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.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic disorder associated with premature aging in various tissues and organs of the afflicted individuals, including accelerated skeletal muscle atrophy. Classical HGPS manifests due to single-base substitution in the LAMNA gene which encodes Lamin A/C proteins. As a result of the mutation, a truncated form of Lamin (known as Progerin) is produced which undergoes persistent farnesylation during post-translational modification. Accumulation of Progerin in the nucleus has been linked to various cellular abnormalities including abnormal nuclear morphologies and altered chromatin organization, among others. However, the exact molecular mechanisms leading to skeletal muscle atrophy have not yet been elucidated. In this study, the iPSC approach was implemented in order to study the skeletal muscle phenotype of HGPS by generating and characterizing a population of Pax7 positive skeletal muscle precursor cells (SMPs). During the course of this project, we have demonstrated the need for excessive optimization of the previously developed directed differentiation protocol for successful application on induced Pluripotent Stem Cells. Furthermore, we have successfully modified the protocol to allow for a more rapid expansion of the SMPs through regular passaging of the myogenic cells starting on day 20 of differentiation. Additionally, this new method produced more uniform distribution of the myogenic cells and allowed for successful freezing/thawing of the myogenic cells. When compared to the controls, the HGPS-derived SMPs did not appear to be defective in formation, proliferation or differentiation. Abnormal nuclear morphology and DNA damage, documented in HGPS fibroblasts and vascular smooth muscle cells, were not detected the in myogenic cells. Furthermore, we were not able to detect Progerin protein accumulation in the generated myogenic cultures, offering an explanation for the absence of these phenotypes in the skeletal muscle system.

Professional development has been deemed ineffective for several decades. This ineffectiveness could stem from the one-size-fits-all professional development designs, and the inconsistencies and contradictions pointed out in professional development research (which is used to create these designs). Investigating how subject taught (STEM and non-STEM), Title I status of the school (Title I and non-Title I), and grade level of instruction (elementary, middle and high) could influence teachers’ preferences regarding components included in an effective design is a step toward resolving some of these inconsistencies. The research design was an embedded mixed method – an overall causal-comparative design embedded with interviews. Interviews determined teachers’ perceptions of an effective professional development design. The survey investigated preferences for nine components: content knowledge, pedagogical knowledge, active learning, duration, alignment with goals and policies, follow-up, collaboration, support, and resources (tangible and intangible). In the interviews, teachers communicated a need for differentiation based on grade level of instruction, Tittle I status of the school, and subject taught, with high percentages of agreement with the final questions of the survey. The ordinal logistic regression indicated that subject taught and Title I status of the school did not have a statistically significant effect on the dependent variable. Breaking up participants according to grade level of instruction (elementary versus secondary) had a statistically significant effect on teachers’ preferences regarding the components included in an effective professional development design. This indicated that professional development should be differentiated based on elementary and secondary instruction. When the researcher reviewed the components, some showed that the independent variables, Title I status of the school and grade level of instruction had a statistically significant effect. Although the ordinal logistic regression revealed a lack of statistical significance, percent differences indicated that factors such as subject taught, Title I status of the school, and grade level of instruction influenced teachers’ preferences regarding specific components in an effective professional development design. These findings illustrate promise that in a larger study, statistical significance might be present. Thus, professional development should be differentiated based on subject taught, Title I status of the school, and grade level of instruction. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

The purpose of this qualitative study was to understand the feelings and experiences of state college transfer STEM students regarding transfer from the state college to the university. The 25 participants in this study were asked to describe their feelings about the transfer process. Data were gathered from the interviews and from a review of short journals and documents. It was hoped that the experiences of successful transfer students would help illustrate the feelings present when these STEM transfer students enter the university as juniors and compete with native juniors or juniors who began their collegiate experiences as freshmen at the university. Each participant had varied experiences and feelings from the experience. There was no one process that each student traveled through. Findings included: financial influences on decision-making, inconsistencies in advising causing anxieties, hands on learning impacts persistence, negative connotations for transfer students, class size impacts difficulty, the importance of university transfer orientation, and areas in need of improvement. During participant interviews, the issue of finance and budgeting was the most prevalent topic. Participants found that their state college advisors did not always understand their intended career paths causing confusion and anxiety. They valued hands on experiences provided by state college professors who encouraged them to continue their education. The increased class size at the university was disconcerting to many study participants. At the state college professors had open-door policies for students with questions or in need of extra assistance with course materials. Participants felt that the classification of transfer student had some negative connotations at the university. Students were reluctant to be singled out as state college transfer students. In addition, transfer orientation was viewed by older participants as demeaning. Participants were asked about changes they would make in the transfer process. The link programs seemed to be under promoted. A database for immigration information was suggested. Participants also suggested changes to transfer student orientation. This study was limited to 25 successful transfer students. The results should not be generalized to a larger population, but instead provide ideas for future policy discussions and research. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

No description available.

Tsang Kam Sze Kent. / "March 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 277-339). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

In the mid-gestation embryo, the first definitive transplantable hematopoietic stem cells (dHSCs) emerge by embryonic day E10.5-E11 in the aorta-gonadomesonephros (AGM) region, as a result of a step-wise maturation of precursors called pre-HSCs. The analysis of several Notch mutants suggests that Notch signalling is essential for the execution of the definitive hematopoietic programme in the AGM. Mouse embryos deficient for Notch1, RBP-Jk or Jagged1 cannot efficiently generate intra-embryonic hematopoeitic progenitors. It has also been reported that knockdown of Notch target genes (Hes1, Hes5) results in hematopoietic impairment. However a clear picture of the role of Notch pathway in HSC development is still missing. In this work we characterised precise stages and cell types during HSC development in which Notch signalling is involved. First we used a Hes1-dEGFP reporter mouse line that allowed us to monitor Notch pathway activity in a narrow window of time. The results suggest that the level of Notch activity fluctuates in HSC lineage in the AGM region and is down-regulated in dHSCs in the foetal liver (where dHSCs migrate after generation in the AGM region). By using transplantation assay, we further showed that fluctuations of Notch activity are essential for HSC development, and that this pattern in the HSC lineage might work as a switch between maturation and proliferation of PreHSC1, PreHSC2 and dHSC, in which temporary decrease might be required to mature from one type to another, both in vitro and in vivo. These findings might need to be taken into consideration for in vitro generation of haematopoietic stem cells, where a fine tuning of Notch signalling activity could greatly improve their emergence.

One of the requisites for a growth factor and a biomaterial in tissue engineering, cell therapy and regenerative medicine is the ability to control cell fate. Cells exist in a complex micro-environment consisting of extra-cellular matrix, growth factors, together with adjacent cells. Stem cell culture and modulation remains a challenge due to insufficient, undefined and costly culture systems. This thesis describes synthetic approaches that can modulate stem cell fate by the identification of new synthetic substrates for the growth of cancer, embryonic stem cells and potential short peptide sequences that can mimic the biological functions of the native cytokine used to culture stem cells. Glioma cancers exist as a heterogeneous population of cancer stem cells and cancer progenies. Scale up and spin coating of a polyurethane and polyacrylate polymers was done on agarose for the enrichment of the cancer stem cell population from glioma cells. A polyurethane, synthesised from poly(tetramethylene glycol) and 1,3-bis(isocyanatomethyl)cyclohexane spin (PU10) coated on an agarose surface, was identified to have a higher affinity for the cancer stem cell population over its progenies. By using this polymer to study the mechanism of the cancer stem cell adhesion, two niche components i.e. galectin, transferrin that are enriched by the polymer that contributed to the growth of the cancer stem cells were identified. A synthetic hydrogel (HG21) was identified as substrate for the culture of mouse embryonic stem cells (mESC) as a replacement for gelatin. mESCs were cultured on the hydrogel under undefined and defined conditions. Under both culture conditions, mESC pluripotency and naïve phenotype markers were verified. Marker profiles by immunostaining (Oct-4, Nanog), flow cytometry (SSEA-1) and qPCR (14 gene markers) of mESC grown on the hydrogel were comparable to gelatin, while enabling thermo-detachment for enzyme free passaging of mESC. To identify alternative substances to the cytokines used in stem cell culture, a microarray system was developed. The microarray system was developed initially with adhesion cellulose peptides printed onto polyacrylamide coated microscope slides. These slides were then screened for interaction with human embryonic stem cells (hESCs). After successful development of the cell based cellulose peptide microarray system, overlapping 25-mer peptides based on of basic fibroblast growth factor were synthesised, printed onto the same type of slide and screened with hESC. The screen identified “hit” peptides, which could potentially mimic the biological effects of the native cytokine on hESCs. These “hit” peptides were scaled up and tested in solution with hESC. In the linear form the peptides were not sufficient to sustain pluripotency and further optimisation is needed.

The liver has the ability to regenerate rapidly during acute liver injury by activating mature hepatocytes to divide and restore the damaged liver mass. In contrast, the liver relies on hepatic progenitor cells (HPCs) which have the ability to differentiate into both hepatocytes and biliary cells for regeneration during chronic liver injuries. Whole organ transplant is the most effective treatment for end stage liver diseases. However, there is a constant shortage of donor organs causing the death of many patients while waiting for suitable donor organs. HPC transplant is a potential alternative for whole organ transplant. However the isolation of HPC which is scarce in the liver and the expansion of these cells to a number that is suitable for transplant have been challenging. To investigate the plausibility of using HPCs as an alternative for liver transplant, I developed a protocol to isolate and expand HPC in vitro. Using this system, I investigated the complex hierarchy of HPCs in aid to select a defined population of HPC that is suitable for transplant. I found the EpCAM+ CD24+ population marks a naïve population of HPC that might be suitable for cell therapy. I further investigate the liver repopulating capacities of these cells by isolating EpCAM+CD24+ HPC population by Fluorescence Activated Cell Sorting (FACS) from a hepatocellular injury model. Surprisingly, a subpopulation of the EpCAM+ CD24+ HPCs which are also CD133+ possesses a higher colony forming capacities has been identified. Most importantly, this population can be expanded to a large scale in vitro and able to repopulate the injured liver after transplant. This defined population of HPCs can also be isolated from a mouse model of fatty liver disease and the isolated HPCs can be expanded in vitro. These cells are able to repopulate the liver after cell transplantation. The presence of HPCs that are capable of being isolated from the fatty liver proved the potential of using HPCs for transplant in a clinical setting by using cells isolated human fatty liver that are from rejected for transplant to overcome the shortage of donor organs.

One-third of science, technology, engineering, and mathematics (STEM) jobs are unfilled, resulting in less revenue for companies. The purpose of this qualitative single case study, grounded in the human capital theory, was to explore strategies that 2 hiring managers used to recruit STEM professionals in an organization in central Virginia. Data were collected from semistructured interviews, member checking, and company documents on STEM recruitment. The 4 themes that emerged from the data, analyzed using Yin's 5-step process, were as follows: partnerships with state government and local colleges, leadership and employee development programs used as recruitment incentives, competitive compensation package and work-life balance to entice new hires, and meticulous investigation, evaluation, and trial periods. The findings of this study could provide hiring managers with strategies to improve recruitment of skilled STEM professionals. The implications for positive social change could include a reduction in employee turnover, a decrease in employee recruiting costs, an increase in employee retention, more motivated and engaged employees, and an increase in employee production, all of which could increase profits and positively influence the company and the health of the community.