Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (doctoral)--University of Lund, 1996. / Added t.p. with thesis statement added.

Thesis (doctoral)--University of Lund, 1996. / Added t.p. with thesis statement added.

Experiments were done to determine the effect of recombinant interleukin 2 (rIL-2) on mononuclear cells (MC) of newborns and adults. MC were tested for (1) ability to lyse Daudi cells in a 51Cr release assay, (2) cell surface markers using monoclonal antibodies and flow cytometer analysis, and (3) cell types as determined by differential cell counts. Without rIL-2 adults show greater cytotoxicity than newborns in vitro. Incubation with rIL-2 dramatically increased the cytotoxicity expressed with cord blood and adult MC showing equivalent responses. Differences in cell surface markers between newborns and adults prior to rIL-2 exposure were in agreement with those previously published. This study did not demonstrate changes in phenotypes after exposure to rIL-2. Slight changes in differential cell counts occurred after increased incubation periods and rIL-2 exposure.

Oesophageal adenocarcinoma (OAC) is a leading cause of cancer death in the UK and current systemic therapies are ineffective for the majority of patients. The central aim of this work was to explore strategies to identify novel therapeutic targets. Research has failed, thus far, to identify a dominant oncogene in OAC, although the tumour suppressor p53 is frequently mutated. Inhibiting the mitotic kinase, polo-like kinase 1 (PLK-1), was proposed as a synthetic lethal strategy. PLK-1 was demonstrated to be over-expressed in both verified OAC cell lines and human OAC tissue compared to non-transformed cells and epithelium. Mutation of p53 was associated with over-expression of PLK-1 in both OAC and ovarian cancer tissue. Using a carefully validated viability assay, both an established and novel PLK-1 inhibitor were demonstrated to induce a G2/M arrest and reduce OAC cell proliferation. Relative selectivity was demonstrated for OAC compared to non-transformed cells. This therapeutic window could be enhanced with the induction of cancer cell cytotoxicity by pulsed administration of a short half-life inhibitor. Immunotherapeutics offer potential tumour-selectivity but no OAC-specific proteins have been defined. A comparative proteomic approach was employed to identify OAC-specific proteins as potential therapeutic targets. A tissue resource was established and methods to lyse fresh frozen biopsies optimised. An isobaric quantitative proteomic workflow was applied to OAC and matched normal biopsies and quantitative accuracy confirmed for 6 candidate proteins by immunohistochemistry. Proteome coverage and quantitative dynamic range were compared between isobaric and label-free systematic sequencing proteomic strategies applied to further patients’ tissues. The challenges of combining incomplete datasets were approached with a Bayesian framework to estimate the probability that a protein was missed during an experiment compared to not being present in the sample. This method was applied to generate a complete set of protein identifications and relative tissue expression. To gain insight into the dysregulated cellular processes in human OAC tissue, a network analysis was applied to the quantitative proteomic data. Enriched functional clusters were identified suggesting deranged glucose metabolism, potentially due to the Warburg effect. These findings were duplicated and candidate tumour-specific proteins identified in a further set of biopsies using the optimised quantitative proteomic method. The combined quantitative oesophageal proteomic dataset represents the largest in OAC to date. This thesis demonstrates a hypothesis-driven, synthetic lethal approach can yield cancer-selective therapeutic effects. Novel candidate therapeutic targets are also revealed through the development of quantitative proteomic methods and the application of network analysis.

This thesis describes a new method for the synthesis of pseudodisaccharides containing a carbasugar analogue attached to a "true" sugar. The methodology is based on a Diels-Alder cycloaddition of vinyl sugars and appropriately substituted pyran-2-ones, followed by chemical manipulation of the resulting cycloadducts. The thesis also describes the synthesis of inhibitors of Golgi α-mannosidase II and glucokinase. The first chapter is a comprehensive survey of the reported synthetic routes to pseudodisaccharides from the literature. The results and discussions are presented in chapter 2. This chapter starts by discussion of the preparation of vinyl sugars and pyran-2-ones and the regio- and stereoselectivity of their cycloadditions. This is followed by reporting the chemical manipulations of these cycloadducts and the synthesis of a pseudodisaccharide. Cycloadducts are shown to lose carbon dioxide at elevated temperatures to afford dihydrobenzenes. The loss of the bridging carbon dioxide from the cycloadducts is experimentally and computationally investigated. The resulting dihydrobenzenes are shown to also be useful as precursors in the synthesis of pseudodisaccharides. The chemical manipulation of these dihydrobenzenes is used towards the synthesis of a pseudodisaccharide. The third and fourth chapters focus on the synthesis of new inhibitors of Golgi α-mannosidase II and glucokinase respectively. A range of 6-aminoglucose and mannose derivatives were prepared and tested for the inhibition of Jack bean α-mannosidase, but were found to lack any inhibition. Similarly, a range of 6-triazologlucose derivatives were prepared but were found to lack any cytotoxicity. The fifth chapter contains the details of the preparation, experimental procedures and spectroscopic characterisation of the synthesised chemical compounds. Rate calculations are reported in Appendix I and the X-ray crystallographic data are presented in the Appendix II.

HIV-1 has evolved a number of strategies in response to current anti-retroviral drugs and the selection pressure of humoral and cellular immunity. In particular, R5 viral strains that are essential for AIDS pathogenesis are very resistant to neutralization by antibodies. Therefore, the aim of this thesis was to develop synthetic nucleic acid ligands, aptamers, against gp120 of an R5 strain of HIV-1, with a view of using aptamers as novel neutralization molecules and analytical tools to study HIV-1 entry into target cells. The central hypothesis of this thesis was that aptamers by virtue of their small size and slow dissociation rates, compared to antibodies, would easily access and bind occluded gp120 neutralization sites. Using the SELEX protocol and SPR technology, I isolated 2'-Fluoro-pyrimidine-RNA aptamers against HIV-l_Ba-L monomeric gp120. Most of these aptamers not only bound gp120 with high affinities but also neutralized R5 primary isolates in human PBMC by 1,000 to 100,000-fold, truly unprecedented when compared with natural ligands such as antibodies. Some aptamers, like B4, defined a conserved site of gp120 that could not mutate to escape neutralization following stringent selection, in vitro, for breakthrough virus. This was consistent with subsequent findings that B4 aptatope (binding site) overlaps a poorly immunogenic but highly conserved CD4-induced epitope as determined by competition with 17b and 48d mAbs that map to this neutralization epitope on the gp120. This study was thus the first of its kind to describe neutralization of HIV-1 primary isolates by a ligand against the CD4-induced epitope. Most intriguing, although B4 potently neutralized HIV-1_Ba-L infection in PBMC, which is a mixed T cell and macrophage population, it modestly neutralized infection of the same virus in a purified culture of macrophages. These findings are intriguing in that they suggest that aptamers could be used to dissect unique sites on the virus that interact with target cell surface in ways that have not been revealed heretofore, and would help understand better HIV-1 entry pathways, especially in macrophages. Thus neutralizing aptamers such as these could be exploited to provide leads in developing alternative anti-HIV-1 drugs and a deeper understanding of the molecular interactions between the virus and its host cell.

This study examined the cytotoxic effects of STERIPLEX™ HC (sBioMed, Orem, UT) and sodium hypochlorite (NaOCl) on human fibroblast cells in vitro. Fibroblasts exposed to various concentrations of NaOCl or STERIPLEX™ HC were visualized via light microscopy. Dilutions of either NaOCl or STERIPLEX™ HC that did not appear to disrupt the integrity of the cells were recorded for further analysis. Cells were then cultured and grown to confluence in five separate plates. A void was created down the middle of each plate. If the cells were viable, cellular confluence was seen. If nonviable, confluence of the cells did not occur. Both disinfectants showed absolute kill at all concentrations above 1%. The cells treated with 0.1% NaOCl were found to be nonviable. However, at 0.1% STERIPLEX™ HC, the cells were viable and able to replicate, filling the void and returning to confluence.

Charles University in Prague Faculty of Pharmacy in Hradec králové Department of Pharmaceutical Botany and Ecology Author: Lenka Nechanická Supervisor: RNDr. Jitka Vytlačilová, Ph.D. Title of diploma thesis: Antiprotozoal activity of natural substances The discovery of new active substances and plants with a potential antiprotozoal effect nowadays is the aim of many studies and is required for obtaining more active drugs in a number of protozoal disease. In this study was investigated antiprotozoal activity of extracts obtained from various plant parts Salvia officinalis, Apium graveolens L. var. rapaceum, Evodia rutaecarpa, Coptis chinensis, Zanthoxylum nitidum and Ziziphus jujuba. Effect of the tested extracts was evaluated in a typical unicellular organism Tetrahymena thermophila using MTT method. From the values obtained the percent inhibition was detected Tetrahymena thermophila and for each extract value calculated median inhibitory concentrations IC50. Of the extracts tested had the greatest antiprotozoal activity of the extract of C. chinensis, further extracts activity decreases in the order C. chinensis > Z. nitidum > Z. jujuba > S. officinalis > E. rutaecarpa > A. graveolens L. var. rapaceum Key words: antiprotozoal activity, Tetrahymena thermophila, cytotoxicity, natural substances

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Graphene oxide (GO) is a single carbon layer, oxygen bearing graphene derivative, containing hydroxyl and carboxyl groups. Graphene oxide nanoparticles (GONPs) are promising nanomaterials for a variety of applications such as electrochemical analysis, adsorption of biomolecules, biosensors and drug and vaccine delivery systems. While these newly engineered nanoparticles hold great potential for developments in industry and medicine, the widespread use of these material will inevitably result in GO residues in the environment where they could possibly pose a risk to human and wildlife health. Interaction of the nanoparticles and biota can affect numerous biological processes. In humans they can affect any of the physiological systems such as the immune, endocrine, reproductive and cardiovascular systems. Although studies have indicated that GO exposure cause increased reactive oxygen species in cells, they mechanisms whereby GO act on the cell are still poorly understood. A few studies have investigated the effects of GONP and other graphene nanoparticle derivatives on the immune system. The aim of this study was to investigate the in vitro effects of GONPs on the immune system by the exposure of the murine macrophage cell line, RAW 264.7, to different concentrations of GONPs.