Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (M.S.)--California Polytechnic State University, 2009. / Mode of access: Internet. Title from PDF title page; viewed on November 13, 2009. Major professor: Susan Hawk. "Presented to the faculty of California Polytechnic State University, San Luis Obispo." "In partial fulfillment of the requirements for the degree [of] Master of Science in Agriculture, with specialization in Food Science and Nutrition." "October, 2009." Includes bibliographical references (p. 40-46).

Abstract All-trans retinoic acid (ATRA) is a derivative of vitamin A. It is able to stimulate neutrophilic differentiation of normal progenitors and acute promyelocytic leukemia (APL) cells. Although ATRA-induced differentiation is not observed in any other acute myeloblastic leukemia (AML) subtypes, ATRA is known to be able to inhibit AML blast cell proliferation. The present in vitro study using AML cell lines representing subtypes other than APL focuses on the following questions: (1) Is the inhibitory effect of ATRA on AML cell growth related to apoptosis of cells? (2) Are the effects of ATRA dependent on two important regulators of apoptosis, p53 and Bcl-2? (3) Do mitochondria have any role in mediating the effects of ATRA? ATRA-induced apoptosis in AML cells was observed by morphology, DNA fragmentation, phosphatidylserine externalization, and poly(ADPribose)polymerase (PARP) cleavage. It was a slow event, manifested as DNA cleavage after 48 hours exposure and as morphological apoptosis after 72 hours exposure. The AML cells expressed constitutively p53 as determined by immunohistochemistry, Western blotting and flow cytometry. However, no mutation of TP53 was observed in exons 5 to 8 as analysed with a single strand conformation polymorphism technique. As the flow cytometer analysis showed, most of p53 was in a aberrant conformation, which was not changed into a wild type conformation by ATRA. Two of the cell lines were analysed more specifically in relation to Bcl-2 and mitochondral function: ATRA-induced apoptosis of the cell lines was associated with down-regulation of Bcl-2. Western blotting showed ATRA-induced apoptosis also to be related to the release of cytochrome c from mitochondria into cytosol, resulting in the activation of caspase-3, an apoptotic effector, which was manifested as a cleavage of its substrate PARP. The process was also accompanied by disruption of the mitochondrial membrane potential as determined fluoricytometrically. These results show that ATRA is able to induce apoptosis in AML cells other than APL, and ATRA-induced apoptosis in the AML cells studied is related to the down-regulation of Bcl-2 and the disruption of mitochondrial function, but is independent of the p53 pathway.

Bladder cancer is currently the 6th most common cancer in the United States, resulting in 17,000 deaths annually. Clinically, bladder cancers are mostly urothelial carcinoma, classified as either non-muscle invasive bladder cancer (NMIBC) or muscle-invasive bladder cancer (MIBC), with the latter having a 5-year survival rate of merely 50%. With recent advances in next-generation sequencing, several international consortia have elucidated molecular subtypes of MIBC. The two major subtypes of MIBCs are basal and luminal; the basal subtype frequently exhibits hallmarks of squamous differentiation and highly expressed basal markers (CD44, KRT14, KRT6A, KRT6B). Tumors of the luminal subtype have papillary morphology and highly express differentiation-associated luminal markers (e.g., KRT20, PPARG, UPKs, and FOXA1). Notably, the transcription factor Peroxisome Proliferator Activated Receptor Gamma (PPARG) gene is frequently amplified in luminal MIBC. And recurrent activating mutations have been reported for its obligatory functional partner Retinoic X Receptor (RXR). In addition, the basal subtype is immune-infiltrated and is postulated as more likely to respond to immunotherapies. In contrast, the luminal subtype is immune-cold. Despite these advances in recent years, the molecular driver of subtype determination, specifically in luminal MIBC, remains poorly understood. Furthermore, subtype-specific targeted therapy for MIBCs is still in its infancy. Our previous work determined that Pparg activation can drive luminal tumor formation. We generated a novel Krt5CreERT2; VP16;Pparg transgenic mouse model, where Pparg expression is constitutively active in basal urothelial cells upon tamoxifen induction. During homeostasis, constitutive Pparg promoted luminal differentiation and cell cycle exit in basal cells but did not produce tumors. However, increased Pparg signaling in activated basal cells following 1-month exposure to bladder-specific carcinogen BBN produced luminal tumors. These tumors are similar both in morphology and molecular markers to human luminal MIBCs. The resulting VP16;Pparg luminal tumors have reduced Nf-kb expression and are immune cold compared to basal tumors. These findings suggest that Pparg is a driver of luminal tumor formation and a suppressor of immune infiltration in bladder cancer. In Chapter 2 of the thesis, I focus on the therapeutic potential of activating Pparg in basal MIBC. We treated mice bearing BBN-induced, Pparg-negative basal tumors with synthetic Pparg ligand - Rosiglitazone (Rosi) and Mek1/2 inhibitor Trametinib (Tram), both of which have been shown to induce Pparg signaling in vitro and in vivo. The combined RosiTram treatment induced apoptosis and significantly reduced tumor burden. The post-treatment urothelium appeared similar in morphology to a healthy urothelium. RosiTram treatment also restored normal urothelial differentiation and generated resident cell types (e.g., superficial cells, intermediate cells, Keratin5+ (K5), basal cells, and Keratin14+ (K14) basal cells) that are normally seen in a healthy urothelium. In contrast, basal tumors are almost entirely composed of K14-Basal cells. Mechanistically, RosiTram treatment partially restores differentiation through retinoic acid signaling and Ezh2 inhibition. Together, our study established targeted transcriptionally and epigenetically reprogramming as a promising differentiation therapeutic approach for basal bladder tumors.

To investigate the changes in glycolipid biosynthesis during early embryogenesis mouse F9 teratocarcinoma cells were induced to differentiate in vitro in the presence of retinoic acid. Control embryonal carcinoma cells and their differentiated derivatives, RNF9 cells, were metabolically-radiolabeled with [6-3H]galactose or [6-3H]glucosamine, and their glycolipids were compared. The neutral and acidic glycolipid fractions from both cell lines were subjected to ozonolysis and alkali fragmentation or endoglycoceramidase digestion to release the glycolipid-derived oligosaccharides. The neutral oligosaccharides were separated according to size by gel filtration and high performance liquid chromatography. These analyses indicated that differentiated F9 cells synthesized less high molecular weight oligosaccharides (containing more than 5 sugar residues) relative to controls. Serial lectin affinity chromatography on columns of immobilized Helix pomatia. Wisteria f1oribunda. Griffonia simplicifolia-I and Ricinus communis-' agglutinins followed by reduction and permethylation revealed that globoside (GaINAcβ1, 3Galα1, 4Galβ1, 4Glc) and lactose (Galβ1,4Glc) are the principal glycolipid-derived oligosaccharides synthesized by F9 and RNF9 cells. An increased biosynthesis of these components was observed in RNF9 cells relative to controls. These changes paralleled the reduced biosynthesis of Forssman pentasaccharide (GaiNAcα 1 ,3GaINAcβ1 ,3Galα1 ,4Galβ1 ,4Glc) reported previously. Normalization of the incorporation of 3H-monosaccharides in glycolipid-derived oJigosaccharides to the number of cells indicated a 2-6 fold increase in the incorporation of radioactive precursors in RAlF9 cells relative to F9 controls, suggesting that an enhancement in glycosphingolipid biosynthesis accompanies the differentiation of F9 cells. The monosialylganglioside-derived oligosaccharides obtained from F9 and RAlF9 cells were separated by anion exchange chromatography. A reduced biosynthesis of high molecular weight components was observed in RAlF9 cells when compared with undifferentiated F9. Lectin affinity chromatography on immobilized Maackia amurensis agglutinin followed by reduction and permethylation indicated a dramatic increase in the synthesis of GM1 (Galβ1,3Ga1NAcβ1,4[NeuAcα2,3] Galβ1,4Glc) and GM3 (NeuAcα2,3Galβ1,4Glc) in RAlF9 cells relative to controls. These changes were accompanied by a decrease in the synthesis of sialyltetrasaccharide a (NeuAcα2,3Galβ1 ,3GlcNAcβ1 ,3Galβ1,4Glc) and sialylparagloboside (NeuAcα2,3Galβ1 ,4GlcNAβ1 ,3Galβ1 ,4Glc) in the differentiated cells. These observations are in agreement with previous reports in leukemic and human embryonal carcinoma cell lines and may be related to the growth arrest and antigenic changes associated with F9 differentiation. In the work reported herein, serial lectin affinity chromatography in concert with permethylation analysis prove to be powerful methods for the isolation and characterization of glycolipid-derived oligosaccharides. The application of these methods has allowed the unequivocal identification of main glycosphingolipid components as well as of some representing less than 1 % of the total glycolipids synthesized by two cell lines. This information should provide the basis for further studies involving glycosyltransferas. / Master of Science

Lee, Man Yuen. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 191-215). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 4 uppsatser.

by Tse Kam Wah. / "September 1998." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (p. 123-145). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

The splotch (sp) mutation has been identified as a mutation in the paired box gene, Pax-3. Heterozygous mice carrying this mutation are phenotypically normal, with the exception of a white spot on their bellies. Homozygous embryos do not live to birth, and suffer from a wide range of developmental defects, all of which result from delayed neural tube closure, or inadequate neural crest cell migration. Most notably, homozygotes have an increased rate of spina bifida with or without exencephaly. Retinoic acid (RA), which has been shown to be very important in the development of a number of systems, was shown to cause a selective mortality of the homozygous splotch embryos when administered maternally at day 9 p.c. (Moase and Trasler, 1987). Since cellular retinoic acid binding protein (CRABP) is localized to the tissues which are affected by both the splotch gene, and retinoic acid teratogenesis, its expression patterns in the developing splotch embryo were examined. It was found that the distribution of CRABP mRNA is normal, but its expression levels are excessive in splotch homozygous day 9 mouse embryos.

A two by six factorial design was used to investigate the effect of zinc and all-trans retinoic acid (RA) on the growth of two human breast cancer cell lines differing in their expression of metallothionein (MT) and of estrogen receptors; MCF7 cells express estrogen receptors, BT-20 cells do not. Cells were treated with zinc to induce MT then treated with six concentrations of RA. Cell proliferation, lipid peroxidation, MT protein, MT mRNA and glutathione concentrations were measured. / BT-20 cells expressed higher constitutive MT concentrations than MCF7 cells. MT was significantly induced by zinc treatment in BT-20 cells but not in MCF7 cells. Low RA concentrations stimulated growth proliferation but higher concentrations inhibited cell proliferation. High RA concentrations increased lipid peroxidation. There was a significant negative correlation between lipid peroxidation and cell proliferation. Growth inhibition and lipid peroxidation were reduced by zinc in BT-20 cells but not in MCF7 cells. Glutathione did not appear to be a significant factor. / Induction of metallothionein by zinc may modulate the growth inhibitory effects of all-trans retinoic acid in human breast cancer cells. One mechanism of growth inhibition may be through increased lipid peroxidation.