A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of requirements for the degree of Master of Science. Johannesburg 2015. / Ovarian cancer is the most lethal gynaecological cancer. About 90% of ovarian cancers are epithelial (ovarian carcinomas), thought to arise from the ovarian surface epithelium. Diagnosed usually at clinically advanced stages, many patients show poor response to chemotherapy, with resistance and recurrent disease being prevalent. siRNA technology is currently being explored in clinical trials as a form of targeted therapeutic strategy in the disease. RBBP6 is a 250kD protein that enhances MDM2-mediated ubiquitination of p53 and also plays a role in cell cycle regulation and cell differentiation. It is upregulated in numerous cancers such as lung, oesophageal, colorectal and cervical cancer. RBBP6 suppresses p53 binding to DNA thereby inhibiting p53-dependent gene transcription. RBBP6 was knocked down using 30 nM siRNA in RMG-1 cells for 48 hours, after which the cells were treated with 50 nM paclitaxel and 0.5μM camptothecin for 24 hours. xCELLigence real time cell analysis was used to evaluate cell proliferation. qPCR and western blot were used to evaluate both gene expression and protein expressions respectively, of Bax, Bcl-2, MDM2, p53 and p21. Flow cytometry was used to determine the mode of cell death elicited apoptosis and also analyse changes in cell cycle progression. qPCR and Western blot analyses showed that RBBP6 expression reduced by approximately 57%. There was a significant upregulation of p53 and a significant downregulation of Bcl-2 in siRBBP6 transfected cells (p<0.05). Knockdown of RBBP6 resulted in a 37±5.8% cell death. There was a significant increase in cell death in paclitaxel and siRBBP6 co-treated cells (81.6±0.79%) as compared to cells treated with paclitaxel only (76.±1.14%). siRNA-mediated knock down of RBBP6 induces cell death in RMG-1 ovarian carcinoma cells. In addition, paclitaxel-induced cell death in RMG-1 cells is potentiated by RBBP6 siRNA transfection. A combination of chemotherapy with paclitaxel or camptothecin and RBBP6 siRNA could be a possible therapeutic strategy in combatting ovarian carcinomas.

La thérapie photodynamique (PDT) est un traitement minimalement invasif basé sur une approche photochimique. Ce traitement est prometteur en oncologie et pour d’autres pathologies. La plupart des drogues utilisées en PDT (photosensibilisateurs) y compris le PS de seconde génération, la méta-tétra (hydroxyphényl)chlorine (mTHPC), sont hautement hydrophobiques et nécessitent un système de transport. Pour améliorer la solubilité de la mTHPC et ses propriétés pharmacocinétiques, les dérivés des β-cyclodextrines (β-CDs) ont été proposés. L’étude présente a analysé l’effet des β-CDs sur le comportement de la mTHPC à différentes étapes de sa distribution in vitro et in vivo. L’interaction de la mTHPC avec les β-CDs conduit à la formation de complexes d’inclusion qui abolissent complètement son agrégation après introduction dans le sérum. Il a été démontré que les β-CDs ont un effet lié à la concentration sur le processus de distribution de la mTHPC dans le sérum sanguin et les cultures cellulaires in vitro. L’étude in vivo confirme le fait que l’utilisation de β-CDs permet de modifier les processus de distribution de la mTHPC chez les animaux porteurs de tumeurs, ce qui se traduit par un taux moins élevé d’accumulation du PS dans la peau et les muscles, ainsi qu’une accumulation plus élevée du PS dans la tumeur. En conclusion, l’application des dérivés β-CDs peut ouvrir de nouvelles possibilités pour modifier et contrôler la biodistribution et les pharmacocinétiques de la mTHPC au cours de la PDT / Photodynamic therapy (PDT) is a minimally invasive photochemical treatment with a promising clinical track record for oncological and some other diseases. Most PDT-drugs (photosensitizers) including a second-generation PS meta-tetra(hydroxyphenyl)chorin (mTHPC) are highly hydrophobic and require delivery systems. To improve mTHPC solubility and pharmacokinetic properties, β-cyclodextrins (β-CDs) derivatives were proposed. The present study investigates the effect of β-CDs on mTHPC behavior at various stages of its distribution in vitro and in vivo. Interaction of mTHPC with β-CDs leads to the formation of inclusion complexes that completely abolishes its aggregation after introduction into serum. It was demonstrated that the β-CDs have a concentration-dependent effect on the process of mTHPC distribution in blood serum and cellular cultures in vitro. In vivo study confirms the fact that the use of β-CDs allows modifying mTHPC distribution processes in tumor bearing animals that is reflected in the decreased level of PS accumulation in skin and muscles, as well as in the increased PS accumulation in tumor. In conclusion, application of β-CD derivatives can open up new possibilities to modify and control biodistribution and pharmacokinetics of mTHPC in the course of PDT

by Wong Ping Shing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 97-112). / ACKNOWLEDGMENTS --- p.i / ABSTRACT --- p.ii / ABBREVIATIONS --- p.viii / Chapter 1. --- INTRODUCTION --- p.1 / Chapter 1.1 --- General properties of zinc / Chapter 1.1.1 --- Biochemistry of zinc --- p.2 / Chapter 1.1.2 --- Distribution of zinc in body --- p.3 / Chapter 1.1.3 --- Roles of zinc in protein function --- p.4 / Chapter 1.2 --- Zinc and zinc-binding proteins in brain / Chapter 1.2.1 --- Distribution of zinc in brain --- p.7 / Chapter 1.2.2 --- Metabolism of zinc in brain --- p.9 / Chapter 1.2.3 --- Compartments of zinc in brain --- p.10 / Chapter 1.2.4 --- Zinc-binding proteins in brain --- p.12 / Chapter 1.3 --- Pathological conditions of brain in relation to zinc --- p.15 / Chapter 1.4 --- Aim of the project --- p.20 / Chapter 2. --- MATERIALS AND METHODS --- p.22 / Chapter 2.1 --- Detection of zinc-binding proteins / Chapter 2.1.1 --- Sodium-Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.22 / Chapter 2.1.2 --- Electroblotting --- p.24 / Chapter 2.1.3 --- Radioactive zinc blotting --- p.25 / Chapter 2.1.4 --- Autoradiography --- p.25 / Chapter 2.2 --- Subcellular fractionation of porcine brain --- p.26 / Chapter 2.3 --- Purification and structural characterization of a 19 kDa zinc-binding protein / Chapter 2.3.1 --- Purification of a 19 kDa protein --- p.27 / Chapter 2.3.2 --- Sequencing of N-terminal blocked 19 kDa protein --- p.30 / Chapter 2.4 --- Characterization of the binding and biological properties of the 19 kDa zinc-binding protein / Chapter 2.4.1 --- Effect of divalent metal ions on zinc binding to the 19 kDa protein --- p.33 / Chapter 2.4.2 --- Effect of pH on the dissociation of radioactive zinc from the19 kDa protein --- p.34 / Chapter 2.4.3 --- Radioactive calcium blotting --- p.34 / Chapter 2.4.4 --- Interaction of radioactive zinc and radioactive calcium binding to the 19 kDa protein --- p.35 / Chapter 2.4.5 --- Calmodulin activity assay --- p.35 / Chapter 3. --- RESULTS / Chapter 3.1 --- Specificity of radioactive zinc-blot on zinc-binding protein detection --- p.38 / Chapter 3.2 --- Zinc-binding proteins in porcine brain --- p.38 / Chapter 3.3 --- Purification and identification of a cytosolic 19 kDa zinc- binding protein in porcine brain / Chapter 3.3.1 --- Zinc-dependent hydrophobic interaction chromatography --- p.44 / Chapter 3.3.2 --- N-terminal amino acid sequencing --- p.51 / Chapter 3.3.3 --- High pH native gel electrophoresis of 19 kDa protein --- p.51 / Chapter 3.4 --- The zinc and calcium binding properties of the 19 kDa protein / Chapter 3.4.1 --- Effect of pre-exposure to divalent cations on zinc binding --- p.54 / Chapter 3.4.2 --- Competition by divalent cations for zinc binding --- p.56 / Chapter 3.4.3 --- pH dependency of zinc dissociation --- p.56 / Chapter 3.4.4 --- Effect of zinc on radioactive calcium binding --- p.61 / Chapter 3.5 --- The biological activity of the 19 kDa protein / Chapter 3.5.1 --- Effect of the 19 kDa protein on the activity of calmodulin- dependent phosphodiesterase --- p.66 / Chapter 3.5.2 --- Effect of zinc on calmodulin-dependent phosphodiesterase activity --- p.69 / Chapter 3.5.4 --- "Effect of zinc on calcium-deficient, calmodulin-dependent phosphodiesterase activity" --- p.72 / Chapter 4. --- DISCUSSION / Chapter 4.1 --- Detection and Purification of zinc-binding proteins / Chapter 4.1.1 --- Strategy for the detection of zinc-binding proteins --- p.77 / Chapter 4.1.2 --- Purification of zinc-binding protein --- p.79 / Chapter 4.2 --- Amino acid sequencing of the 19 kDa protein --- p.82 / Chapter 4.3 --- Binding properties of the 19 kDa zinc-binding protein --- p.86 / Chapter 4.4 --- Effect of zinc and 19 kDa zinc-binding protein on calmodulin dependent phosphodiesterase --- p.92 / Chapter 4.5 --- Effect of zinc on the properties of calmodulin --- p.90 / Chapter 4.6 --- Significance of the ability of zinc to affect calmodulin activity --- p.94 / Chapter 5. --- CONCLUSION --- p.95 / Chapter 6. --- REFERENCES --- p.97

Antibodies are soluble proteins produced by the adaptive immune system to bind and counteract invading pathogens. The binding properties of a typical human antibody are determined by the structure of its variable domain, composed of two chains – heavy and light and by the conformation of six loops located on the surface of the variable domain, known as Complementarity Determining Regions (CDRs). In the first chapter, we describe our analysis of the conformational space occupied by five out of six antibody CDRs (L1, L2, L3, H1 and H2) and the development of a novel, length-independent method for grouping these CDRs into structural clusters (canonical forms). We show that using our method we can increase coverage and precision of assigning CDR sequences into clusters. In the next chapter, we describe a method for ranking structural decoys of the CDR-H3 loop. We show that by computationally perturbing CDR-H3 decoys we can improve the performance of existing ranking methods. In the same chapter, we discuss the development of a method for high-throughput assignment of heavy-light chain orientation. The power of the method was demonstrated by assigning orientation to billions of potential Fv sequences. The third Chapter describes the analysis of a large dataset of CDR sequences with the aim of identifying sequence patterns responsible for the loops' structure. Using a neural network methodology, we found several groups of CDR sequences which might be indicative of previously-unseen conformations. In the final results Chapter, we describe how we used the structural knowledge developed throughout the rest of the thesis to create a novel pipeline for computational antibody design. We show that the binders developed using our methodology had similar features to available antibody therapeutics and low predicted propensity to cause an immunogenic response. These results demonstrate the potential for using computational methods for designing high affinity therapeutics with human properties.

Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants that have been associated with a myriad of negative human health effects. These man-made compounds were used throughout most of the 20th century and although their intentional production has since been banned and their use limited to closed systems, their prevalence in the environment remains a factor in disease states for exposed populations. The worldwide levels of PCBs has been declining, however, there is evidence for renewed sources of these compounds. The presence of PCBs with lower numbers of chlorine atoms (LC-PCBs) have been verified as unintentional byproducts in paints and pigments, the decomposition of PCB waste, or the recycling or disposal attempts of PCB-laden materials. While exposure to the higher chlorinated congeners (HC-PCBs) is often attributed to the consumption of contaminated water or fatty animal meat, a significant route of exposure to the airborne LC-PCBs is through inhalation. These semi-volatile compounds have been detected in high quantities in both indoor and outdoor air in urban and rural communities, and their presence is pronounced in older buildings (e.g., homes and schools). When compared to HC-PCBs, LC-PCBs are more highly susceptible to metabolic transformations, and recently their sulfated metabolites have gained much interest. Although the sulfation of xenobiotics often is considered a route for their removal from the body, a previous study of Sprague-Dawley rats treated with 4-chlorobiphenyl (PCB 3) resulted in the substantial formation of sulfated metabolites (i.e., hydroxylation followed by sulfation of the LC-PCB). This metabolic route accounted for more than half of the treatment dose. Furthermore, LC-PCB sulfates have been shown to bind to the human serum protein, transthyretin, in vitro. Of the health effects associated with PCB exposure, neurotoxicity has been well established through various laboratory and epidemiological studies. It is proposed that the dopaminergic system lies at the core of the observed cognitive, motor, and intellectual dysfunction observed in exposed populations, especially in children exposed perinatally. Interestingly, PCB exposure has been linked to Parkinson's disease (PD) etiology, which is marked by a substantial loss of dopaminergic neurons. This thesis describes studies on the binding of selected LC-PCBs and their hydroxylated and sulfated metabolites to human serum albumin (HSA), the most abundant protein in human serum. The displacement of fluorescent probes, selective for the two major drug binding sites of HSA, indicates that LC-PCB sulfates generally bind to HSA with such affinity that is equal to or greater than that for the LC-PCBs or OH-LC-PCBs This work also included a study of the selective toxicity of these compounds to dopaminergic neuronal cells. The selective toxicity of these compounds was studied in a series of immortalized cell lines (i.e., two neuronal cell lines: the rat midbrain-derived N27 cell line, the human neuroblastoma-derived SH-SY5Y cell line, and the human liver-derived HepG2 cell line). The assessment of toxicity by MTT reduction and LDH release in these cellular models indicated that hydroxylated and sulfated metabolites of LC-PCBs exhibited toxicity that was selective to neuronal cells and, in most cases, selective for the dopaminergic neuronal cells. Furthermore, HPLC analysis of the distribution of the compounds from the extracellular medium into the cellular milieu indicated that the observed toxicity may be due in some cases to selective transport and further metabolism. This work contributes to understanding the neurotoxicity of LC-PCB hydroxylated and sulfated metabolites and the role that binding to serum proteins may play in it. Furthermore, it emphasizes the need for future studies on the effects that metabolism, particularly sulfation, may play in the disposition of LC-PCB congeners as it pertains to their metabolism, retention, and toxic effects.

Abstract not available

[Truncated abstract] Regulation of mRNA stability is an important posttranscriptional mechanism involved in the control of gene expression. The rate of mRNA decay can differ greatly from one mRNA to another and may be regulated by RNA-protein interactions. A key determinant of mRNA decay are sequence instability (cis) elements often located in the 3' untranslated region (UTR) of many mRNAs. For example, the AU rich elements (AREs), are such well characterized elements, and most commonly involved in promoting mRNA degradation, and specific binding of proteins to these elements leading to the stabilization of some mRNAs. Other cis-elements have been described for mRNA in which mRNA stability is a critical component of gene regulation. This includes the androgen receptor (AR) UC-rich cis element in its 3'UTR. The AR is a key target for therapeutics in human prostate cancer and thus understanding the mechanism involved in regulating its expression is an important goal. The [alpha]CP1 protein, a KH-domain containing RNA-binding protein has been found to bind this UC-rich region of the AR and is thought to play an important role in regulating AR mRNA expression. [alpha]CP1 protein is a triple KH (hnRNP K homology) domain protein with specificity for Crich tracts of RNA and ssDNA (single stranded DNA). Relatively little is known about the structural interaction of [alpha]CP1 with target RNA cis elements, thus the present study aimed to better understand the nature of interaction between 30 nt 3'UTR UC-rich AR mRNA and [alpha]CP1 protein using various biophysical techniques, in an attempt to determine which [alpha]CP1 domain or combination of domains is involved in RNA-binding. These studies could ultimately provide novel targets for drugs aimed to regulate AR mRNA expression in prostate cancer cells. At the commencement of this study little was known about the structure of the [alpha]CP1- KH domains and their basis for poly (C) binding specificity. ... Additional studies addressed the significance of the four core recognition nucleotides (TCCC) using a series of cytosine to thymine mutants. The findings verified some of the results predicted from structural studies, especially the need for maximum KH binding to a core tetranucleotide recognition sequence. Our mutational studies of the four core bases confirmed the importance of cytosine in positions two and three as no binding was observed, while some binding was observed when the fourth base was mutated. In summary, the work presented in this thesis provides new detailed insight into the molecular interactions between the [alpha]CP1-KH domain and AR mRNA. Furthermore, these studies shed light on the nature of protein/mRNA interactions in general, as well as the specific complex that forms on AR mRNA. These studies have provided new understanding into the mode of [alpha]CP1 binding at a target oligonucleotide binding site and, provide a foundation for future studies to define structure of multiprotein/oligonucleotide complexes involved in AR mRNA gene regulation. Understanding the detailed interaction between the AR mRNA and [alpha]CP1 could provide possible targets for drug development at reducing AR expression in prostate cancer cells by interfering with the interaction of [alpha]CP1 and AR-mRNA.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on December 18, 2007) Vita. Includes bibliographical references.

Graduation date: 2005

The influence of hyperlipidemia on the pharmacodynamic and pharmacokinetic aspects of lipophilic drugs was explored. The antiarrhythmic, amiodarone, and the antifungal, ()-ketoconazole, were used as model drugs. Experimental hyperlipidemia was induced in rat using poloxamer 407 and two sensitive novel HPLC assays were developed. In a multiple dosing study, hyperlipidemia increased amiodarone plasma concentrations, heart concentrations and electrocardiographic changes. The amiodarone heart uptake could not be totally attributed to its unbound fraction, where the cardiac very low density lipoprotein receptors seemed to play a role in the uptake of bound drug. Amiodarone liver metabolism in presence and absence of hyperlipidemia was studied using isolated primary rat liver hepatocytes. The metabolism of amiodarone was lower in hepatocytes isolated from hyperlipidemic than those from normolipidemic rats. Hyperlipidemic serum resulted in a decrease in amiodarone metabolism and when coincubated, the expected decrease in unbound fraction seemed to resulted in greater inhibition of metabolism. ()-Ketoconazole showed stereoselectivity in its pharmacokinetics in rat with (+)-ketoconazole showing higher plasma concentrations than its antipode. This was attributed to its higher protein binding. There was no difference in the total bioavailability of the two enantiomers. Ketoconazole enantiomers exhibited nonlinear pharmacokinetics. In normolipidemic rat plasma ketoconazole enantiomers were more than 95% bound to lipoprotein deficient fraction. Hyperlipidemia resulted in shifting both enantiomers 20% to very low density and low density lipoprotein fractions. In a pharmacokinetic assessment, hyperlipidemia was found to increase ketoconazole enantiomer volume of distribution. Moreover, the stereoselectivity ratios of most pharmacokinetic parameters were changed. After oral dosing, the uptake of (-)-ketoconazole was significantly decreased. Since ketoconazole is used as a potent CYP3A inhibitor, alteration in liver concentrations of (-)-ketoconazole, the more potent inhibitory enantiomer, could decrease its CYP inhibitory potential. Hyperlipidemia potentiated the CYP-mediated interaction between ketoconazole and midazolam with significantly higher midazolam AUC and lower clearance. This was attributed to the inhibitory action of ketoconazole and the effect of hyperlipidemia on the binding of midazolam. Hyperlipidemia was found to unexpectedly decrease midazolam unbound fraction in plasma. In conclusion, the findings could explain some unexpected dose versus effect outcomes in hyperlipidemic patients receiving amiodarone or ketoconazole. / Pharmaceutical Sciences