Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 183-214). Also available in electronic version.

Thesis (Ph. D. in Molecular Physiology and Biophysics)--Vanderbilt University, Aug. 2007. / Title from title screen. Includes bibliographical references.

The respiratory system represents a major interface between the body and the external environment. Its design includes a tree-like network of conducting tubules (airways) that carries air to millions of alveoli, where gas exchange occurs. The conducting airways are characterized by their great diversity in epithelial cell types with multiple populations of secretory, multiciliated, and neuroendocrine cells. How these different cell types arise and how these populations are balanced are questions still not well understood. Aberrant patterns of airway epithelial differentiation have been described in various human pulmonary diseases, chronic bronchitis, asthma, neuroendocrine hyperplasia of infancy, and others. The goal of this thesis is to investigate mechanisms of regulation of airway epithelial cell fate in the developing lung epithelium. More specifically, these studies focus on Notch signaling and address a long unresolved issue whether the different Notch ligands (Jagged and Delta) have distinct roles in the epithelial differentiation program of the extrapulmonary and intrapulmonary airways. Moreover, these studies investigate the ontogeny of the bHLH transcription factor Ascl1 and identify its targets in the developing airways as potential regulators of neuroepithelial body (NEB) size and maturation. My studies provide evidence that the Notch ligand families Jag and Dll are required for the specification and formation of different cell lineages in the developing airway epithelia. Jag ligands regulate multiciliated versus secretory (club) cell fates but also controls abundance of basal cell progenitors in extrapulmonary airways. Dll ligands regulate pulmonary neuroendocrine versus club cell fates in intrapulmonary airways. Analysis of mouse mutants showed that loss of Jag ligands has minimal impact on the size or abundance of NEBs and their associated secretory cells while loss of Dll ligands results in an expansion of NEB size and associated cells. To gain additional insights into the potential mechanisms of how neuroendocrine cells develop and undergo aberrant hyperplasia, I characterized the global transcriptional profile of embryonic lungs from mice deficient in Ascl1, which lack NEBs and neuroendocrine cells and identified a number of genes associated with neuroendocrine cell development, maturation, and the NEB microenvironment. Among these genes, components of the catecholamine biosynthesis pathway, such as tyrosine hydroxylase (Th), a key enzyme for catecholamine production, were downregulated in Ascl1 null lungs. Subsequent functional analysis using a pharmacological inhibitor of this pathway in lung organ cultures showed expansion of pulmonary neuroendocrine cells and NEB size, an observation of potential relevance in human diseases in which neuroendocrine cells are aberrantly expanded. Together these studies highlight the distinct role of Notch ligands and further implicate Ascl1 targets, as illustrated by catecholamine pathway components, in regulating epithelial cell fate. Further examination of these pathways may provide insights into the pathogenesis and ultimately therapeutic approaches for airway diseases.

No description available.

Indiana University-Purdue University, Indianapolis / This dissertation examines the molecular mechanisms regulating Rac2 gene expression during cell differentiation and identification of a minimal cis-element required for the induction of Rac2 gene expression during K562 cell differentiation. The Rho family GTPase Rac2 is expressed in hematopoietic cell lineages and is further up-regulated upon terminal myeloid cell differentiation. Rac2 plays an important role in many hematopoietic cellular functions, such as neutrophil chemotaxis, superoxide production, cytoskeletal reorganization, and stem cell adhesion. Despite the crucial role of Rac2 in blood cell function, little is known about the mechanisms of Rac2 gene regulation during blood cell differentiation. Previous studies from the Skalnik lab determined that a human Rac2 gene fragment containing the 1.6 kb upstream and 8 kb downstream sequence directs lineage-specific expression of Rac2 in transgenic mice. In addition, epigenetic modifications such as DNA methylation also play important roles in the lineage-specific expression of Rac2. The current study investigated the molecular mechanisms regulating human Rac2 gene expression during cell differentiation using chemically induced megakaryocytic differentiation of the human chronic myelogenous leukemia cell line K562 as the model system. Phorbol 12-myristate 13-acetate (PMA) stimulation of K562 cells resulted in increased Rac2 mRNA expression as analyzed by real time-polymerase chain reaction (RT-PCR). Luciferase reporter gene assays revealed that increased transcriptional activity of the Rac2 gene is mediated by the Rac2 promoter region. Nested 5’- deletions of the promoter region identified a critical regulatory region between -4223 bp and -4008 bp upstream of the transcription start site. Super shift and chromatin immunoprecipitation assays indicated binding by the transcription factor AP1 to three distinct binding sites within the 135 bp minimal regulatory region. PMA stimulation of K562 cells led to extensive changes in chromatin structure, including increased histone H3 acetylation, within the 135 bp Rac2 cis-element. These findings provide evidence for the interplay between epigenetic modifications, transcription factors and cis-acting regulatory elements within the Rac2 gene promoter region to regulate Rac2 expression during K562 cell differentiation.

H1 linker histones are structural components of chromatin, generally implicated in the formation of “higher order” chromatin states. With eleven non-allelic subtypes in mammals, the H1 family is highly diverse. While they are commonly associated with chromatin compaction and transcription repression, these histones also play crucial roles in mouse development and stem cell differentiation. Although the prevailing belief is that H1 subtypes have redundant functions, their distinct amino acid composition and differential expression throughout development suggest subtype-specific roles. Previous studies have explored the roles and interactions of linker histones, but limitations in model systems, cell types, and subtypes studied have hindered our comprehensive understanding of the implications and synergy of multiple H1 linker histones. To gain insight into the individual and combined roles of linker histones in human embryonic stem cells (hESCs), we conducted an extensive study in which we systematically removed each somatic linker histone and looked at all potential combinations. Using RNA-seq and in-depth bioinformatic analysis, we discovered that linker histones in hESCs exhibit partial non-redundancy. We classified them into three main groups associated with distinct biological processes, particularly related to development and stem cell differentiation. We observed that depleting H1.1 or H1.5 influenced the proportion of mesodermal progenitor cells, with further impact when combined with specific H1 subtypes, resulting in changes in ectodermal progenitor cells. Additionally, we demonstrated that linker histones synergistically regulate interconnected biological pathways, potentially affecting early stem cell differentiation. Based on our findings, we propose that H1 subtypes regulate specific transcriptional programs, which in conjunction, are fundamental in the coordination of essential cellular processes involved in early human embryonic development, both in the ground state of hESCs and during stem cell differentiation. We anticipate that the generation of the H1 KO library described in our study will provide a novel tool for studying the role of linker histones in later stages of human development and will facilitate the comprehension of specific roles of these chromatin proteins in other relevant cellular processes.

No description available.

No description available.

CD4+ T cells are a group of lymphocytes that play critical roles in the immune system. By releasing cytokines, CD4+ T cells regulate other immune cells for maximizing the efficiency of the system. Naive CD4+ T cells are activated and become mature upon engagement with antigens, and the mature CD4+ T cells have several subsets, which play diverse regulatory functions. For the past two decades, our understanding of CD4+ T cells has been advanced through the studies on the differentiation process and the lineage specification of various subsets of these cells. Although in most experimental studies of CD4+ T cells, researchers focused on how transcription factors and signaling molecules influence the differentiation of a particular subset of these cells, many evidence have shown that the differentiation of CD4+ T cells can be heterogeneous in terms of the phenotypes of the cells involved. This dissertation describes a framework that uses mathematical models of the dynamics of the signaling pathways to explain heterogeneous differentiation. We show that the mutual inhibitions among the master regulators govern the formation of multi-stability behavior, which in turn gives rise to heterogeneous differentiation. The framework can be applied to systems with two or more master regulators, and models based on the framework can make specific predictions about heterogeneous differentiations. In addition, this dissertation describes an experimental study on CD4+ T cell differentiation. Being part of the adaptive immune system, the differentiation of CD4+ T cells was previously known to be induced by the signals from the innate immune cells. However, the expression of Toll-like receptor in CD4+ T cells suggests that microbial products can also influence the differentiation directly. Using an in vitro cell differentiation approach, we show that the differentiation and proliferation of CD4+ T cells can be influenced by lipopolysaccharide under the condition that would favor the differentiation of induced regulatory T cells. These theoretical and experimental studies give novel insights on how CD4+ T cells differentiate in response to pathogenic challenges, and help to gain deeper understanding of regulatory mechanisms of the complex immune system. / Ph. D.

Introduction: MSCs are multipotent progenitor cells that can differentiate into various cell lineages, such as osteoblasts, chondrocytes and adipocytes. MSCs synthesize abundant secretion factors to extracellular matrix which contain a variety of growth factors, cytokines and microRNAs. Secretion factors could stimulate the regeneration and differentiation of surrounding cells, but their underlying mechanism still remains elusive. We hypothesized that secretion factors from different tissues derived MSCs had potential to promote MSCs differentiation and musculoskeletal tissue regeneration. We also suggested that microRNAs played an essential role in the effects of secretion factors. In present study, we investigated the effects of secretion factors obtained from different tissues derived MSCs (umbilical cord, dental pulp, gingiva and adipose tissue) on multi-differentiation potentials of MSCs, including osteogenesis, chondrogenesis, tenogenesis, neurogenesis and adipogenesis. Moreover, we illustrated the effects of umbilical cord derived MSC (UCMSC) secretion on bone, cartilage and tendon tissue repair. We further revealed that microRNAs may impact the effect of secretion factors on MSCs osteogenic differentiation. / Methods: Human bone marrow MSCs (hBMSCs) were incubated with various differentiation induction media. Secretion factors were used as supplement. Different animal models of tissue repair (bone, cartilage and tendon) were employed for study of the effects of secretion factors on tissue healing. miRNA microarray was performed to find the potential effective miRNAs in secretion factors. Real time qRT-PCR, microCT, mechanical test, immunohistological analysis and various staining methods were employed as outcome measurements. / Results: We found that both UCMSC and dental pulp derived MCS secretion could initiate osteogenic differentiation of hBMSCs without osteogenic induction medium. UCMSC secretion had positive effect on chondrogenic and tenogenic differentiation of MSCs and inhibitory effect on adipogenesis of hBMSCs. Our results showed that UCMSC secretion in HA/TCP scaffolds with hBMSCs promoted ectopic bone formation in nude mice. UCMSC secretion with rat BMSCs in hyaluronic hydrogel significantly enhanced the bone repair of rat calvarial bone critical defect. To reveal the underlying mechanism, secretion factors were analyzed by miRNA microarray. Among the differentially expressed microRNAs, we found miR-1237 could promote osteogenesis while miR-3676 could inhibit osteogenic differentiation of MSCs. / Conclusions: This study indicated that among secretion factors from MSCs form four types tissues, UCMSC secretion could initiate osteogenesis of MSCs and promote bone repair. We also demonstrated that microRNAs from secretion had impact on osteogenic differentiation of MSCs. Our study showed clinical potential of UCMSC secretion in bone regeneration, and more research are needed for optimizing the preparation and delivery of the MSCs secretive factors, as well as to understand their mechanisms of action. / 前言：間充質幹細胞是具有強大分化潛能的始祖細胞。間充質幹細胞可以分化為多種細胞系，例如成骨細胞，軟骨細胞和脂肪細胞。間充質幹細胞合成并釋放大量分泌素到細胞外基質中。這些分泌素包括多種生長因子，細胞因子和微小核糖核酸。分泌素能夠刺激周圍細胞的再生和分化，但是分泌素的作用機理還不是很清楚。我們認為，不同組織來源的間充質幹細胞分泌素有可能會促進間充質幹細胞的多系分化和骨骼肌肉組織的再生，並且微小核糖核酸在分泌素的效應中發揮了重要作用。我們首先研究了臍帶，牙髓，牙齦和脂肪來源的間充質幹細胞分泌素對于間充質幹細胞的分化能力的作用。我們還對臍帶幹細胞分泌素在骨，軟骨和肌腱修復的效果做了進一步的研究。另外，我們還發現分泌素中的微小核糖核酸在間充質幹細胞的成骨分化方面有一定的效果。 / 方法：我們用人間充質幹細胞來進行誘導分化實驗。臍帶，牙髓，牙齦和脂肪來源的間充質幹細胞的分泌素用於細胞培養基的補充。在體內實驗中我們用了不同的動物模型，把填充物和分泌素一起種植在動物體內。我們利用微小核糖核酸陣列技術來檢測分泌素中的有效微小核糖核酸。我們使用了定量聚合酶鏈反應技術，微型計算機斷層掃描成像，力學測試，免疫組織分析和多種染色方法。 / 結果：我們發現臍帶和牙髓間充質幹細胞分泌素可以在沒有成骨誘導培養基的情況下啟動骨髓間充質幹細胞的成骨分化。臍帶間充質幹細胞對成軟骨和成肌腱分化起到積極作用，而且可以抑制脂肪分化。我們發現在羥基磷灰石/磷酸三鈣材料中，臍帶間充質幹細胞分泌素與人骨髓間充質幹細胞可以共同促進裸鼠的異位成骨。臍帶間充質幹細胞分泌素與鼠骨髓間充質幹細胞一起用於透明質酸水凝膠中能夠加快大鼠頭骨缺損的修復。為了揭示分泌素的作用機理，我們用微小核糖核酸陣列技術來檢測分泌素。在表達不同的微小核糖核酸之中，我們發現miR-1237可以促進間骨髓間充質幹細胞的成骨分化，而miR-3676能夠抑制骨髓間充質幹細胞成骨分化。 / 結論：本研究表明，在四種不同來源的分泌素中，臍帶間充質幹細胞分泌素可以啟動骨髓間充質幹細胞的成骨分化，同時加快骨組織修復。我們發現微小核糖核酸在分泌素的促進間骨髓間充質幹細胞成骨分化的效果中發揮了一定的作用。我們的研究表明，使用臍帶間充質幹細胞分泌素修復骨組織具有廣泛的臨床應用前景。間充質幹細胞分泌素的生產，使用過程和作用機理還有待于進一步的優化和研究。 / Wang, Kuixing. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 131-147). / Abstracts also in Chinese. / Title from PDF title page (viewed on 01, November, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.