2.8 --- RNA Extraction --- p.58 / Chapter 2.9 --- Reverse Transcription --- p.59 / Chapter 2.10 --- Quantitative Real-time Polymerase Chain Reaction --- p.59 / Chapter 2.11 --- Statistical Analysis of Real-time PCR Data --- p.61 / Chapter 2.12 --- MicroRNA Profiling --- p.61 / Chapter 2.12.1 --- Reverse Transcription --- p.62 / Chapter 2.12.2 --- Quantitative Real-time Polymerase Chain Reaction --- p.62 / Chapter 2.13 --- mRNA Target Prediction of MicroRNA --- p.63 / Chapter 2.14 --- MicroRNA Knockdown Assay --- p.63 / Chapter 2.15 --- MicroRNA Over-expression Assay --- p.64 / Chapter 2.15.1 --- Vector Amplification --- p.64 / Chapter 2.15.1.1 --- Transformation --- p.64 / Chapter 2.15.1.2 --- Purification of Plasmid DNA --- p.65 / Chapter 2.15.1.3 --- Confirmation of Construct Insertion --- p.66 / Chapter 2.15.2 --- Transfection of Plasmid and Establishment of MicroRNA Precursor Expressing Cell Lines --- p.66 / Chapter 2.16 --- Gene Expression Microarry --- p.67 / Chapter 2.16.1 --- Preparation of Amplification and Labeling Reaction --- p.67 / Chapter 2.16.2 --- Purification of the Labeled/Amplified RNA --- p.68 / Chapter 2.16.3 --- RNA Fragmentation --- p.68 / Chapter 2.16.4 --- Hybridization --- p.69 / Chapter 2.16.5 --- Array Washing and Scanning --- p.69 / Chapter 2.16.6 --- Statistical Analysis of Microarray Data --- p.69 / Chapter CHAPTER 3 --- RESULTS --- p.71 / Chapter 3.1 --- Isolation and Characterization of ATSCs --- p.71 / Chapter 3.2 --- ATSCs Exhibited Multilineage Differentiation --- p.75 / Chapter 3.2.1 --- Chondrogenic Differentiation --- p.75 / Chapter 3.2.2 --- Expression of Chondrogenic Markers --- p.76 / Chapter 3.2.3 --- Neural Differentiation --- p.80 / Chapter 3.2.4 --- Expression of Neural Markers --- p.83 / Chapter 3.3 --- Effect of Donor's Reproductive Status on the Proliferation and Differentiation Capacity of ATSCs --- p.83 / Chapter 3.3.1 --- Expression of Stem Cell Makers --- p.86 / Chapter 3.3.2 --- Cell Proliferation Assay --- p.86 / Chapter 3.3.3 --- Differentiation Capacity of ATSCs --- p.89 / Chapter 3.4 --- Effect of E2 Treatment on the Proliferation Rate of ATSCs --- p.89 / Chapter 3.5 --- MicroRNA --- p.91 / Chapter 3.5.1 --- MicroRNA Expression Profile of Undifferentiated and Chondrogenic Differentiated ATSCs --- p.91 / Chapter 3.5.2 --- Clustering Analysis Identified MicroRNAs Segregate with ATSCs --- p.91 / Chapter 3.5.3 --- Identification of Differentially Expressed MicroRNAs in Chondrogenic-induced ATSCs --- p.95 / Chapter 3.5.4 --- mRNA Target Prediction for miR-199a --- p.97 / Chapter 3.6 --- Correlating MicroRNA Expression and mRNA Levels: Clues to MicroRNA Function --- p.97 / Chapter 3.6.1 --- Effect ofmiR-199a RNAi in Phenotypic Changes of Chondrogenic-induced ATSCs --- p.97 / Chapter 3.6.2 --- Identification of Potential Target Genes by Microarray Analysis of ATSCs with miR-199a Over-expression and Knockdown --- p.102 / Chapter CHAPTER 4 --- DISCUSSION --- p.104 / Chapter CHAPTER 5 --- CONCLUSIONS --- p.115 / APPENDICES --- p.117 / REFERENCES --- p.120

abstract: There has been growing interest among learning scientists in the design and study of out-of-school time (OST) learning environments to support equitable development of science, technology, engineering, and math (STEM) interests among youth from groups that are underrepresented in STEM fields. Most of these design studies assumed the youth came to the learning environments without well-developed STEM interests. I challenged this assumption by enacting a social design participatory study to engage youth (aged 11 to 14), from groups that are underrepresented in STEM fields, as partners in designing an OST networked club to support the youth in growing their own STEM interests. Based on longitudinal ethnographic data, I report a three-year iterative design of this networked club. I characterize the heterogeneity of STEM interests that emerged and grew across the networked club. Building on ecological theories of interest development, and leveraging the cultural assets of the nondominant community, I argue that heterogeneity of interests, resources, and practices served as a design tool and a catalyst for the development of STEM interest in the OST networked club. / Dissertation/Thesis / Doctoral Dissertation Learning, Literacies and Technologies 2019

Les recherches sur les nanocristaux semiconducteur (NCs) ont conduit à des résultats scientifiques fascinants, spécialement pour l'application en dispositifs optoelectroniques. Afin de répondre à certaines exigences comme des coûts mineurs, des gains d'efficacité, des composants respectueux de l'environnement, etc., des nouvelles méthodes sont explorées: dans les procédés en solution, dans l'ingénierie de bande et des niveaux d'énergie. En particulier, la méthode de synthèse peut influencer les propriétés optoélectroniques. Par conséquent, une meilleure compréhension des facteurs complexes pendant la synthèse entraînera une amélioration des performances.La microscopie électronique avancée fournit un moyen précis de recueillir des informations sur la morphologie, la structure cristalline et la composition chimique des matériaux avec une résolution spatiale au niveau atomique. La première partie de cette thèse traite de la synthèse et de la préparation des échantillons pour la microscopie électronique à transmission en haute résolution (HRTEM).La deuxième partie traite du mécanisme de croissance des NCs Cu2ZnSnS4 synthétisés par une méthode colloïdale. La morphologie et la stoechiométrie des intermédiaires de réaction extraits après différents intervalles de temps sont déterminés par HRTEM et analyse dispersive en énergie (EDS).Deux méthodes complémentaires, la diffraction par nanofaisceau d’électrons en précession (NPED) et la microscope électronique en transmission par balayage à haute résolution avec imagerie en champ sombre avec détecteur annulaire à grand angle (HRSTEM-HAADF) permettent une profonde caractérisation de la structure cristalline.En outre, la structure cristalline de NCs CsPbBr3 est résolue avec simulations de STEM-HAADF. Cet approche peut différencier entre structures cristallines cubiques et orthorhombiques, impossible avec techniques de diffraction traditionnelles. Enfin, l'influence des méthodes de synthèse sur la morphologie et sur la structure cristalline de NCs CuFeS2 pour applications dans le domaine de la thermoélectricité est analysée par HRTEM. / The investigations of semiconductor nanocrystals (NCs) led to fascinating scientific results in optoelectronic devices. In order to fulfill certain requirements, i.e. cheaper costs, higher efficiencies, environmental friendly components etc., new methods are explored in solution-processing, band gap and energy level engineering. Particularly, the method of synthesis can alter the optoelectronic properties. Therefore, a better understanding of the intricate factors during synthesis will lead to improved performances. Advanced electron microscopy provides a precise way to gather information about morphology, crystal structure and chemical composition of materials with a spatial resolution down to the atomic level. The first part of this thesis deals with the optimization of the synthesis and sample preparation for high resolution transmission electron microscopy (HRTEM).The second part deals with the growth mechanism of Cu2ZnSnS4 NCs synthesized by a colloidal method. The morphology and stoichiometry of the samples extracted after different time intervals are characterized by HRTEM and electron dispersion spectroscopy (EDS). Two complementary methods, Nanobeam Precession Electron Diffraction (NPED) and High Resolution Scanning Transmission Electron Microscopy by High Angle Annular Dark-Field Imaging (HRSTEM-HAADF), provide an in-depth crystal structure characterization.Moreover, the crystal structure of CsPbBr3 NCs is solved by probing STEM-HAADF simulations. This approach is able to differentiate cubic and orthorhombic crystal structures, which is otherwise impossible by diffraction techniques. Finally, the influence of synthesis methods on the morphology and crystal structure of CuFeS2 NCs is investigated by HRTEM for thermoelectric applications.

The regenerative tissues such as hair follicles, intestine and teeth have a particular microenvironment known as “stem cell niche” which houses stem cells and act as a signaling center to control stem cell fate. The precise and timely regulation of stem cell renewal and differentiation is essential for tissue formation, growth and homeostasis over the course of a lifetime. However, the molecular underpinning to control this regulation is poorly understood. To address this issue, we use the continuously growing mouse incisor as a model to study the gene regulatory network which controls dental epithelial stem cell (DESC) maintenance, growth and craniofacial morphogenesis. We found FoxO6, a transcription factor mainly expressed in the brain and craniofacial region, control DESC proliferation by regulating Hippo signaling. FoxO6 loss-of-function mice undergo increases in cell proliferation which finally leads to lengthening of the incisors, expansion of the face and skull and enlargement of the mandible and maxilla. We have screened three human FOXO6 single nucleotide polymorphisms which are associated with facial morphology ranging from retrognathism to prognathism. Our study also reveals that Sox2 and Lef-1, two markers for early craniofacial development, are regulated by Pitx2 to control DESC maintenance, differentiation and craniofacial development. Conditional Sox2 deletion in the oral and dental epithelia results in severe craniofacial defects, including ankyloglossia, cleft palate, arrested incisor development and abnormal molar development. The loss of Sox2 in DESCs leads to impaired stem cell proliferation, migration and subsequent dissolution of the tooth germ. On the other hand, conditional overexpression of Lef-1 in oral and dental epithelial region increases DESC proliferation and creates a new labial cervical loop stem cell compartment in dental epithelial stem cell niche, which produces rapidly growing long “tusk-like” incisors. Interestingly, Lef-1 overexpression rescues the tooth arrest defects but not the ankyloglossia or cleft palate in Sox2 conditional deletion mice. Our data also reveal that miRNA and histone remodeler are involved in regulating DESC proliferation and craniofacial morphogenesis. We describe a miR-23a/b:Hmgn2:Pitx2 signaling pathway in regulating dental epithelial cell growth and differentiation. Pitx2 activates expression of amelogenin which is the major protein component for enamel deposition. This activation can be repressed by the chromatin-associated factor Hmgn2. miR-23a and miR-23b directly target Hmgn2, leading to the release of the Hmgn2 inhibition of Pitx2 transcriptional activity and thus enhance Amelogenin production. Phenotypically, ablation of Hmgn2 in mice results in an overgrowth of incisors with increased Amelogenin expression. The findings in this study increase our current understanding of the molecular regulation of dental epithelial stem cell fate. It not only highlights new gene regulatory network that controls dental stem cell maintenance, growth and craniofacial morphogenesis, but also sheds new light on developing novel stem cell therapy or gene therapy for tooth regeneration and dental diseases.

Type 1 Diabetes (T1D) is an autoimmune disorder in which the pancreatic β-cells are destroyed by the body's immune system. The reduced number of β-cells leads to inadequate insulin secretion and high glucose levels in the body. The requirement of insulin injection throughout life and lack of donors for islet transplantations has prompted a search for more accessible and available sources of insulin producing cells that can be transplanted in T1D patients. To that end, the discovery of induced pluripotent stem (iPS) cells has provided a potential source of precursors for cell therapy for T1D. iPS cells are reprogrammed somatic cells which can be transplanted back into the patient from whom the somatic cells were initially derived, thus potentially avoiding immune rejection when transplanted. As a potential therapy for T1D, we aim to derive insulin producing cells (IPCs) from human iPS cells. In contrast to the conventional two dimensional (2D) cell culture systems used in many iPS derived IPC studies, the inner cell mass (ICM) from which various organs differentiate during embryogenesis is a cluster of cells that enables signaling crosstalk between cells of different types. Three dimensional (3D) cell culture systems allows cells to form cell clusters that promote cell - cell signaling. Hence, we hypothesized that 3D cell culture systems will yield better efficiency of differentiation to functional IPCs in vitro than 2D cultures. Initially, the synthetic polymers sodium alginate and matrigel were analyzed for their ability to enable cell clustering to establish 3D cell culture systems. The 3D cell environment established using matrigel was used for the differentiation of human iPS cells to Insulin Producing Cells (IPC). The cells were first converted to endodermal cells. A mixture of growth factors then induced the differentiation of endodermal cells to pancreatic cells. The pancreatic cells were converted to IPCs that resemble pancreatic β-cells. Our 3D differentiated IPCs strongly expressed pancreatic endocrine transcription factors and pancreatic hormones. The IPCs also produced insulin when exposed to a high glucose environment. But the number of IPCs obtained at the end of the differentiation was low. Hence, our results demonstrate that 3D differentiation generates functional IPCs in vitro unlike 2D differentiation. In the future we aim to improve the percentage of IPCs that we generate from the 3D differentiation. Our expectation is that these cells will be able to cure hyperglycemia in diabetic mice more rapidly compared to the 2D differentiated cells owing to their proven insulin production in the presence of a high glucose environment in vitro.

This research is focused on the intersection of secondary teachers’ data-use to inform instructional decisions and their teaching of STEM in STEM-focused high schools. Teaching STEM requires presenting more than just the content knowledge of the STEM domains. The methods of inquiry (e.g., scientific inquiry, engineering design) are skills that should be taught as part of STEM activities (e.g., science labs). However, under the data- and standards-based accountability focus of education, it is unclear how data from STEM activities is used in instructional decision-making. While teachers give tremendous weight to the data they collect directly from their observations of their classrooms, it is data from standardized testing that strongly influences practices through accountability mandates. STEM education alters this scenario because, while there is a growing focus on teaching STEM, important aspects of STEM education are not readily standardized. This mixed-methods study will examine the perspectives of 9th through 12th grade science and mathematics teachers, in STEM-focused schools, on data-use and STEM teaching. We developed a framework, adapted from existing frameworks of data-use, to categorize these perspectives and outline contexts influencing them. Through a concurrent triangulation design we will combine quantitative and qualitative data for a comprehensive synthesis of these perspectives.

Researchers have shown rural elementary and middle-grade science teachers' inability to integrate STEM career-related lessons into their curricula despite engagement in professional development linked to the teachers' intent-driven beliefs. Researchers, however, have not investigated the influence of intentions on teachers' abilities to integrate STEM career-related lessons into science instruction. The purpose of this transcendental phenomenological study was to understand how intentions impacted rural elementary and middle-grade teachers' ability to integrate STEM career-related lessons during science instruction. Guided by Ajzen's (1988) theory of planned behavior, this study was designed to examine teachers' intentions to integrate STEM career-related lessons during science instruction and the underlying causes of such intentions. In this transcendental phenomenological study, reflective journal entries and interview data were collected through purposeful sampling of 10 rural elementary and middle-grade science teachers. Data were analyzed using a modification of the Van Kaam method of analysis. Findings showed that teachers intended to regularly integrate STEM career-related lessons, but needed more support from their administrators, colleagues, and community partners in fulfilling their intents to integrate STEM career-related lessons. Additional studies are needed for an increased understanding of how teachers in rural areas intend to integrate STEM career-related lessons amid challenges rural teachers face. This study may be of benefit to administrators and teachers who want to unite efforts in constructing a positive climate of integrating STEM career-related lessons during science instruction.

A number of studies have sought to identify factors influencing STEM students’ success in colleges and universities (Crisp et al., 2009; Excelencia, 2011; Hagedorn & Purnamasari, 2012). However, there are few qualitative studies focusing on students’ perspectives and how they make meaning of their experiences as participants in a mentoring program. The main purpose of this research was to explain the perceptions of Latino students in a STEM Mentoring Program at Miami Dade College. Because this study sought to gain an in-depth understanding of how students involved in a mentoring program make meaning of their experiences, the type of qualitative research that fits this inquiry was a single case study. This study was undertaken to address these questions: (a) How do STEM students make meaning of mentorship? (b) How do STEM students construct their experiences in the Program? (c) To what extent do gender and ethnicity play a role in how students make meaning of their mentoring experiences? (d) What do students identify as important for succeeding in a mentorship program? The major findings of this study were: (1) For the participants, a formal mentoring programs offers various forms of academic support, but they found interpersonal support with informal mentors; (2) For the participants, in a formal mentoring program a career match between mentor and mentee is essential; (3) For the participants, the required number of meetings in a formal mentoring program was burdensome, but other required activities were important; (4) For the participants, the peer mentoring experience was important and self-fulfilling; (5) For the participants, the gender or race of the mentor was insignificant, but some believed that sharing the same cultural background made them feel more connected with their majors; and (6) For the participants, encouragement and emotional support from their families was important, but only those with college-educated parents received the academic and financial support necessary important to succeed in college; (7) For the participants, a mentoring program will be successful if there are opportunities for building community among students and faculty, but ultimately, what matters for success are the personal characteristics of students. This study was significant because it provided insight into what students understand are key experiences of being in a mentoring program, and it also identified the kinds of institutional support students themselves thought would help in STEM careers. This information can help institutions of higher education plan and administer effective mentoring programs in STEM or even other fields.