Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 148-171). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 148-171). Also available on the Internet.

Sigma receptors originally thought to be an opioid receptor is now categorized as a distinct class of receptor. There are two main subtypes, the sigma-1 receptor and an uncharacterised binding site, named the sigma-2 binding site. The presence of the sigma-2 binding site shows high correlation with proliferation of cells and is associated with cancer. I have categorized sigma-1 and sigma-2 binding sites in 11 human tumour cell lines. I have demonstrated that tumour cell lines from a range of tissues express both sigma-1 and sigma-2 binding sites. One exception is the MCF7 breast cancer cell line, which lacks sigma-1 receptors. I show that the quantitation of sigma-2 binding sites using the "masking" protocols are flawed, significantly overestimating levels of sigma-2 binding sites. I propose novel protocols to determine levels of sigma-1 receptors and sigma-2 binding sites in cell lines and tissue. Using radioligand binding assays in MCF7 cells, I have characterised novel sigma-2 ligands. These ligands are simple ammonium salts containing a single nitrogen atom. They are simpler than the previously recognised pharmacophore for the sigma-2 site. I have shown that these simple ammonium salts show graded affinity for the sigma-2 binding site. The highest affinity ligands were dihexylammonium (pKi 7.58) and dioctylammonium (pKi 7.9). I have used these ammonium salts and previously characterised ligands to determine sigma-2 binding site biology. I have shown that the biological activity of these drugs is related neither to their hydrophobicity nor their ability to effect calcium signalling in cells. I propose that the Hill slope of binding is inversely related to the efficacy of a ligand to inhibit metabolic activity of cancer cells. Furthermore, I offer an explanation as to why concentrations of sigma-2 ligands far higher than their determined binding affinities are required to inhibit metabolic activity.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2005. / Bibliography: leaves 70-81.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 95-113). Also available on the Internet.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--Ohio State University, 2007. / Full text release at OhioLINK's ETD Center delayed at author's request

The stem cell-like seam cells of the nematode, Caenorhabditis elegans, represent a tractable and powerful model for studying stem cell biology. rnt-1, the worm homologue of the mammalian RUNX family of transcription factors, together with the CBFβ homologue bro-1, is essential for the proliferation of the seam cells. RUNX genes and CBFβ are important regulators of stem cell development in mammals, and are associated with a variety of cancers. The worm seam cell model offers an opportunity to examine how these genes function in stem cell biology. The aim of this work was to shed light on the genetic network in which bro-1 and rnt-1 function, and to reveal the identity of regulators of these genes as well the downstream targets of the bro-1/rnt-1 pathway. Here, a number of genes that interact with bro-1 and rnt-1 have been identified. ELT-1, a GATA transcription factor, is shown to be a direct regulator of bro-1. Findings which show that the MEIS gene unc-62 acts upstream of bro-1/rnt-1 and regulates the symmetry of seam cell divisions are also presented. The seam cell marker, scm::gfp, is widely used in studies of the seam cells; here the results of an investigation into its identity and functional links are described. In addition, the mechanism underlying spatial regulation of rnt-1 was examined; this led to the discovery of distinct tissue-specific enhancer modules within an intron of this gene. Finally, interactions between pal-1 and bro-1/rnt-1 are reported and described. Together, these findings provide a framework for furthering our understanding of the mechanisms and genes associated with the functioning of bro-1 and rnt-1 in the worm.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A vitamina A (retinol) à um nutriente essencial necessÃrio em pequenas quantidades para o funcionamento normal do sistema visual, funÃÃo imune e reproduÃÃo. Nosso grupo de pesquisa investigou o efeito de altas doses por via oral em crianÃas com diarrÃia em estudos prospectivos em comunidade em Ãreas endÃmicas no Nordeste do Brasil e encontrou benefÃcios terapÃuticos com a utilizaÃÃo do retinol na reduÃÃo da mÃdia de duraÃÃo da diarrÃia, porÃm nÃo houve diminuiÃÃo da incidÃncia de episÃdios diarrÃicos. Esse estudo explorou o papel da suplementaÃÃo do retinol em linhas de cÃlulas intestinais frente ao dano citotÃxico da Toxina A do C. difficile (TxA). O C. difficile à principal patÃgeno causador da diarrÃia associada ao uso de antibiÃticos e da colite pseudomembranosa. Investigou-se alteraÃÃes da resistÃncia elÃtrica transepitelial (TER) em cÃlulas Caco-2, e nos modelos de proliferaÃÃo, migraÃÃo e morte celular em IEC-6, apÃs a injÃria induzida pela TxA. Os resultados mostraram que a suplementaÃÃo do retinol, principalmente no meio sem glutamina, aumentou a TER, migraÃÃo celular, como tambÃm promoveu uma reduÃÃo significativa da apoptose e necrose, porÃm este efeito nÃo foi visto no modelo de proliferaÃÃo celular. Para estudar a modulaÃÃo do retinol na diminuiÃÃo da TER induzida pela TxA, as cÃlulas foram expostas a TxA (0,1&#956;g/mL) durante 24h. O retinol aumentou a TER (% do valor inIcial) nas concentraÃÃes de 0,1 e 0,3nM Ãs 3h (59,3Â1,3; 69,8Â0,6vs 59,3Â1,3&#937;cm2,respectivamente), e Ãs 4h (36,1Â0,02; 33,5Â1,8 vs 27,3Â0,2&#937;cm2,respectivamente) em relaÃÃo ao controle sem retinol e com TxA. Nesses intervalos de tempo nÃo houve influÃncia da proliferaÃÃo celular nas cÃlulas Caco-2. O retinol aumentou a proliferaÃÃo celular na lesÃo induzida pela TxA (0,1&#956;g/mL) em 14,2; 23,8; 59,8; 8,4; 30,2; 44,1% (0,01; 0,03; 0,1; 1,0; 10; 100nM de retinol, respectivamente), apÃs 24h comparado com controle com TxA. Depois de 12 e 24h de exposiÃÃo a TxA (0,01&#956;g/mL), seguido do arranhÃo na monocamada da cÃlulas IEC-6, a suplementaÃÃo do retinol aumentou significantemente a migraÃÃo nas concentraÃÃes de 0,1-100nM em uma taxa de 30-80% e 60-100%, nas 12h e 24h, respectivamente. O retinol reduziu a apoptose e a necrose induzida pela TxA nas concentraÃÃes de 0,03-100nM, em comparaÃÃo com controle com a TxA. Os resultados sugerem que o retinol exerce um importante papel na reduÃÃo da apoptose, em aumentar a migraÃÃo e proliferaÃÃo e em prevenir a reduÃÃo da TER, na lesÃo pela TxA, sugerindo que a vitamina A à um nutriente essencial na proteÃÃo na barreira funcional epitelial / Vitamin A (retinol) is an essential nutrient that is necessary in small amounts for normal functioning of the visual system, immune function and reproduction. Our group has investigated the effect of oral dosis of vitamin A on the early childhood diarrhea in our prospective community-based studies in high endemic areas in the Northeast of Brazil and has found a benefit of retinol therapy in reducing the mean duration but not the incidence of diarrheal episodes. In this study, we have explored the role of retinol supplementation in intestinal cell lines, following Clostridium difficile toxin A (TxA) cytotoxic challenge. C. difficile is the most common anaerobic pathogen that causes antibiotic-associated diarrhea and pseudomembranous colitis. We have focused on changes in transepithelial electrical resistance (TER) in Caco-2, a more differentiated intestinal cell line, and on models of proliferation, migration and cell death in IEC-6 cells, an undifferentiated crypt cell line, following or not by TxA-induced cell injury. The results showed retinol alone increased the TER, cell migration and proliferation, and also promoved significant reduction of apoptosis and necrosis, however this effect was not observed at the cell proliferation. To study the retinol effect on the TxA-induced loss of TER, cells were exposed during 24h to 0,1&#956;g/mL TxA. Retinol improved TER (% of initial value) at the concentrations of 0,1nM and 0,3nM at 3h (59,3Â1,3; 69,8Â0,6 vs 59,3Â1,3&#937;cm2, respectively), and at 4h (36,1 0,02; 33,5 1,8 vs 27,3Â0,2&#937;cm2, respectively) in relation to the untreated control challenged with TxA. During this time there was no influence of the cell proliferation in the Caco-2 cells. Retinol increased cell proliferation after TxA-induced cell damage (0,1&#956;g/mL) at a rate of 14,2%, 23,8%, 59,8%; 8,4%; 30,2%; 44,1% after 24h (doses of 0,01; 0,03; 0,1; 1,0; 10; 100nM of retinol, respectively), compared to controls only with TxA. After 24h of TxA exposure (0,01&#956;g/ml), following plate scraping, the retinol supplementation improved significantly IEC-6 migration at the concentrations of 0,1-100nM in rate of 30-80% and 60-100%, in 12h and 24h, respectively. Retinol reduced TxA-induced apoptosis and necrosis at the concentration of 0,03: 0,1; 1; 10 and 100nM, p<0.05, in comparison to the control with TxA. These results suggest that retinol has a critical role in reducing apoptosis, improving cell migration and proliferation and preventing the reduction in TER, following TxA challenge, suggesting that vitamin A is an essential nutrient to protect the intestinal epithelial barrier function