The process of ubiquitination plays an essential role in numerous cell functions, including apoptosis and the induction of immune responses. Ariadne 2 is a RING finger E3 ligase and is part of the highly conserved RBR (RING-B-Box-RING) superfamily, however, little is known of its function in mammalian systems. To further examine the physiological role, Ariadne 2 deficient mice were generated. In a mixed background, Ariadne 2 deficient (Arih2-/-) mice die prematurely after birth however lethality is not fully penetrant. Adult mice that escape lethality have lower body weight and reduced viability due to an apparent lymphoproliferative disorder. In a C57BL/6 background, Ariadne 2 deficiency leads to a fully penetrate embryonic lethality, occurring after embryonic day 16.5. Arih2-/- foetal liver have reduced cellularity and increased apoptosis, however haematopoietic cells are capable of differentiating into myeloid and granulocytic progenitors and can fully reconstitute lethally irradiated Rag1-/- recipient mice. These Rag1-/-Arih2-/- chimeras recapitulate the lymphoproliferative disorder observed in the mixed background Arih2-/- mice. Further analysis show Rag1-/-Arih2-/- chimeras display increased number of lymphocytes, granulocytes, macrophages and dendritic cells, increased serum immunoglobulin levels and pro-inflammatory cytokines, and dramatic heterogeneous cellular organ infiltration, consisting mainly of T cells. T cell homeostasis is also altered, as seen by increased activated and ‘memory-like’ T cells, elevated TH1 and TH2 cytokines, increased regulatory T cells (Treg), and increased T cell proliferation. This may be due to an observed premature maturation of Arih2-/- dendritic cells. Arih2-/- foetal liver derived dendritic cells (FLDC) express high levels of maturation markers CD80/B7.1, CD86/B7.2, CD83, CD40 and MHCII and are capable of activating T cells in the RIP-GP model of induced diabetes. This may be linked to modulation of the NFκB and ERK pathways, in particular increase in nuclear p65/RelA and phospho-p65/RelA leading to an increase in NFκB and AP-1 binding to DNA and sustained and hyperactive NFκB response in Arih2-/- dendritic cells. Overall, Ariadne 2 is shown to be a negative regulator in the activation of immune cells, in particular dendritic cells, and is a novel regulator in the maintenance of peripheral tolerance and the pathogenesis of autoimmunity.

The process of ubiquitination plays an essential role in numerous cell functions, including apoptosis and the induction of immune responses. Ariadne 2 is a RING finger E3 ligase and is part of the highly conserved RBR (RING-B-Box-RING) superfamily, however, little is known of its function in mammalian systems. To further examine the physiological role, Ariadne 2 deficient mice were generated. In a mixed background, Ariadne 2 deficient (Arih2-/-) mice die prematurely after birth however lethality is not fully penetrant. Adult mice that escape lethality have lower body weight and reduced viability due to an apparent lymphoproliferative disorder. In a C57BL/6 background, Ariadne 2 deficiency leads to a fully penetrate embryonic lethality, occurring after embryonic day 16.5. Arih2-/- foetal liver have reduced cellularity and increased apoptosis, however haematopoietic cells are capable of differentiating into myeloid and granulocytic progenitors and can fully reconstitute lethally irradiated Rag1-/- recipient mice. These Rag1-/-Arih2-/- chimeras recapitulate the lymphoproliferative disorder observed in the mixed background Arih2-/- mice. Further analysis show Rag1-/-Arih2-/- chimeras display increased number of lymphocytes, granulocytes, macrophages and dendritic cells, increased serum immunoglobulin levels and pro-inflammatory cytokines, and dramatic heterogeneous cellular organ infiltration, consisting mainly of T cells. T cell homeostasis is also altered, as seen by increased activated and ‘memory-like’ T cells, elevated TH1 and TH2 cytokines, increased regulatory T cells (Treg), and increased T cell proliferation. This may be due to an observed premature maturation of Arih2-/- dendritic cells. Arih2-/- foetal liver derived dendritic cells (FLDC) express high levels of maturation markers CD80/B7.1, CD86/B7.2, CD83, CD40 and MHCII and are capable of activating T cells in the RIP-GP model of induced diabetes. This may be linked to modulation of the NFκB and ERK pathways, in particular increase in nuclear p65/RelA and phospho-p65/RelA leading to an increase in NFκB and AP-1 binding to DNA and sustained and hyperactive NFκB response in Arih2-/- dendritic cells. Overall, Ariadne 2 is shown to be a negative regulator in the activation of immune cells, in particular dendritic cells, and is a novel regulator in the maintenance of peripheral tolerance and the pathogenesis of autoimmunity.

The tyrosine kinase receptor Tie-2 is expressed on the surface of endothelial cells and is necessary for angiogenesis and vascular stability. To date, the best characterized ligands for Tie-2 are Angiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2). Ang-1 has been identified as the main activating ligand for Tie-2 while the role of Ang-2 has been controversial since its discovery; some studies reported Ang-2 as a Tie-2 antagonist while others described Ang-2 as a Tie-2 agonist. The purpose of this thesis was to understand: (1) how the receptor Tie-2 is regulated by Ang-1 and Ang-2 in endothelial cells, (2) to compare the effects of Ang-1 and Ang-2, and (3) to determine the arrangement and distribution of Tie-2 in endothelial cells. The research presented in this thesis indicates that Tie-2 is arranged in variably sized clusters on the endothelial cell surface. Clusters of Tie-2 were expressed on all surfaces of cells: on the apical plasma membrane, on the tips of microvilli, and on the basolateral plasma membrane. When endothelial cells were stimulated with Ang-1, Tie-2 was rapidly internalized and degraded. Upon Ang-1 stimulation, Tie-2 localized to clathrin-coated pits on all surfaces of endothelial cells indicating that one pathway mediating Tie-2 internalization is through clathrin-coated pits. After activation of Tie-2, Ang-1 dissociates from the endothelial cell surface and accumulates in the surrounding medium. When experiments were repeated with Ang-2, it was discovered that Ang-2 induced all of the same effects on Tie-2 as Ang-1 but at a much reduced level and rate, indicating that Ang-2 likely functions as a partial agonist for Tie-2 in endothelial cells. / PhD

The metastatic spread of tumor cells is the major lethal aspect of cancer, and lymphatic metastasis is one of the most important routes. Recent studies indicated that cyclooxygenase-2 (COX-2) expression is frequently associated with lymph node metastasis and over-expression of COX-2 can enhance lymphatic invasion of cancer cells. The interaction of chemokines and their cognate receptors also plays a critical role in cancer metastasis. Previous results of our laboratory demonstrated that CCR7 is a downstream target for COX-2 and COX-2 up-regulated CCR7 expression via the EP2 and EP4 receptor. We also found that protein kinase A (PKA) and AKT kinase are involved in COX-2-induced CCR7. In this study, we provided further evidences that COX-2 directly stimulates CCR7 expression via promoter activation. Promoter deletion and mutation assay indicated that COX-2 stimulated CCR7 promoter via the Sp1 binding site located at the -61/-52 bp region upstream of the transcription start site. Increase of Sp1 binding to CCR7 promoter by COX-2 was confirmed by chromatin immunoprecipitation (ChIP) assay. Furthermore, knockdown of Sp1 expression resulted in inhibition of PGE2-induced CCR7, and over-expression of Sp1 potently up-regulated CCR7 in MCF-7 cells. In vitro kinase assay indicated that AKT could directly phosphorylate Sp1 at S42, T679 and S698 sites. And the phosphorylation of Sp1 by AKT led to enhanced protein stability and DNA binding affinity of Sp1. The results of immunohistochemistry indicated that CCR7 expression was significantly associated with Sp1 and phosphor-AKT. Taken together, COX-2 may act via the EP receptor/PKA/AKT/Sp1 signaling pathway to stimulate CCR7 expression in breast cancer cells to promote lymphatic spread.

(¤@) Pyrolysis of 1,2-bis(3-methoxy-2-naphthyl)ethene (35) gave polycyclic aromatic hydrocarbons (PAH) 42¡B43¡B44 and 45. In addition, photolysis of 35 gave photocyclic products 50. (¤G) Photolysis of £\-azidotoluene (35) and 2-azido1-(2-furanyl)ethanone(45) gave dimer products benzylbenzene (51)¡B2-(furan-2-carbon-yl)-amino-1-(2-furyl)ethan-one (60) and 2-(2-formylfuranyl)-4-(2-furan-yl)-imidazole (48).

In this study, we collected 18 plasma samples from NPC patients, and 10 plasma samples from healthy person for plasma proteomic analysis. We classified these samples into 3 groups: treatment, pretreatment and recurrent. Two-dimensional electrophoresis (2-DE) and MALDI-TOF were performed followed by comparative and statistic analysis. In conclusions, we totally identified 30 proteins in Nasopharyngeal carcinoma (NPC) plasma, and found 10 proteins with expression level down regulated (p<0.001). These proteins were characterized to be Serotransferrin, Vitamin D-binding protein (VDB), alpha-1 antitrypsin (AAT), Haptoglobin, Apolipoprotein B fragment, Syntaxin-7, Apolipoprotein, A-I, PRO1779, Transthyrin, MDN1 protein, respectively. Consequently, we established a protocol to remove high abundant proteins (e.g., albumin, immunoglobin etc.) in plasma. We are especially interested in ATT and VDB. Western blotting assay was performed to confirm ATT and VDB expression. Furthermore, the quantity of ATT and VDB were measured by ELISA to obtain the threshold value of these proteins. Finally, we want to realize the relationship between these down regulation proteins and clinical parameters in NPC malignancy and tumor progression. Since there are few protein expression research of NPC in clinical studies, our works will provide insights in NPC studies for tumor progression with potential to elevate treatment efficiency.

The effectiveness of most chemotherapeutic regimens is limited by the toxicity of the therapy to normal healthy cells. Therapies to selectively modulate abnormal T cells bearing the interleukin 2 receptor (IL-2R) have been developed to treat diseases associated with aberrant immune response. This study describes the development and optimization of a targeted gene or oligonucleotide therapy vehicle to IL-2R bearing T cells for selective elimination of these cells. In this work, a monoclonal antibody to the IL-2R was used to target the oligonucleotide delivery vehicle which consisted of a polyamidoamine dendrimer. Optimization of the delivery vehicle involves understanding the factors that govern its association with oligonucleotide, the pathway of IL-2R endocytic trafficking, and the stability of the oligonucleotide in the biological milieu. Oligonucleotide stability in a cellular environment was examined intra- and extracellularly. Results showed that the rate of intracellular degradation of oligonucleotides was much greater than extracellular degradation. Binding of oligonucleotides to dendrimers was demonstrated as a function of dendrimer generation. The total binding capacities for dendrimers differed depending upon dendrimer size and surface group, whereas equilibrium binding affinity was comparable for all dendrimers tested. Binding of oligonucleotide delivery vehicle to the cell surface and subsequent internalization was inversely related to dendrimer size, and in all cases, significantly less than binding and internalization of the natural ligand for the IL-2R. Based on experimental results, a kinetic model of the delivery vehicle was derived which includedthe dependence of binding and internalization on dendrimer size and surface charge and intracellular degradation of oligonucleotide. Based on model predictions, we show that larger dendrimers carry more oligonucleotide than the smaller dendrimer vehicles, and delivery is more effective with larger vehicles. This work establishes our ability to predict the effects of different delivery vehicle properties on oligonucleotide delivery and aids in the development of design criteria for new vehicles for delivery of antisense, siRNA, or genes to IL-2R bearing cells.

Proefschrift--Universiteit Leiden, 2003. / Textes en anglais et en néerlandais. Bibliogr. en fin de chap.

Giessen, Universität, Thesis (doctoral), 2002.

München, Universität, Thesis (doctoral).