Epidemiologic data suggest that soy consumption may protect against cancer induction in several tissues in humans, including urothelial carcinoma. Genistein have been reported to regulate genes that are involved in several cellular events. However, the molecular mechanism of genistein -induced upregulation of epithelial membrane protein 2 (EMP2), candidate urothelial tumor suppressor, is not entirely understood. At first, we found that the mRNA and protein expression levels of EMP2 were significantly greater in the normal urothelial tissues and human urothelial cells than those in urothelial bladder carcinoma tissues and urothelial cell carcinoma-derived cell lines. Second, EMP2 knockdown via RNA interference markedly enhanced cell proliferation, colony formation, migration and invasiveness. By contrast, EMP2 overexpression suppressed these malignant behaviors. Third, we showed that genistein-induced inhibition in cell proliferation is associated with an increase in EMP2 expression. Using various deleted EMP2 promoter constructs, we defined that the EMP2 core promoter is enough to observe the genistein-induced upregulation of EMP2 transcriptional activity. Using site direct mutagenesis and chromatin immunoprecipitation assays demonstrated that cyclic-AMP response element binding protein 1 (CREB1) acts as a positive regulator of EMP2 transcription by directly binding to its promoter. These results showed EMP2 suppressed urothelial cell carcinoma-derived cell growth, motility and invasion and for the first time that genistein promoted EMP2 expression in urothelial cell carcinoma-derived cells by inducing EMP2 transcriptional activity via CREB1 binding.

Expressed sequence tags (ESTs) are derived from the developing tilapia brain was established in our lab. There are 9 transcripts were identified as thermosensitive/Neural development-related gene. The effects of different temperatures on the ontogenetic expression of these thermosensitive/Neural development-related gene during the critical period of brain sexual differentiation were investigated in the present study. The ontogenetic expression of inhibitor of DNA binding/differentiation protein 2 (Id2), thermosensitive/Neural development-related gene, were enhanced by both lower (20¢J) and higher (32¢J) temperatures before 10 days post-hatching. In this study, bioinformatics were searched for Id2, which is a gene with 738 bp of patial cDNA sequence, open reading frame (ORF) is 411bp, and deduced 137 amino acids of protein sequence. The protein of Id2 was expressed in a prokaryotic system, BL21 (Escherichia coli) and purified with Ni-NTA affinity chromatography. Also, the ORF of Id2 was cloned into pEGFP vector, and plasmid (pEGFP-Id2) was transfected into the eukaryotic system, mouse neuroblastoma cell (Neuro-2a cell). The distribution of Id2 expressed in the Neuro-2a cell was identified by fluorescence microscopy.

Cancer immunotherapy seeks to boost the host’s immune system to respond to tumor antigens. The adaptive immune system comprises of two arms, one that elicits a cellular immune response and one that elicits a humoral immune response. Cytotoxic T lymphocytes (CTL) recognize short antigenic peptides presented to them in the context of class I major histocompatibility complex (MHC) molecules and are capable of killing tumor cells. CTL are educated to discriminate between foreign and self-antigen. Tumors frequently express self-antigen which usually makes them poorly immunogenic. Because tumors are genetically unstable, they may present excess self peptides and/or peptides in a reading frame different from wild type self proteins. These frameshift (FS) peptides, are caused by an insertion or deletion of nucleotides that disrupt translation of the normal reading frame and alters the protein produced such that it is non-self. Binding affinity, dissociation rate and the overall stability of the peptide/MHC/β₂-microglobulin complex are important considerations in determining the immunogenicity of a given peptide. Interaction between the anchor residues in a peptide and binding pockets in MHC are essential, but this interaction is not always strong enough to stimulate T cell responses. This indicates that not all amino acids of the peptide ligand bound to MHC are equally important for the functional outcome of the receptor engagement and that other amino acid residues in the sequence are important for binding. Optimized peptide ligands (OPL) are analogues derived from natural wild type antigenic peptides that contain amino acid substitutions at anchor and auxiliary residues. OPL can be rationally designed to generate a more robust immune response compared to that of the wild-type peptide. Active immunotherapy using OPL of tumor antigen epitopes are designed to elicit tumor-specific CTL that can overcome tolerance and either re-awaken or elicit new T-cell responses to an antigen. The work and principles presented here using brain tumor-derived peptides demonstrates that HLA-A*0201-restricted CTL generated against wild type, frameshift and OPL peptides elicit CTL that were able to recognize and respond to wild type, tumorderived peptides. The response was donor dependent in that not all individuals responded more strongly to OPL; a minority responded better to wild type peptide. This data further suggests that the rational design and testing of multiple peptides for the same epitope should elicit a broader response among different individuals than single peptide immunization.

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Repair of bone defects is a major challenge in orthopaedic surgery. Current bone graft treatments, including autografts, allografts and xenografts, have many limitations making it necessary to develop a biomaterial to be a bone graft substitute. One such biomaterial is bioactive resorbable silica-calcium phosphate nanocomposite (SCPC). SCPC was processed using a 3D rapid prototyping technique and sintered at different temperatures to create porous scaffolds. SEM analyses and mercury intrusion porosimetry showed SCPC to be highly porous with micro- and nanopores. BET analysis indicated that SCPC had high surface area. Mechanical testing demonstrated that SCPC had a compressive strength similar to trabecular bone. Analysis of different thermal treatment temperatures indicated as the temperature was increased, the porosity decreased and the mechanical strength increased. When loaded with rhBMP-2 (SCPC-rhBMP-2), SCPC provided a sustained release profile of rhBMP-2 for 14 days. This was shown to be a greater release than hydroxyapatite (HA)-rhBMP-2. After immersion in SBF, ICP analyses showed the calcium concentration of SBF dropped drastically after one day of immersion. In conjunction, FTIR showed the formation of a hydroxyapatite layer on the SCPC surface and was confirmed by SEM. SCPC thermally treated at 850 ??C demonstrated the greatest dissolution/precipitation reactions when immersed in SBF. Processing the SCPC-rhBMP-2 hybrid using a rapid prototyping technique allowed for an exact replica of the rabbit ulna to be fabricated. This was implanted into a 10 mm segmental defect in the rabbit ulna. CT scans during the healing of the defect showed intimate union between SCPC-rhBMP-2 and the bone and about 65% healing of the defect after 4 weeks. Rabbits were euthanized after 12 and 16 weeks. Digital images show almost complete healing of the defect after 16 weeks. Torsional testing of the ulna after 12 weeks demonstrated restoration of maximum torque and angle at failure. Histological evaluation after 12 weeks showed the regenerated bone has all the morphological characteristics of mature bone. Through in-vitro and in-vivo testing, it can be recommended that the porous bioactive SCPC can serve as a successful delivery system for biological growth factors and serve as an alternative to autologous bone grafting.

Thesis (Ph. D.)--Cornell University, August, 2008. / Vita. Includes bibliographical references (leaves 194-227).

Thesis (Ph. D.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains v, 42 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Demineralized bone matrix (DBM) is an allograft material used in orthopedics that promotes endochondral bone formation. While the placement of DBM on either the periosteal surface of a bone or within a skeletal muscle promotes the recruitment of stem cells that can form skeletal tissues through the temporal progression of endochondral bone development, it remains unclear to what degree these processes are different between the two sites. In this study, we utilize a comparative in vivo model of endochondral ossification by implanting the DBM on the periosteum and in the muscle. Within the muscle we further compared the effects of DBM with and without Bone morphogenetic protein-2 (BMP-2), a primary morphogenetic factor involved in the differentiation of skeletal stem cells. The mice were harvested at various time points after DBM implantation in order to analyze the development of the bone. Analysis included X-ray imaging, microCT imaging, and mRNA expression. Plain x-ray and micro-CT imaging analysis showed mineralized bone formation in the implant on the periosteum and in the muscle with BMP-2, but no growth in the muscle when BMP-2 was not added to the DBM. The mechanisms for bone development were further analyzed by qRT-PCR to determine temporal patterns and levels of expression of various stem cell and differentiated skeletal cell associated genes. The stem cell gene expression varied between implant placement locations suggesting different mechanisms for stem cell recruitment. Interesting, while DBM implants in the muscle without BMP did not induce mineralized tissue specific mRNA expression; specific stem cell and early skeletal cell lineage commitment genes were present. These results suggest that while DBM in muscle is capable of recruiting stem cells that higher BMP-2 levels are needed to promote the progression of cartilage to mineralized bone in muscle tissues.