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Small diameter vascular grafts comprised entirely from cells and cell-derived extracellular matrix (ECM) have shown promise in clinical trials and may have potential advantages as in vitro vascular tissue models. A challenge with current cell-derived tissue engineering approaches is the length of time required to generate strong, robust tissue. There is a lack of alternative methods to rapidly assemble cells into a 3D format without the support of a scaffold. Toward the goal of engineering a new approach to rapidly synthesizing vascular tissue constructs entirely from cells, we have developed and characterized a strategy for creating cell-derived tissue rings by cellular self-assembly. The focus of this thesis was to develop the system to rapidly generate engineered tissue rings, and to evaluate their structural and functional properties. To generate tissue rings, rat smooth muscle cells (SMCs) were seeded into round-bottomed, ring-shaped agarose wells with varying inner post diameters (2, 4, and 6 mm). Within 24 hours of seeding, cells aggregated, contracted, and formed robust tissue that could be removed from their wells and handled. If kept in culture, the thickness of these tissue rings increased with time. Mechanical analysis of the tissue showed that it was stronger after only 8 days in culture than engineered tissues generated by other approaches (such as seeding cells in biopolymer gels) cultured and tested at similar time points. Histological staining of the tissue rings revealed high cell densities throughout, along with the presence of glycosaminoglycans and some collagen. We also found that we could use the tissue rings as building blocks to generate larger tubular structures. Briefly, tissue rings were removed from the agarose wells and transferred onto silicone tubing mandrels. Once the rings were placed in contact with each other on the mandrel, they were cultured to allow the rings to fuse together. We found that the ability of tissue rings to fuse decreased with increasing ring “pre-culture� duration, and that we were able to generate fully fused tissue tubes in as little as 8 days (with only one day of ring pre-culture and seven days of fusion). In the last section of this thesis, we established the feasibility of using primary human SMCs to generate self-assembled tissue rings, similar to the self-assembled rings generated with rat SMCs. Compared to the rat SMC rings, human SMC rings were stronger, stiffer and appeared to contain increased levels of collagen. These data showed that human SMCs are capable of self-assembling into tissue rings similar to rat SMCs, and may therefore be used to create engineered human vascular tissue. Overall, we have developed a platform technology that can be used to screen the effects of culture parameters on the structure, mechanics, and function of vascular tissue. We anticipate that through the use of this technology, we can further improve vascular grafts by better understanding factors which promote ECM synthesis and SMC contraction. We can use these results directly toward the generation of vascular grafts by fusing self-assembled cell rings together to form tissue tubes. These novel bioengineered vascular tissues may also serve as a method to produce in vitro models to help further our understanding of vascular diseases, as well as facilitate pre-clinical screening of vascular tissue responses to pharmacologic therapies.

This thesis examines the costs and benefits of maternal allocation to both mother and offspring in gamebirds, specifically ring necked pheasants (Phasianus colchicus) and the Chinese painted quail (Coturnix chinensis). Maternal allocation of compounds from the mother to the young during early development can potentially have both positive and negative effects. For example the transfer of nutrients to the offspring can help to increase post-partum survival and subsequent life time reproductive success. In contrast, the transfer of stress hormones can potentially have long term negative effects on the offspring’s development. The first part of this thesis investigates the effects of the transfer of immune components from mother to offspring, which are known to have positive short term effects (protection against pathogens in early development). There is therefore considerable interest in whether this response can be induced by maternal vaccination to confer protection to young birds in commercial situations. However, it is unclear how this affects life-history trade offs in the mother and the development of the offspring post-hatching. These effects are especially important in the gamebird industry where birds are commercially reared on a large scale before being released into the wild. Chapter 2 therefore examines the costs and benefits to the mother of producing a maternally transmitted immune response to the CoxAbic vaccine, such as the impact of vaccination on egg laying characteristics and body condition. Chapter 3 looks at these costs and benefits to the chicks by examining growth rates and survival after a challenge of live coccidia in offspring from vaccinated and unvaccinated mothers. Chapter 4 examines the immune response to vaccination in adult pheasants. Finally, chapter 5 considers other factors that have been shown to affect the allocation decisions of females, specifically the effect of male characteristics on female reproductive effort.

Inhibitors of apoptosis (IAPs) modulate cell death and play critical role in signal transduction that promotes inflammation. Recently, Smac mimetics, which are IAP antagonists, have attracted attention as novel cancer therapeutics. Cellular Inhibitor of Apoptosis 2 (cIAP2), a member of IAP family, positively affects both NF-κB and MAPK activation in response to many inflammatory stimuli. We observed that the lack of cIAP2 ablates LPS-induced neuroinflammation. Also, cIAP2-/- macrophages demonstrated diminished antigen presentation potential that could contribute to ablated immunity. In addition to these functions, we have previously reported that cIAP2 also regulates the activation of Interferon Regulatory Factor 1 (IRF1). Since IRF1-/- mice are resistant to experimental autoimmune encephalomyelitis (EAE), we hypothesized that cIAP2-/- mice will be protected from the disease. Surprisingly, induction of EAE in cIAP2-/- mice resulted in exaggerated infiltration of immune cells increased expression of proinflammatory cytokines and demyelination within CNS. We found that the lack of cIAP2 induces caspase-8 expression in microglia derived macrophages, contributing to their activation and polarization towards M1 phenotype, and exacerbates the symptoms of EAE. These findings suggest that cIAP2 limits neuroinflammation in the CNS and thus the use of Smac mimetics as chemotherapeutics needs to be reevaluated.

<p>In this thesis we try to construct a stability condition on the quintic threefold. We have not succeeded in proving the existence of such a stability condition. However we have constructed a stability condition on a quotient category of projective space that approximates the quintic. We conjecture the existence of a stability condition on the quintic threefold generated by spherical objects and explore some consequences.</p> / Dissertation

Hepatocellular carcinoma (HCC) is the most common and devastating malignant tumor in Taiwan. The major factors involved in the molecular pathogenesis for the development of HCC have been explored in recent years. An extensive array of growth factors and their receptors have been identified and may act as positive and negative modulators in different stages of hepatocarcinogenesis. HDGF (hepatoma-derived growth factor) is a novel growth factor, identified from conditioned medium of hepatoma cell line. HDGF has growth stimulating activity for fibroblast and some hepatoma cells. The high homology of protein sequence to HMG (high mobility group) protein but with distinct structure indicate it is a novel growth factor with mitogenic effect. Recently, elevated HDGF expression was found in developing kidneys but little in adult kidney. Besides, HDGF expression was found to be correlated with angiogenic status of tissues. Thus, it is speculated that HDGF plays a role during embryonic development and angiogenesis. Besides, HDGF also plays a role in cell-to-cell interaction and cell movement. HDGF is a growth factor that is involved in stimulating vascular smooth muscle cells (SMCs) proliferation during development and in disease. HDGF contains a true bipartite nuclear localization sequence necessary for nuclear targeting. It is required for HDGF stimulation of DNA replication and cell proliferation in vascular smooth muscle cell. In present studies, have transfected HDGF cDNA to 3T3 cell and the HDGF expression was verified by Western blot analysis. HDGF expression altered the morphologies and growth rate of 3T3 cells by 2-fold. Besides, HDGF-expressing cells were more resistant to serum-starvation. Injection of 3T3-HDGF cells, but not 3T3 cells, resulted in tumor formation in nude mice, suggesting that the angiogenic and mitogenic functions of HDGF might contribute to carcinogenesis. By using various reagents including H2O2, dexamethasome, taxol, cisplatin, CoCl2 and KCN, the cellular stress studies revealed differential responses between in 3T3 and 3T3-HDGFH. analyzed dose and time-dependent effects of UV irradiation and found that 3T3-HDGF cells are more sensitive to UV irradiation than 3T3 cells and susceptible to apoptosis. hope these experiments will bring further insights into the cellular function of HDGF, particularly during angiogenic process, thereby enable to evaluate its role during HCC progression and its potential as clinical marker and therapeutic target for HCC.

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Investigations on platelet-derived growth factor (PDGF) and serotonin (5-HT), molecules stored in platelet granules, imply their potential effects in regulating megakaryopoiesis, which also intimates the existence of an autocrine and/or paracrine loop constructed by megakaryocytes/platelets and their granular constituents. In addition, numerous reports indicate that melatonin, a derivative from serotonin effectively enhances platelet counts in patients with thrombocytopenia. However, their exact roles on human megakaryocytes and the underlying mechanisms remain unknown. Present studies showed that PDGF, like thrombopoietin (TPO), significantly promoted platelet recovery and the formation of bone marrow colony-forming unit-megakaryocyte (CFU-MK) in an irradiated-mouse model. An increased number of hematopoietic stem/progenitor cells and a reduction of apoptosis were found in the bone marrow aspirate. In the M-07e apoptotic model, PDGF had a similar anti-apoptotic effect as TPO on megakaryocytes. Our findings demonstrated that PDGF activated the PI3-k/Akt signaling pathway, while addition of imatinib mesylate reduced p-Akt expression. Our findings suggested that the PDGF-initiated radioprotective effect is likely to be mediated via PDGF receptors (PDGFRs) with subsequent activation of the PI3-k/Akt pathway. We also provide a possible explanation that blockade of PDGFR may reduce thrombopoiesis and play a role in imatinib mesylate-induced thrombocytopenia. We explored how serotonin regulated megakaryopoiesis and proplatelet formation. Our results indicated that serotonin (5-HT) significantly promoted CFU-MK formation and reduced apoptosis on megakaryocytes through phosphorylation of Akt. These effects were attenuated by addition of ketanserin, a 5-HT2 receptor inhibitor. In addition, serotonin was able to stimulate the F-actin reorganization in megakaryocytes through activating the p-Erk1/2 expression. Bone marrow mesenchymal stromal cells (MSCs) are important in regulating megakaryopoiesis through stimulating the release of thrombopoietic growth factor, such as TPO. Our studies suggested that when activated by serotonin, bone marrow MSCs were induced to release significant amount of TPO. Furthermore, thousands of membrane-derived microparticles (MPs) arose from MSCs and the TPO RNA/proteins contained within MPs were also considerably increased under serotonin treatment. In summary, our findings demonstrated an important role serotonin played on megakaryopoiesis. This effect was likely mediated via 5HT2 receptors with subsequent activation of Akt and Erk 1/2 phosphorylation, which led to survival of megakaryocytes and proplatelet formation. Serotonin also stimulated TPO released from MSCs in both dissociative and MP-encapsulated form, which indirectly promoted megakaryopoiesis. The effects of melatonin on megakaryopoiesis were also determined in our studies. Our findings showed that melatonin enhanced proliferation and reduced doxorubicin-induced toxicity on MKs. We further demonstrated the mechanism for melatonin-mediated protection on MKs maybe via repair of G2/M phase cell cycle arrest and inhibition of cell apoptosis on MK cells. The effects of melatonin on megakaryopoiesis were also determined in our studies. Our findings showed that melatonin enhanced proliferation and reduced doxorubicin-induced toxicity on MKs. We further demonstrated the mechanism for melatonin-mediated protection on MKs maybe via repair of G2/M phase cell cycle arrest and inhibition of cell apoptosis on MK cells. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy