Upper airways are lined with a pseudostratified mucociliary epithelium maintained by basal cells. To investigate functional and phenotypic heterogeneity within the human basal cell compartment, we used a combination of limiting dilution assays and surface marker profiling on primary cultures of basal cells with verified progenitor activity. The limiting dilution assay suggested functional heterogeneity in the ability of basal cells to repopulate a filter and maintain a barrier at ALI. The frequency of cells with this activity varied between patient strains and ranged from 0.08%-1% of basal cells. Validation of large-scale comprehensive surface marker profiling on basal cells led to identification of 74 antigens demarking consistent subpopulations. Preliminary functional analyses suggest differences in differentiation potential of some subpopulations. This work supports the idea that the basal cell compartment may be functionally heterogeneous, and provides new molecular tools for interrogation of human basal cells.

Engineering epithelium with correct structure is essential for generating functional tissue. During tissue development, cells organize in defined patterns through cellular signalling. Artificial generation of the signalling that organizes cells within the tissue offers a novel approach for engineering tissues with appropriate structure. Planar cell polarity (PCP) is a cellular signalling pathway involved in the organization of epithelial cells. Our goal is to study the effect that co-culturing genetically distinct populations of epithelial cells, with variable levels of one of the core PCP proteins, has in epithelial cell sheet organization. MDCK cells transduced with a tagged PCP core protein (GFP-Vangl2) and wild type MDCK cells were co-cultured side-by-side. The effect of tight junction and cilia formation, and localization of the GFP-Vangl2 protein were evaluated. The results suggest that tight junction and cilia formation are not affected. On the other hand, the GFP-Vangl2 protein seems to be affected at some level.

Upper airways are lined with a pseudostratified mucociliary epithelium maintained by basal cells. To investigate functional and phenotypic heterogeneity within the human basal cell compartment, we used a combination of limiting dilution assays and surface marker profiling on primary cultures of basal cells with verified progenitor activity. The limiting dilution assay suggested functional heterogeneity in the ability of basal cells to repopulate a filter and maintain a barrier at ALI. The frequency of cells with this activity varied between patient strains and ranged from 0.08%-1% of basal cells. Validation of large-scale comprehensive surface marker profiling on basal cells led to identification of 74 antigens demarking consistent subpopulations. Preliminary functional analyses suggest differences in differentiation potential of some subpopulations. This work supports the idea that the basal cell compartment may be functionally heterogeneous, and provides new molecular tools for interrogation of human basal cells.

The epithelial sodium channel (ENaC) is the central component of the sodium absorption pathway in epithelia. It is critical for sodium homeostasis and blood pressure control, which is demonstrated by rare genetic disorders such as Liddle�s syndrome and pseudohypoaldosteronism type I, that are associated with hyper- and hypotension, respectively. ENaC is mainly regulated by mechanisms that control the expression of active channels at the cell surface. Ubiquitin ligases of the Nedd4-like family, such as Nedd4 and Nedd4-2 decrease epithelial sodium absorption by binding to and targeting ENaC for endocytosis and degradation. This is most likely achieved by catalyzing the ubiquitination of ENaC. Conversely the serum- and glucocorticoid regulated kinase (SGK) increases ENaC activity. This effect is partly mediated by the interaction of SGK with the ubiquitin ligases Nedd4 and Nedd4-2. SGK is able to bind to both Nedd4 and Nedd4-2, however only Nedd4-2 is phosphorylated by SGK. The phosphorylation of Nedd4-2 inhibits its interaction with ENaC, thus reducing ENaC ubiquitination, thereby increasing surface expression and sodium absorption. Nedd4-like proteins interact with ENaC via their WW-domains. These domains bind PY-motifs (PPXY) present in ENaC subunits. Nedd4 and Nedd4-2 both have four highly similar WW-domains. Previous studies have shown that interaction between Nedd4 and ENaC is mainly mediated by WW-domain 3. SGK also has a PY-motif; therefore it was analyzed whether the WW-domains of Nedd4 and Nedd4-2 mediate binding to SGK. Here, it is shown that single or tandem WW-domains of Nedd4 and Nedd4-2 mediate binding to SGK and that, despite their high similarity, different WW-domains of Nedd4 and Nedd4-2 are involved. These data also suggest that WW-domains 2 and 3 of Nedd4-2 mediate the interaction with SGK in a concerted manner, and that in vitro the phosphorylation of SGK at serine residue 422 increases its affinity for the WW-domains of Nedd4-2. The stimulatory effect of SGK on ENaC activity is partly mediated via Nedd4-2 and will decrease if competition between Nedd4 and Nedd4-2 for binding to SGK occurs. Here it is shown that Nedd4 and Nedd4-2 are located in the same subcellular compartment and that they compete for binding to SGK. Besides its function in the proteasomal degradation pathway ubiquitination is involved in the regulation of membrane protein trafficking, including their endocytosis. ENaC was shown previously to be ubiquitinated. Here, we provide evidence that ENaC can be ubiquitinated differentially depending on its cellular location. Channels residing in the plasma membrane are multiubiquitinated and we suggest that this serves as an internalization signal for ENaC and a control for further trafficking. Cytosolic ENaC is mainly polyubiquitinated, and therefore probably targeted for proteasomal degradation. However, mono- and multiubiquitination of ENaC located within the cytosol is very likely to occur as well. In addition, it is shown that both proteasomal and lysosomal pathways are involved in the regulation of ENaC.

[Truncated abstract] Asthma affects millions of people worldwide and places a substantial burden on the healthcare system. Despite advances in our understanding of disease mechanisms and the role of respiratory viruses in asthma exacerbations, there is little known regarding the role of the epithelium in commonly observed structural changes in the airway wall. The epithelium of the airways provides an essential protective barrier between the environment and underlying structures and is responsible for the secretion of diverse compounds. Since it is likely that dysregulated epithelial characteristics and function in childhood asthma are critical determinants of disease progression in adults, it is pertinent to investigate the cellular mechanisms involved in paediatric asthma. However, full comprehension of paediatric respiratory diseases and the childhood antecedents of adult respiratory disease are currently hampered by the difficulty in obtaining relevant target organ tissue and most of the data to date have been generated from studies involving adults or commercially derived cell lines. This laboratory has successfully developed methodologies of obtaining and studying samples of paediatric primary airway epithelial cells (pAECs) and has identified significant biochemical and functional differences between healthy non-atopic (pAECHNA) and atopic asthmatic (pAECAA) airway cells, which have assisted in the identification of potential mechanisms responsible for abnormal epithelial function. Stevens 2009 ... Exposure of pAECs with RV resulted in elevated PAI-1 mRNA expression and reduced MMP-9 release in both pAECAA and pAECHNA samples. Collectively, the data presented indicate that RV exposure induces a pronounced antiproliferative and retardative repair effect in pAECAA and that the presence of virus may have a role in the PAI-1 and MMP expression witnessed in these cells. In conclusion, this investigation has further characterised the essential role the airway epithelium plays in childhood asthma by demonstrating for the first time that pAECs from asthmatic children lack the ability to successfully repair mechanically induced wounds. This investigation also showed that PAI-1 is elevated in pAECAA and has a functional role in the pAEC proliferative and regenerative processes. It was demonstrated that MMP-2 and MMP-9 activities and the MMP-9/TIMP-1 as well as MMP2/TIMP2 ratios were significantly reduced in pAECAA thereby providing additional evidence that there is a dysregulation in the mechanisms that monitor the turnover of the ECM in childhood asthma. Furthermore, this study has shown for the first time that pAECs from untreated mild atopic-asthmatic children are more sensitive to the pathogenic effects of RV than healthy control cells and that RV exposure delays cellular proliferation and repair. Ultimately, these findings support the hypothesis postulated and provide evidence that indeed the dysregulated epithelial functional characteristics seen in childhood mild asthma may be a critical determinant of disease progression in adults.

Diss. Stockholm : Karol. inst.

Thesis (M.D.S.)--University of Adelaide, Dental School, 2001. / Includes bibliographical references (leaves 68-76).

Thesis (M.Sc. (Forensic Science))--Mahidol University, 1982.

Thesis (M.Sc. (Anatomy)) -- Mahidol University, 1976.

Thesis (Ph.D.)--Cleveland State University, 2010. / Abstract. Title from PDF t.p. (viewed on April 15, 2010). Includes bibliographical references (p. 151-187). Available online via the OhioLINK ETD Center and also available in print.