Zugl.: Mainz, Univ., Diss., 2006. / Also available in print.

Thesis (Ph. D.)--Ohio State University, 2007. / Full text release at OhioLINK's ETD Center delayed at author's request

The aim of this project was to study ATP binding cassette (ABC) transporters in the human placenta, in particular their regulation and role in trophoblast differentiation and survival. The presence and localisation of four major placental drug transporters, multidrug resistance gene product 1 and 3 (MDR1 and 3)/ABC subfamily B members 1 and 4 (ABCB1 and 4), multidrug resistance associated proteins 1 and 2 (MRP1 and 2)/ABCC1 and 2 and breast cancer resistance protein (BCRP)/ABCG2 was initially studied in term human placenta, cultured primary trophoblast and BeWo and Jar trophoblast-like cell lines. Jar cells were found to be more similar to nondifferentiated cytotrophoblast with respect to their ABC protein expression profile, whereas BeWo cells more closely reflected differentiated syncytiotrophoblast. Treatment of primary term trophoblasts in vitro with cytokines (TNF- or IL-1) decreased expression and activity of apical transporters ABCB1/MDR1 and ABCG2/BCRP. Growth factors, on the other hand, increased BCRP expression and activity, while estradiol stimulated BCRP, MDR1 and MDR3 expression MDR1/3 functional activity. The ability of BCRP/ABCG2 to abrogate the apoptotic effects of TNF- and ceramides was studied in primary trophoblast and BeWo cells using pharmacological and molecular (siRNA) approaches. The results suggest that BCRP/ABCG2 contributes to the resistance of trophoblast cells to cytokine-induced (extrinsic) apoptosis, whereas its effects on apoptosis activated via the intrinsic mitochondrial pathway is minimal. This altered resistance was associated with increased intracellular accumulation of ceramides and reduced ability to maintain phosphatidylserine in the inner leaflet of the plasma membrane. A role for BCRP/ABCG2 in cell protection from differentiation-induced stressors was also demonstrated during the process of cell fusion associated with transient loss of plasma membrane lipid asymmetry. Finally, expression of BCRP/ABCG2 (and 9 other genes) was studied in 50 placentas from normal pregnancy and pregnancies complicated with fetal growth restriction (FGR). A marked reduction of BCRP/ABCG2 and MDR1/ABCB1 expression was observed in FGR placentas, while other transporter genes were unaffected. Collectively these data suggest that BCRP/ABCG2 and probably other ABC transporters may play a hitherto unrecognised survival role in the placenta, conferring a “stress resistance” to trophoblast cells.

Thesis (Honors)--College of William and Mary, 2009. / Includes bibliographical references (leaves 75-79). Also available via the World Wide Web.

BACKGROUND: Fat is stored in adipose tissue. In obesity, differentiation of preadipocytes to new adipocytes (fat cells) is required for energy storage. Otherwise fat accumulation in non-adipocytes contributes to fatty liver and diabetes. Our goal was to assess subject characteristics associated with poor in-vitro differentiation capacity of preadipocytes from omental (OM) and abdominal subcutaneous (SC) fat. APPROACH: A convenience sample of, 4 males and 20 females, age 39±2 (range 20-56) years, BMI 42 ± 2 (23-63) kg/m2 (i.e. from lean to obese), 7 Caucasian, 8 Hispanic, 1 other and 8 African Americans) undergoing elective surgery was studied. Fat samples collected during surgery were used for histology and preadipocyte isolation. Fat cell diameters and their distribution (normal or bimodal) were analyzed from histology. Preadipocyte differentiation capacity was measured in vitro. RESULTS: In the OM depot, no effect of ethnicity, sex or HbA1c was found. Unexpectedly, subjects with preadipocytes with poor differentiation capacity tended to be younger (poor differentiation group 36 ± 2 years versus high 43 ± 3 years, p=0.09) and to have lower fasting glucose (poor 97 ± 3.65 mg/dl versus high 111 ± 7.08 mg/dl, p=0.06). In SC, no differences were noted. Fat cell size was not associated with differentiation capacity in either depot. Bimodal distribution, which may show formation of new adipocytes, was seen mostly in Caucasian subjects (5 out of 7) compared to Hispanic (3 out of 8) and African Americans (2 out of 8). CONCLUSION: It is important to investigate the associations between age/ethnicity and OM preadipocyte differentiation/cell distribution in adequately powered cross-sectional studies.

Osteoblastic differentiation was studied in vitro using primary cultures of bone cells derived from neonatal mouse calvaria. Using alkaline phosphatase as a marker, maintenance of the osteoblastic phenotype was found to be dependent upon the presence of ascorbic acid. No toxic effect due to ascorbic acid was seen. Insulin and dexamethasone were found to stimulate alkaline phosphatase expression, the former only in the absence of ascorbic acid. Two growth factors, epidermal growth factor and platelet-derived growth factor, were found to inhibit alkaline phosphatase expression in the presence of ascorbic acid. Osteogenesis was most pronounced in cultures supplemented with ascorbic acid. The osteoblasts formed multilayers of cells and secreted an organic extracellular matrix composed mainly of type I collagen. Matrix vesicles were found among the collagen fibres. In the presence of 6-glycerophosphate, calcium phosphate crystals were deposited in discrete patches forming a mineralisation front which progressively engulfed osteoblasts. The type of matrix formed and the pattern of mineralisation resembled those of lamellar bone. Insulin at 5000ng/ml stimulated matrix calcification in the absence of ascorbic acid. Dexamethasone, EGF and PDGF inhibited calcification. The extent of calcification was dependent upon the concentration of glycerophosphate in the culture medium. Conditioned medium from osteogenic cultures contained a GM-CSF which was secreted constitutively by the osteoblasts. Preliminary experiments with a mesenchymal stem cell line, Balb/c 3T3, showed the existence of a factor(s) with mitogenic activity in bone cell conditioned medium. No inducer of osteogenic differentiation was found.

For the successful establishment of secondary tumours, blood-borne metastatic tumour cells must adhere and spread on the vascular endothelium before they can migrate through it to form secondary growths in the tissue beneath. In this study an in vitro assay was developed to study the behavourial interactions between B16F10 cells and Bovine aortic endothelial cells. It was hypothesized that molecules synthesized by the endothelial cells may be involved in the mediation of the adhesion, spreading and migration events and hence that such molecules may possibly be involved in the process of haematogenic metastasis. Endothelial derived extracts were obtained from the cell surface and from conditioned medium. The extracts were tested for their adhesion promoting abilities in a quick dot blot adhesion assay. To verify that these molecules promoted adhesion, antibodies were raised against the extracts. Partial characterisation of the molecules was achieved using SDS-PAGE and immunoprobing. The extracts were also tested for their spreading and migration promoting properties. An attempt was made to block the adhesion, spreading and migration events using antibodies directed against components of the extracts. Clearly, if endothelial-derived molecules are involved in metastasis, then preventing the mediation of adhesion, spreading and migration may ultimately have relevance in the clinical situation.

The investigation into the mechanisms by which compounds such as butyrate induce differentiation in HT-29 cells, is lacking. The colonic carcinoma cell line, HT-29, undergoes differentiation induction in the presence of butyrate and acetoacetate. The Caco-2 cell line spontaneously differentiates on contact inhibition. In this study, a signal transduction pathway involving ATP, cAMP, Ca2+ and the transcriptional factor CREB was investigated following suggestions that the energy state of the cell and diffferentiation are linked. The activity of the MAP kinase cascade, including possible crosstalk that may exist between these pathways was determined. The HT-29 cells were exposed to 5 mM acetoacetate, butyrate, DMSO and propionate. The results of this differentiation induction were compared to Caco-2 and HeLa cells, which are cervical carcinoma cells. It was found that ATP levels are decreased on differentiation induction in HT-29 cells, which, in turn affected the cAMP concentrations. Theoretically, the inducers do not have any effect on PDE 4 activity, and may facilitate the interaction between cAMP and PKA. Influx of Ca2+ into the cells was inhibited to a degree by the inducers, which was possibly overcome by crosstalk between the cAMP and Ca2+ pathways. CREB activation, lineage-specific gene expression, ERK activity and c-myc expression were all dependent on both the inducers used and the cell-type. PKA played a major role in CREB activation in acetoacetate- and butyrate -induced HT-29, Caco-2 and HeLa cells, while a2+/Calmodulin-dependent kinases I/IV may have a secondary role. Alkaline phosphatase expression in HeLa cells was independent of CREB. Evidence that crosstalk between the MAP kinase cascade and the REBactivation pathways exist, was illustrated by increased CREB activation on ERK inhibition in acetoacetate- and butyrate-induced HT-29 and HeLa cells. Also, the role that ERK played in the cells differed with inducer and cell-type. The dependence of cmyc expression on c-jun and c-fos, appeared to be differentiation induction- and celltype specific. Results from this study indicate the potential use of acetoacetate and butyrate as anti-cancer compounds.

The effects of stalk cell differentiation inducing factor (DIF) on stalk cell induction from vegetative cells and prestalk cells of Dictyostelium discoideum were investigated. It was found that the major DIF component DIF-1 is a poor inducer of stalk cell formation from prestalk cells, but a minor component, DIF-2 is a more important inducer for the conversion of prestalk to stalk cells. Evidence is presented that DIF-2 synergizes the activity of the other DIF components for prestalk to stalk cell conversion. In contrast DIF-5 inhibits the activity of DIF-1 and DIF-3/4. A model is proposed to explain those results. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Cell membranes are made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as neuronal differentiation, cell membranes undergo dramatic structural changes induced by proteins such as ARC and Cofilin among others in the case of synaptic modification. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. As expected, NIH3T3 cells have more rigid membrane at earlier stages of their development. On the other hand neurons tend to have the highest membrane fluidity early in their development emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines.