<p>The number one cause of death in the industrialized world, atherosclerosis, can be treated through a variety of methods: angioplasty, stenting, vein graft bypass, synthetic grafts, and maybe one day tissue engineering vessels (TEBVs). The long term goal that motivated this research is the delivery of umbilical cord blood derived endothelial progenitor cells (CB-EPCs) to damaged arteries and possibly reducing the rate of re-occlusion by re-establishing a healthy, functional, intact endothelium. The proposed research tested the following hypotheses: (1) Mild trypsinization methods produces strong endothelial cell (EC) adhesion strength, (2) CB-EPCs are functionally similar to native ECs (specifically human aortic endothelial cells (HAECs)) and exhibit similar anti-thrombotic and anti-inflammatory behavior compared to HAECs, (3) CB-EPCs are capable of adhering to smooth muscle cells (SMCs) and extracellular matrix (ECM) proteins under flow conditions, (4) CB-EPCs can be used to prevent thrombosis in mice that have undergone vein bypass grafts through re-endothelialization of the vessel, and (5) CB-EPCs are capable of proliferating under flow conditions. In order to produce supraphysiological adhesion strengths of HAECs or CB-EPCs, the cells must be detached using 0.025% trypsin for 5 minutes prior to adhesion to adsorbed ECM proteins or SMCs. CB-EPCs have a high proliferation rate and express similar levels of important anti-thrombotic genes and inflammatory proteins compared to HAECs. CB-EPCs and HAECs produce similar levels of nitric oxide and alignment in the direction of flow when exposed to laminar shear stress for at least 24 hours. CB-EPCs are capable of adhering to many different substrates under flow conditions. The adhesion of CB-EPCs with response to shear stress appears to be biphasic and increases with shear stress up to 0.75 dyn/cm2 and then decreases above this value. CB-EPC adhesion is much greater than HAECs and EPCs isolated from peripheral blood (PB-EPCs) of healthy individuals, which can be related to their higher expression level of adhesion integrin &#945;5&#946;1 and their smaller size. When seeded onto FN coated plastic, CB-EPCs proliferated under flow conditions and had a much shorter doubling time than PB-EPCs and HAECs. Proliferation of CB-EPCs and HAECs on SMCs was limited. Further, Cb-EPCs formed network-like structures except when growth factors were removed and a shear stress of at least 5 dyne/cm2 was applied. To assess whether CP-EPCs could promote vessel repair in vivo, human CB-EPCs were injected into SCID mice that received a carotid interpositional vein grafts, resulting in 100% patency. In contrast, only 2 of the 8 saline injected mice had a patent vein graft 2 weeks post surgery. We found that CB-EPC injected mice had roughly 55% endothelialization compared to less than 20% for the patent saline controls, with CB-EPCs making up approximately 33% of this coverage. These results suggest that CB-EPCs could be used as a therapeutic method to prevent vessel re-occlusion in patients undergoing treatment for atherosclerosis.</p> / Dissertation

Liver fibrosis is a wound healing process in liver with¡@chronic injury and is characterized by the excess production and accumulation of extracellular matrix (ECM) component. Liver injury of any etiology may lead to activation of hepatic stellate cells (HSCs), which are trans-differentiated from lipocyte-like cells to highly proliferative myofibroblast-like cells. Activation of HSCs is considered a crucial event that promotes increased ECM production and consequently hepatic fibrosis. Liver fibros is resulted from a net increased synthesis and decreased degradation of ECM proteins. Pluchea indica (Less) has been reported to have antipyretic, anti-ulcer, anti-inflammatory, anti-oxidant, diuretic and anti-amoebic activities. Our previous studies showed that the aqueous extract of roots from P. indica (PIRAE) showed that it can suppress the growth and migration of HeLa and GBM8401 cancer cell lines, and also significantly reduce serum glutamate pyruvate transaminase (GPT), alpha-smooth muscle actin (£\-SMA) and collagen type I expression in animal model of liver fibrosis induced by thioacetamide (TAA). In this study, we plan to investigate the effects of PIRAE on activation, proliferation and migration of rat culture activated HSCs. The results indicated that protein expression of £\-SMA and collagen type I of HSCs was decreased followed by treatment of either 0.5 or 1.0 mg/ml PIRAE for 48 hours. In addition, the effects of PIRAE on proliferation in culture activated HSCs were assessed by analyses of cell growth curve, MTT, WST-1 and BrdU, respectively. The results showed that PIRAE inhibited HSCs proliferation in a dose- and time-dependent manner. Moreover, wound healing assay and transwell assay showed that PIRAE prevented migration in activated HSCs. In conclusion, PIRAE may suppresse culture activated HSCs proliferation, migration, and activation of culture activated HSCs, as well as accumulation of collagen type I.

C1 inhibitor (C1 INH) is a member of the serine protease inhibitor (serpin) superfamily. It is the only physiological inhibitor of protease C1r and C1s in the complement system. C1 INH is a single chain glycoprotein with apparent molecular weight of 105 KDa, consisting of 478 amino acids. C1 INH N-terminal domain includes first 98 amino acids with 10 definite and 7 potential glycosylation site. Various of carbohydrates are present on the cell surface and component of ECM (extracellular matrix) in every eukaryotic cell, including both cancer cells and cells that are important for tumur survival. Carbohydrates on the cancer cell surface have been shown to be important in many aspects of cancer cell physiological processes, involved in cell growth and cell adhesion. Carbohydrates are also able to bind and interact with growth factors and other proteins that trigger signal transduction. Interfere carbohydrates maybe offer a useful therapeutic approach for treating cancers. In order to understand whether the C1 INH NT98 polypeptides can influences cancer or not, we amplified a DNA fragment encoding C1 INH N-terminal domain 98 residues (C1 INH NT98) by PCR, and transfer to the plasmid pGEX-2T, than use E.coli (BL21 strain) to express the non-glycosylated polypeptides, and further analyze the influence of the effective roles exhibited by the polypeptides non-glycosylated on breast cancer cell MDA-MB-435s. Proliferation and migration assays in our experiment showed that non-glycosylated C1 INH NT98 can inhibited breast cancer cell growth and migration, and the mechanism needed to be clarified clearly through extensive research.

The structure and function of brain shows sexual dimorphism in the vertebrates. Sexual differentiation is divided into brain sexual differentiation and gonad sexual differentiation. Brain sexual differentiation is resulted from the neural development. In the present study, the neurosphere cloned from tilapia, Oreochromis mossambicus, was used. The characteristics of neurosphere derived from both primary- and sub- culture, were studied. The effects of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF, FGF2), and temperature on the neurosphere cloned from both primary culture and subculture, were investigated. These results show that the neurospheres, cloned form both primary- and sub- culture, is consist of the nestin-immunoreactive cell. Furthermore, the cell of the neurosphere shows an ability of differentiation. And the diameter of neurosphere in the subculture is significantly larger than that of primary culture. On the other hand, both FGF and temperature have an effect to increase the diameter of neurosphere in the primary culture.

The comparison of nuclear facilities based on their barriers to nuclear material proliferation has remained a difficult endeavor, often requiring expert elicitation for each system under consideration. However, objectively comparing systems using a set of computable metrics to derive a single number representing a system is not, in essence, a nuclear nonproliferation specific problem and significant research has been performed for business models. For instance, Multi-Attribute Utility Analysis (MAUA) methods have been used previously to provide an objective insight of the barriers to proliferation. In this paper, the Proliferation Resistance Analysis and Evaluation Tool for Observed Risk (PRAETOR), a multi-tiered analysis tool based on the multiplicative MAUA method, is presented. It folds sixty three mostly independent metrics over three levels of detail to give an ultimate metric for nonproliferation performance comparison. In order to reduce analysts' bias, the weighting between the various metrics was obtained by surveying a total of thirty three nonproliferation specialists and nonspecialists from fields such as particle physics, international policy, and industrial engineering. The PRAETOR was used to evaluate the Fast Breeder Reactor Fuel Cycle (FBRFC). The results obtained using these weights are compared against a uniform weight approach. Results are presented for five nuclear material diversion scenarios: four examples include a diversion attempt on various components of a PUREX fast reactor cycle and one scenario involves theft from a PUREX facility in a LWR cycle. The FBRFC was evaluated with uranium-plutonium fuel and a second time using thorium-uranium fuel. These diversion scenarios were tested with both uniform and expert weights, with and without safeguards in place. The numerical results corroborate nonproliferation truths and provide insight regarding fast reactor facilities' proliferation resistance in relation to known standards.

Androgen has been shown to stimulate proliferation of osteoblast-like MC3T3-E1 cells. However, the molecular mechanism responsible for this effect remains to be elucidated. In the present study we demonstrate herein the non-genomic effect of androgen on osteoblast-like MC3T3-E1 cells involving activation of a PI(3)K/Akt signaling pathway and stimulating proliferation. In studies of steroids signaling, 5a-dihydrotestosterone (DHT), testosterone and 17b-estradiol but not dexamethasone or progesterone induced a rapid and transient phosphorylation of Akt in MC3T3-E1 cells. The androgen-induced Akt activation reached to the climax after 15 min and gradually diminished to baseline after 60 min. This induction of androgen was unaffected by actinomycin D and was specifically blocked by androgen receptor (AR) antagonist hydroxyflutamide (HF) or transfection of siRNA-AR. Treatment of MC3T3-E1 cells with PI(3)K inhibitor LY294002 or transfection with kinase-deficient Akt blocked androgen-induced cells proliferation. Moreover, androgen-induced activation of Akt was abolished by inhibitors of Src kinase, Gi-protein and phospholipase C showing the involvement of these effectors in androgen signaling pathway. Further, androgen-induced activation of Akt was dependent on intracellular calcium as shown by the effect of EGTA and intracellular calcium chelator BAPTA/AM. Fluorescence microscopy showed translocation of phospho-Akt from cytosol into nucleus after androgen treatment but no change in the subcellular distribution of phospho-Akt when HF or LY294002 pretreatment was administered to the cells. These results strongly suggest that phosphorylation of Akt in osteoblast cells is mediated by androgen receptor and the androgen-induced translocation of Akt is an important step in the androgen/AR signaling pathway that mediates osteoblast cells proliferation.

Immunoglobulin A¡]IgA¡^, the principal antibody class in secretions that bathe mucosal surfaces, acts as an important first line of defense. However, some pathogenic bacteria such as Haemophilus influenzae can produce IgA1 proteases to impair IgA1, especially in human mucosal immune system. IgA1 proteases are characterized by a polypeptide precursor containing four domains, the signal peptide, protease, linking region and the £]-domain. The function of the protease and the £]-domain (£] core) had been studied extensively, but the linking region is less defined, let alone its function. To complete the project, the DNA fragment for linking region was amplified by PCR from iga gene (Gene Bank DQ683353) spanning from NT3130 to NT4686, and then transferred to pGEX-2T for expression. Recombinant linking region-protein was purified using glutathione-Sepharose column. The proliferation assay showed that purified recombinant protein did not enhance cell growth significantly at the concentration of 1 £gg/ml compared to either the negative control or GST control; but when the concentration of the recombinant protein was increased to 5 £gg/ml or 10 £gg/ml, the cell proliferation was significantly stimulated. These results suggest that recombinant linking region-protein contains special element that stimulates the jurkat cell.

Aachen, Techn. Hochsch., Thesis (doctoral), 2001.

Thesis (doctoral)--Technische Hochschule, Aachen, 2000.

Neuroblastoma is a childhood solid tumor of a unique propensity to either regress spontaneously or grow relentlessly. Emerging evidence indicated that neuroblastoma contains heterogeneous populations of cells, and commitment of these cells to neuronal lineage may result in aggressive progression in patients, whereas to fibromuscular lineage may give a favorable outcome. However, mechanism(s) controlling the lineage commitment of neuroblastoma cells remains to be identified. Our preliminary data suggested that Kr?ppel-Like Factor 4 (KLF4) might promote neuroblastoma regression. KLF4 is a transcription factor regulating a variety of cellular functions, including proliferation and cell cycle progression. Recent studies have demonstrated that KLF4 may act as both tumor suppressor and oncogene in a cell-context dependent manner. Importantly, our preliminary data showed that low KLF4 expression is highly associated with poor clinical outcomes of the neuroblastoma patients. In addition, we found that overexpression of KLF4 suppresses neuroblastoma cell growth accompanied with loss of tumorigenicity. Morphologically, KLF4 overexpressing cells changed their morphologies to become epithelial-like, strongly substrate-adherent and expressing smooth muscle marker. Therefore, we hypothesized that KLF4 exerts its effects through two ways, it may (i) function to inhibit cell growth and reduce tumorigenicity; and (ii) promote differentiation of the neuroblastoma cells to the non-tumorigenic, fibromuscular-like cells. RT-PCR data revealed the differential expression of KLF4 in 11 neuroblastoma cell lines. In particular, a modest expression was found in Be(2)C, a cell line which was formerly demonstrated to differentiate and form tumor in mice xenograft assay. It was therefore chosen as the study model. To assess the effects of KLF4 knockdown on tumor growth, stable knockdown clones from Be(2)C cells were established by lentiviral transduction of KLF4-targeting shRNA. In parallel, clones that stably expressed non-target shRNA were used as controls. After the transduction, two stable knockdown clones showing significant KLF4 downregulation were isolated from single colony (monoclonal stable clones) and a pool of cells (polyclonal stable clones) respectively. The cell proliferation and growth rate of the stable clones were then measured by 5-bromo-2’-deoxyuridine (BrdU) proliferation assay and growth curve assay. The results have indicated that both monoclonal and polyclonal stable KLF4 knockdown clones grow faster than the control clones. In order to examine the tumorigenicity in vivo, the stable clones were xenotransplanted to severe combined immunodeficient mice. The stable KLF4 knockdown clones showed a significant higher growth rate and formed a larger tumor. The stable clones were also treated with BrdU for four weeks for differentiation towards fibromuscular lineage. As anticipated, the control clones showed fibromuscular features, like more flattened and epithelial-like morphology. In contrast, the stable KLF4 knockdown clones failed to present the fibromuscular features after treatment. In addition, immunocytochemistry staining of SMA and quantitative analysis of the immunocytochemistry further confirmed that only the control clones showed higher SMA expression after BrdU treatment, while there is no change in the SMA expression in the stable KLF4 knockdown clones. These results demonstrated that KLF4 functioned by inhibiting neuroblastoma cell proliferation and growth, reducing the tumorigenicity, and it was required for fibromuscular differentiation. / published_or_final_version / Surgery / Master / Master of Philosophy