Herein we describe the regulatory significance and molecular targeting of novel non-B-DNA secondary structures formed from the PDGFR-Beta core promoter nuclease hypersensitivity element.

<p>During development of the mammalian embryo, spatial and temporal expression of fibroblast growth factors (FGFs) and their cognate receptors are vital in the regulation of a number of patterning processes. Inappropriate or decreased expression leads to severe malformations and even embryonic death. The objectives of this thesis have been to evaluate the usefulness of differentiating embryonic stem (ES) cells as a model to study FGF and FGF receptors in endothelial and hematopoietic cell function in vitro and in vivo, and the effect of an activating mutation in the platelet-derived growth factor receptor-β (PDGFR-β) on endothelial cells and vessel formation.</p><p>Aggregates of differentiating ES cells, denoted embryoid bodies, faithfully recapitulate many developmental processes. Embryoid bodies cultured in fetal calf serum spontaneously develop cardiomyocytes and endothelial cells. The endothelial cells organize into lumen-containing vessels carrying erythroblasts. Administration of FGF or vascular endothelial growth factor (VEGF)-A promotes development of specific vascular phenotypes. About 20% of endothelial cells in embryoid bodies and teratomas express FGFR-1, and these FGFR-1-expressing endothelial cells are mitogenically active in the absence of exogenous stimuli and respond to VEGF-A to the same extent as endothelial cells lacking FGFR-1 expression. FGFR-1 deficiency leads to arrest in hematopoietic differentiation, whereas endothelial cell development is enhanced. As a consequence, teratomas derived from ES cells lacking FGFR-1 expression display vessels composed of a double layer of endothelial cells. The hyperactivity of endothelial cells derived from FGFR-1-deficient ES cells is suggested to be due to hyperactivity of VEGF receptor-2, as well as to loss of negative regulators of angiogenesis, such as interleukin-4.</p><p>Mutation of platelet-derived factor receptor-β (PDGFR-β) to replace D849 in the activating loop in the kinase domain with V leads to ligand-independent kinase activity, increased basal signal transduction, and enhanced expression of VEGF-A as well as VEGFR-2. As a result, endothelial cell sprouts covered with pericyte-like cells are formed in a VEGF-A/VEGFR-2 dependent manner in ES cells expressing the mutated PDGFR-β.</p><p>In conclusion, embryoid bodies represent a high-quality model for the study of growth factor-regulated vascular development and sprouting angiogenesis.</p>

During development of the mammalian embryo, spatial and temporal expression of fibroblast growth factors (FGFs) and their cognate receptors are vital in the regulation of a number of patterning processes. Inappropriate or decreased expression leads to severe malformations and even embryonic death. The objectives of this thesis have been to evaluate the usefulness of differentiating embryonic stem (ES) cells as a model to study FGF and FGF receptors in endothelial and hematopoietic cell function in vitro and in vivo, and the effect of an activating mutation in the platelet-derived growth factor receptor-β (PDGFR-β) on endothelial cells and vessel formation. Aggregates of differentiating ES cells, denoted embryoid bodies, faithfully recapitulate many developmental processes. Embryoid bodies cultured in fetal calf serum spontaneously develop cardiomyocytes and endothelial cells. The endothelial cells organize into lumen-containing vessels carrying erythroblasts. Administration of FGF or vascular endothelial growth factor (VEGF)-A promotes development of specific vascular phenotypes. About 20% of endothelial cells in embryoid bodies and teratomas express FGFR-1, and these FGFR-1-expressing endothelial cells are mitogenically active in the absence of exogenous stimuli and respond to VEGF-A to the same extent as endothelial cells lacking FGFR-1 expression. FGFR-1 deficiency leads to arrest in hematopoietic differentiation, whereas endothelial cell development is enhanced. As a consequence, teratomas derived from ES cells lacking FGFR-1 expression display vessels composed of a double layer of endothelial cells. The hyperactivity of endothelial cells derived from FGFR-1-deficient ES cells is suggested to be due to hyperactivity of VEGF receptor-2, as well as to loss of negative regulators of angiogenesis, such as interleukin-4. Mutation of platelet-derived factor receptor-β (PDGFR-β) to replace D849 in the activating loop in the kinase domain with V leads to ligand-independent kinase activity, increased basal signal transduction, and enhanced expression of VEGF-A as well as VEGFR-2. As a result, endothelial cell sprouts covered with pericyte-like cells are formed in a VEGF-A/VEGFR-2 dependent manner in ES cells expressing the mutated PDGFR-β. In conclusion, embryoid bodies represent a high-quality model for the study of growth factor-regulated vascular development and sprouting angiogenesis.

In this study the aim was to optimize the production of the Affibody fusion-protein Z03358- ABD094-(S4G)3-IL2 with regard to the amount of soluble protein produced. However, problems with reproducibility with this protein and the chosen expression system were encountered. Therefore, expression of the His-tagged Affibody His6-(Z05477)2 was evaluated using the same expression system as well as expression in another well characterized expression system. Both target proteins are of therapeutic interest. One of the proteins is an IL2 fusion protein (Z03358-ABD094-(S4G)3-IL2) that bind the platelet-derived growth factor receptor β (PDGFR-β). PDGF signaling is of interest in cancer treatment where, among other things, the effects of PDGF on tumor angiogenesis is researched. The His6-(Z05477)2 protein has a classified target but is developed as a therapeutic in the area of inflammation and autoimmune disease. Both model proteins are known to be difficult to purify due to low solubility. The two E. coli expression systems investigated and compared were BL21(DE3) and Lemo21(DE3). The fusion protein Z03358-ABD094-(S4G)3-IL2 was produced in BL21(DE3) in inclusion bodies with a yield of 4.95 g/l. An optimized process for the expression of His6-(Z05477)2 using BL21(DE3) was developed with a yield of 6.6 g/l soluble protein after expression at 30°C for 6 h.