This dissertation mainly discussed the investigation of five different soft corals collected in Green Island and one soft coral collected in Kenting. They were identified as Clavularia viridis, Xenia florida, Cespitularia taeniata, Cespitularia hypotentaculata, and Sarcophyton stolidotum. In the isolation of these corals, there were sixty natural products discovered, including twenty night new compounds and three new derivatives. The research of soft coral Clavularia viridis has result in the isolation of six new prostanoids, designated as 4-deacetoxyl-12-O-deacetylclavulone I (1), 4-deacetoxyl-12-O-deacetylclavulone II (2), bromovulone II (3), iodovulone II (4), 4-deacetoxyl-12-O-deacetylclavuloneIII (5), and bromovulone III (6), together with seven known prostanoids, identified as clavulone I (33), clavulone II (34), chlorovulone II (35), 4-deacetoxylclavulone II (36), clavulone III (37), chlorovulone III (38), and 7-acetoxy-7,8-dihydroiodovulone I (39). In the investigation of the soft coral Xenia florida, three new compounds called florxenilides A-C (7-9), has been purified. The research also obtained seven xenicane-type diterpenes, xeniafaraunol A (40), xeniafaraunol B (41), florlide A (42), florlide C (43), florlide D (44), 9-deoxyxeniolide A (45), and 9-deoxyxeniolide B (46) in addition to two cadinene-type sesquiterpenes, xenitorins A (47) and B (48). Florxenilides A and C also treated with some chemical reagents to afford three new derivatives, 10-benzoylflorxenilide A (10), Florxenilide C monoacetate (11), and 10-dehydroflorxenilide A (12). Otherwise, the study in Cespitularia taeniata has afforded ten new nitrogen-containing compounds, cespitulactams D-K (13-20) and taenialactams A-B (21-22), in addition to four known compounds, atractylenolactam (49), cespitulactam A (50), cespitulactam B (51), and cespitularin F (52). Moreover, there were three new compounds, namely cespihypotins E, F, and H (23, 24, 25) have been discovered from another soft coral Cespitularia hypotentaculata. The study of this coral also observed seven known components called cespihypotin G (53), cespitulactone A (54), cespitularin F (52), cespitularin D (56), cespihypotins A-C (55, 57, 58). Investigation of the non-polar extract of the soft coral Sarcophyton stolidotum collected in Kenting resulted in the isolation of seven new 14-membered carbocyclic cembranes, sarcostolides A-G (26-32), together with two known cembranes isosarcophytoxide (59) and isosarcophine (60). All the structures of above metabolites were elucidated by physical and spectroscopic analyses including IR, Mass, UV, NMR, and optical rotation, and by compared with published data in many previous papers. The stereochemistry of these compounds as determined by NOESY experiments, CD spectroscopic data, and Mosher¡¦s methods. Cytotoxicity tests were measured by Dr. Kuo Yao-Haur and Dr. Guh Jih-Hwa. Isolated marine prostanoids showed potent activities against human prostate carcinoma (PC-3) and colon adenocarcinoma (HT-29) cells. Among them bromovulone III (6) had the most potent cytotoxicity against both cell lines at IC50 0.5

This dissertation illustrates an efficient purification method for citrus limonoids and flavonoids, while examining their effects on cytochrome P450 inhibition and apoptotic induction on human neuroblastoma (SH-SY5Y) and colonic adenocarcinoma (Caco-2) cells. The first study developed a bulk purification method for limonoids, from seeds and molasses of citrus fruits, using a combination of chromatographic techniques. This also resulted in an efficient purification method for naringin and hesperidin from citrus byproducts. The second study investigated the inhibitory effects of purified limonoids and flavonoids on the enzymatic activities of different isoforms of human cytochrome P450. O-Dealkylase and hydroxylase activities of CYP1A2, CYP1B1, CYP3A4 and CYP19, using specific substrates such as ethoxyresorufin (ethoxyresorufin O-dealkylase, EROD), methoxyresorufin (methoxyresorufin O-dealkylase, MROD), and dibenzylfluorescein (DBF), were found to be significantly (P < 0.001) reduced at micromolar levels. A kinetic analysis showed competitive and non-competitive modes of inhibition by limonoids, on CYP19 hydroxylase activity. The results corroborate the active role of limonoids in the redox cycling mechanisms. The third study examined the antioxidant and apoptotic inducing ability of limonoid glucosides on human neuroblastoma cells. Four limonoid glucosides, LG (17beta-D glucopyranoside limonin), OG (obacunone 17beta-D glucopyranoside), NAG (nomilinic acid 17beta-D glucopyranoside), and DNAG (deacetylnomilinic acid 17beta-D glucopyranoside), have shown superoxide scavenging at millimolar levels. Micromolar amounts of LG and OG induced rapid necrosis of SH-SY5Y cells. Cytotoxicity was correlated (P = 0.046) to a concentration and timedependent increase in caspase 3/7 activity. Analyses of DNA content during the S phase of the cell cycle indicated reductions of 86.6% for LG and 82.3% for OG as compared to untreated. The results validate the antineoplastic distinctiveness of limonoid glucosides. In the fourth study, cytotoxic effects of limonoid aglycones and glucosides were assessed on human SH-SY5Y neuroblastoma and colon carcinoma (CaCo-2) cell lines and compared with the non-cancerous Chinese hamster ovary (CHO) cells. Significant (P < 0.001) cytotoxic effects were observed only on cancerous cells, over 24 to 36 h. The study revealed a marked increase in the DNA content of aneuploidic cells, which results in cell cycle arrest. The results confirm that glycosides are the most active apoptotic inducing form.

Tumor necrosis factor's (TNF) cytotoxic mechanism of action was examined using cultured cancer cell lines. TNF demonstrated cytolytic and cytostatic effects on L929 fibrosarcoma and MCF-7 adenocarcinoma cells. TNF failed to show any specific effects on RNA, DNA or protein synthesis or ATP content in tumor cells in vitro. It did not cause DNA single strand breaks. Decreased cellular levels of reduced thiols did not predict sensitivity to the cytotoxic effects of TNF. Depletion of cellular glutathione failed to increase the sensitivity of TNF-sensitive or resistant cells. However, various non-specific and specific lysosomotropic agents lead to an inhibition of TNF's cytotoxic action. Differences in enzyme activity, primarily lysosomal, were noted between TNF-sensitive and resistant cells. These changes involved a general halving of lysosomal proteins and enzymes in the TNF-resistant cells. The antitumor activity of TNF does not involve specific inhibition of macromolecular synthesis but may involve alterations in lysosomes.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

No description available.

The Tim-3+ T cells in HIV infection are dysfunctional in proliferation or cytokine production. Here, we evaluated the effects of Tim-3 expression on the cytotoxicity of CD8+ T cells in HIV infection by examining 1) the ability of Tim-3+ CD8+ T cells to make perforin and 2) the direct ability of Tim-3+ CD8+ T cells to kill HIV infected CD4+ target cells. Tim-3+ CD8+ T cells maintained higher levels of perforin. However, these cells were defective in their ability to degranulate. Blocking the Tim-3 signalling pathway enhanced the cytotoxic capabilities of HIV specific CD8+ T cells by increasing: their degranulation capacity, their ability to release perforin, their ability to target activated granzyme B to HIV antigen expressing CD4+ T cells and their ability to suppress HIV infection of CD4+ T cells. Thus, the Tim-3 receptor can down-regulate the CD8+ T cell cytotoxicity through inhibition of degranulation and perforin/granzyme secretion.

The Tim-3+ T cells in HIV infection are dysfunctional in proliferation or cytokine production. Here, we evaluated the effects of Tim-3 expression on the cytotoxicity of CD8+ T cells in HIV infection by examining 1) the ability of Tim-3+ CD8+ T cells to make perforin and 2) the direct ability of Tim-3+ CD8+ T cells to kill HIV infected CD4+ target cells. Tim-3+ CD8+ T cells maintained higher levels of perforin. However, these cells were defective in their ability to degranulate. Blocking the Tim-3 signalling pathway enhanced the cytotoxic capabilities of HIV specific CD8+ T cells by increasing: their degranulation capacity, their ability to release perforin, their ability to target activated granzyme B to HIV antigen expressing CD4+ T cells and their ability to suppress HIV infection of CD4+ T cells. Thus, the Tim-3 receptor can down-regulate the CD8+ T cell cytotoxicity through inhibition of degranulation and perforin/granzyme secretion.

Knee injuries are a major cause of orthopedic disabilities in the United States. Current reconstruction techniques for torn anterior cruciate ligaments (ACL) require extensive surgery and long physical rehabilitation times since the tissue does not heal upon injury. A common ACL injury occurs where the gap at the rupture site remains open after injury and fails to heal, which can lead to premature osteoarthritis and disability. Hydrogels are a popular material used for tissue engineering applications due to their ability to retain water and good biocompatibility. Previous work has shown that hydrogels can be made through the mixed-mode reaction of radically crosslinked thiol groups and acrylate end groups. This project explores mixed-mode oligo[poly(ethylene glycol) fumarate] (OPF)-based hydrogels as alternate carriers for regeneration of partial tear ligament defects. The main purpose of this project was to determine the degradative properties of and cell response to thiol-PEG-thiol (PEG-diSH), a novel hydrogel material. The swelling and degradative properties of hydrogels containing three components OPF, PEG-diacrylate (PEG-DA), and PEG-diSH were characterized by their fold swelling. In addition, cell viability, morphology changes, proliferation and collagen production were analyzed in tri-ratio hydrogels with and without the presence of RGD over three weeks. Results showed that the hydrogels containing PEG-diSH demonstrated significantly larger fold swelling and promoted cell clustering (as shown by increased area of clusters), probably due to the larger mesh size and possibly due to the presence of free thiol functional groups present in the network from the mixed-mode reaction. However, an increase in cell number was not found in these gels up to eight days, suggesting that cell migration may play a role in the appearance of clusters. Additionally, increased cell spreading in response to RGD was observed inside gels containing PEG-diSH; no spreading was seen in the non PEG-diSH gels (± RGD), possibly because the mesh size was too small to allow for clustering or spreading within the matrix. Results from this work suggest that the presence of PEG-diSH could promote cell-cell contact within the clusters which could be useful in systems where direct contact promotes tissue formation or cell differentiation.

The anticancer properties of dirhodium tetraacetate were discovered in the 1970's, and subsequently motivated the research of several dirhodium paddlewheel derivatives. The promising results of this research led the Dunbar group to investigate the biological properties of dirhodium partial paddlewheel compounds. Previous work in our group has focused on dirhodium carboxylate derivatives with a series of diimine ligands, namely 1,10-phenanthroline (phen), dipyrido[3,2-f:2',3'-h]quinoxaline (dpq), dipyrido[3,2a:2',3'c] phenazine (dppz), and benzo[i]dipyrido[3,2-a:2',3'-c]phenazine) (dppn). Current research has expanded this diimine series by substituting the carobxylate bridging group with p-methoxyphenylphosphine (PMP). This new series of compounds was characterized by several techniques, including: X-Ray crystallography, 1H NMR spectroscopy, and electronic absorption spectroscopy. The cytotoxicity of these compounds towards HeLa cells was investigated in presence and absence of light in an effort to investigate the ability to use these compounds as photodynamic therapy (PDT) agents. Cytotoxicity measurements were carried out using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. It was found that in the dark [Rh2(PMP)2(dppz)2][BF4]2 (the dppz derivative of the dirhodium PMP compound) had no cytotoxicity towards HeLa cells, but experienced a 7 fold increase in cytotoxicity upon irradiation (with lambdai_rr equal to 350 nm). This dramatic increase in cytotoxicity upon irradiation makes this compound a potential PDT agent. Diiridium (II,II) compounds were prepared in a dual attempt to determine how the properties of the dirhodium core effect the biological activities of these compounds, as well as investigate the biological activity of a set of compounds that has yet to be explored. The compound [Ir2(DTolF)2(CH3CN)6][BF4]2 was chosen because it has a well understood dirhodium analogue, and it is a known compound. However, it was discovered that there was a potential silver contamination in the final product, stemming from the silver trifluoroacetate oxidant used during synthesis. Consequently, a new method of preparing this compound was required. The new synthetic pathway for the diiridium compound [Ir2(DTolF)2(CH3CN)6][BF4]2 was devised, and the cytotoxicity and photocytotoxicity studies were performed for the first time (to our knowledge) on a diiridium (II,II) compound. Despite the stability of the compound, it was determined to be highly toxic, both in the dark and upon irradiation.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1994. / Includes bibliographical references (leaves 111-122). / Microfiche. / x, 122 leaves, bound ill. 29 cm