Objective: Endothelial cell (EC)-induced mesenchymal cell (MC) differentiation toward a mural cell phenotype requires transforming growth factor beta (TGF-β), cell contact and connexin 43 (Cx43)- or Cx45- heterocellular gap junction intercellular communication (GJIC). However, the identity of the communicated signal, the features of Cx43 required, and the possible regulatory mechanisms have not been elucidated and were investigated herein. Methods & Results: To determine whether channel functionality and the major regulatory domain (the carboxyl terminus, CT) of connexin Cx43 are necessary to support EC-induced differentiation, Cx43 deficient MCs (incapable of undergoing EC-induced mural cell differentiation without re-expression of Cx43 or Cx45) were transduced with wild-type (Cx43wt), channel dead, or truncated (Cx43tr-residues 258-382 deleted) versions of Cx43 and their ability to support EC-induced differentiation was assessed. Our data indicate that both channel functionality and presence of the CT domain are both necessary for EC-induced mural cell differentiation. Moreover, expression of Cx40 did not restore ability of MCs to undergo EC-induced mural cell differentiation, despite supporting GJIC. To determine whether (and which) specific regulatory sites in the carboxyl terminus are necessary for EC-induced mural cell differentiation, constructs of Cx43 with serine to alanine substitutions at the mitogen activated protein kinase (MAPK) or protein kinase C (PKC) target sites were introduced into Cx43 deficient MCs and their ability to undergo EC-induced differentiation was tested. The data indicated that the MAPK targeted serines (S255,279,2982) are necessary, while the PKC targeted serine (S368) is dispensable, for this process. To determine whether calcium ions might be the messengers communicated between ECs and MCs, we investigated whether elevation in EC free intracellular calcium concentration (with ionomycin treatment) can replace Cx43-mediated GJIC, activate TGF-β and induce differentiation. Conclusions: Channel functionality, CT domain and the MAPK target sites in Cx43 are all necessary, and neither alone is sufficient, for Cx43-mediated, EC-induced mural cell differentiation. Unlike Cx43, Cx40 is not capable of supporting EC-induced differentiation, despite supporting GJIC. Calcium is unlikely to be the messenger critical to TGF-β activation during EC-induced differentiation, but similar signaling pathways can be initiated. Taken together, these data support a role for connexins in EC-induced differentiation that is complex and goes beyond that of a simple conduit.

Vaginal atrophy symptoms such as dryness, irritation, and itching, are common after menopause. Vaginal estrogen therapy is the most effective treatment but not appropriate for all women. Women with estrogen-responsive breast cancer treated with aromatase inhibitor (AI) treatment, suppressing estrogen levels, often suffer from more pronounced vaginal atrophy symptoms. However, vaginal estrogen treatment is not recommended, leaving them without effective treatment options. The aim of this thesis was to study the effect of long-term anti-estrogen therapy on circulating estrogen levels and biochemical factors in vaginal mucosa in relation to morphological changes and clinical signs of vaginal atrophy. Circulating estrogen levels were analyzed by use of mass spectrometry and radioimmunoassay. Immunohistochemistry was used to study vaginal proliferation and steroid hormone receptors in vaginal mucosa. Vaginal gene expression was studied by use of microarray technology and bioinformatic tools, and validated by use of quantitative real-time PCR and immunohistochemistry. An estrogenic regulation of aquaporins and a possible role in vaginal dryness was investigated in vaginal mucosa and in Vk2E6E7 cells. Aromatase inhibitor-treated women had higher than expected estradiol and estrone levels but still significantly lower than other postmenopausal women. Aromatase was detected in vaginal tissue, the slightly stronger staining in vaginal mucosa from AI-treated women, suggest a local inhibition of vaginal aromatase in addition to the systemic suppression. Vaginal mucosa from AI-treated women had weak progesterone receptor, and strong androgen receptor staining intensity. Low estrogen levels lead to low expression of genes involved in cell adhesion, proliferation, and differentiation as well as weak aquaporin 3 protein immunostaining. The higher than expected estrogen levels in AI-treated women suggest that estrogen levels might previously have been underestimated. Systemic estrogen suppression by treatment with AIs, and possibly also by local inhibition of vaginal aromatase, results in reduced cell adhesion, proliferation, differentiation, and weak aquaporin 3 protein staining. Low proliferation and poor differentiation leads to fewer and less differentiated superficial cells affecting epithelial function and possibly also causing vaginal symptoms. Aquaporin 3 with a possible role in vaginal dryness, cell proliferation, and differentiation should be further explored for the development of non-hormonal treatment options for vaginal symptoms.

Cell fate decisions incorporate distinct and overlapping mechanisms. The activity of caspase 3 was initially understood to be a cell death restricted event, however numerous studies have implicated this enzyme in the regulation of both differentiation and proliferation. How the activity of caspase 3 promotes a non-death cell fate remains unclear. Here we examine the role caspase 3 activity plays during skeletal muscle differentiation; in particular we explore the hypothesis that the mechanism of inducing DNA strand breaks during cell death is also a key feature of differentiation, albeit with a distinctly different outcome. We delineate the transient formation of Caspase 3/Caspase activated DNase (CAD) dependent DNA strand breaks during differentiation. The formation of these breaks is essential for differentiation and the regulation of specific genes. In particular expression of the cell cycle inhibitor p21 is related to the formation of a DNA strand break within the gene’s promoter element. Further, we explored the genome wide association of CAD using Chromatin Immunoprecipitation coupled to high through put sequencing (ChIP-seq). This approach identified a potential role for Caspase3/CAD in regulating the expression of Pax7. Here, a CAD directed DNA strand break in the Pax7 gene is correlated with decreased Pax7 expression, an outcome that has been shown to be critical for progress of the myogenic differentiation program. The regulation of Pax7 expression through a CAD induced DNA strand break raises an intriguing connection between this regulation and oncogenic transformation observed in alveolar rhabdomyosarcoma. The putative site of CAD induced DNA strand breaks that promote decreased Pax7 expression during differentiation corresponds to site of chromosomal translocations responsible for Pax7 fusion events in alveolar rhabdomyosarcoma.

Serum-free mouse embryo (SFME) cells are derived in medium in which serum is replaced with growth factors and other supplements. They display unusual properties. They do not lose proliferative potential or show gross chromosomal aberration upon extended culture, they depend on epidermal growth factor (EGF) for survival, and are reversibly growth inhibited by plasma and serum. In the presence of transforming growth factor beta (TGF-β) SFME cells express the astrocyte marker, glial fibrillary acidic protein (GFAP). The growth inhibitory activity of human plasma on serum-free mouse embryo cells was investigated. Human plasma did not inhibit SFME cells transformed with the human Ha-ras oncogene. The activity was present in delipidated plasma and was not dialyzable against 1 M acetic acid. The activity could be precipitated by methanol, bound to concanavalin Aagarose and was retarded by Sephadex G-50 in 200 mM acetic acid. A fifty to hundred fold purification was achieved, although the differential inhibition of untransformed versus transformed cells was lost in the course of the purification. Using the technique of differential screening of a cDNA library a calf serum- and TGF -β-regulated mRNA species was identified in SFME cells. This mRNA was approximately 8.5 kilobases in size and brain-specific. Picomolar quantities of TGF-β caused an increase of this message in SFME cells within four hours. This increase was reversed when TGF-β was removed from the culture medium. / Graduation date: 1993

Copies of author's previously published articles inserted. / Bibliography: leaves 117-130. / v, 130, [51] leaves, [33] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The general aim of this thesis is to develop methods for the identification of markers for pluripotent cell subpopulations in the developing mouse embryo. A screen for mouse embryonic stem (ES) cell markers is carried out, to identify transcripts that are differentially expressed between ES cells and X cells, and to investigate pluripotent cell heterogeneity during early development. The study demonstrates the potential to identify and characterise molecular heterogeneity within the developing pluripotent cell pool in vivo, via the controlled progression and analysis of pluripotent cells in vitro. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1997

Uroplakin 1B (UPKIB) is an integral structural protein interacting with uroplakins 1A, 2 and 3 to form hexameric plaques along the bladder lumen in the asymmetric unit membrane of urothelial umbrella cells in humans and other mammals. UPKIB mRNA expression is deregulated in transitional cell carcinomas (TCCs), however the mechanisms of regulation of UPKIB have not been established. Using genome databases, a Xenopus UPKIB homologue was identified. Maximum Parsimony and BAMBE (Bayesian Analysis in Molecular Biology and Evolution) data support a close evolutionary relationship between mammalian and amphibian UPKIB mRNA. Using Unigene, UPKIB human expressed sequence tags were identified in tissues including brain, skeletal muscle and liver, suggesting the relatively widespread distribution of this membrane protein. The UPKIB genomic structure was also deduced using genome databases. Contig AC083800, identified in a high throughput genomic sequence database, spanned UPKIB and 9 exons and 8 introns were defined. A 67bp 5' untranslated region was identified using 5' rapid amplification of cDNA ends. This product was sequenced and a putative UPKIB promoter and transcription start site was deduced. Contig AC083800 spanned the transcription start site and putative promoter. Transcription factor binding motif prediction programs detected no TATA box, but did predict a CCAAT box and several binding motifs including 4 Sp-1 sites and a NFKB site. A weak CpG island was identified within a 0.5kb region including the putative promoter, exon 1 and intron 1, which was 54% GC rich with CpG:GpC ratio of 0.46, containing 15 CpG dinucleotides. Seven TCC cell lines and five peripheral blood lymphocyte samples were analysed for UPKIB expression using RT-PCR and two cell lines expressed UPKIB transcripts. Eleven CpG sites in the putative promoter were investigated for methylation using bisulfite modification analysis in normal PBL, TCC cell lines and patient TCC samples. An inverse correlation was established in TCC cell lines between UPKIB mRNA expression and degree of methylation. 5-Aza-2'deoxycytidine induced UPKIB mRNA expression in T24 cells, previously observed not to express UPKIB. Sequence analysis of patient samples revealed more complex CpG methylation patterns, reflecting tumour heterogeneity. In summary, the uroplakin 1B gene has been characterised and one mechanism of regulation of gene expression involves methylation. / Thesis (Ph.D.)--Dept of Surgery, 2003.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 4 uppsatser.