Since the discovery of the buckyball in 1985, researchers have imagined its potential in fields ranging from materials science to medicinal chemistry. The unique size, shape and hydrophobicity of C 60 fullerene endow it with the ability to interact with biological superstructures such as enzymes and membranes making it attractive as a potential pharmacophore. In this regard, we have developed a new, simple route to water soluble fullerene amino acids, both alkyl and aryl, through the dipolar addition of azido starting materials. The synthesis of our phenylalanine derivative, including the chromatographic purification, requires only one day for its completion. We have subsequently used our C 60 phenylalanine derivative in the synthesis of a series of C 60 peptides for the purposes of enzyme inhibition, specifically human immunodeficiency virus Type 1 protease, a critical viral enzyme responsible for the maturation of the virus and a popular target of medicinal chemists. We have demonstrated the ability of our C 60 amino acids and peptides to inhibit HIV-1 PR in a cell-free fluorescence based assay at low nanomolar concentrations. Graphite, or specifically graphene, has recently come to the forefront of nanomaterials research due to it similar scale, properties, and reaction pathways as other more costly carbon nanostructures such as carbon nanotubes. We have demonstrated the high yield functionalization of graphitic starting materials through the thermal decomposition of azido amino acids to their corresponding nitrene. The result is an inexpensive, highly functionalized, carbon based scaffold.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2009. / Härtill 6 uppsatser.

published_or_final_version / abstract / Molecular Biology / Doctoral / Doctor of Philosophy

The AIDS epidemic in Africa has reached dramatic proportions. Of the 42 million people infected with HIV worldwide, 30 million are in Africa. Current available therapies have begun to transform this fatal disease into a chronic condition but there are still major obstacles that have resulted in a great demand for new and better drugs. The aim of this study was to synthesize novel and effective HIV protease inhibitors. This work describes the first account of pentacycloundecane (PCU)-peptide and peptoid based protease inhibitors. These inhibitors are proposed to bind the wild type C-South African HIV protease (C-SA) catalytic site via the norstatine or dihydroxylethelene type functional group of the PCU. The desired compounds were synthesized by the coupling of the peptides and peptoids to the PCU cage which resulted in a series of promising and structurally diverse HIV-1 protease inhibitors. The inhibitors were characterized by Nuclear Magnetic Resonance (NMR) and evaluated against the wild type C-SA enzyme for its ability to inhibit 50 % of the enzyme’s activity (IC50). Two of the compounds reported herein, inhibited the enzyme activity at concentrations less than 80 nM. NMR investigations indicated that the activity was related to the chirality of the PCU moiety and its ability to induce conformations of the coupled peptide side chain. Employing the new Efficient Adiabatic Symmetrized Rotating Overhauser Effect Spectroscopy (EASY-ROESY) technique enabled us to obtain vital information about the 3D structure of these small linear peptides and peptoids in solution. This technique is the first example describing the successful through space correlations of such small peptides. Furthermore, docking and a combined quantum mechanics/molecular mechanics (QM/MM) molecular dynamics MD simulation at the AM1 semi empirical level mirrored the observed NMR results and the experimental IC50 activity profile of the considered inhibitors. The combination of these experimental and theoretical methods provided a powerful insight into the interaction mode of these cage peptide and peptoid inhibitors with the enzyme. / Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2011.

Effect of c-Ha-ras(V12) on cathepsin trafficking in invasive breast cancer cells. Various mutations of Ha-ras together with lack of p53-related control over cell cycle progression, result in an immortal, tumorigenic phenotype in 50% human epithelial cancers. Unmutated Ha-Ras transiently mediates external growth factor-related signaling, initiating downstream kinase activity that is normally terminated by p53. This protects the cell from immortalization, i.e. uncontrolled proliferation. An MCF10A breast epithelial cell line, derived from a fibrocystic breast mastectomy specimen, spontaneously immortalized in culture, due to a chromosomal deletion (9p21-/-). This gave rise to a non-malignant and non-invasive cell line in which the effects of deletion of upstream control of both p53 and the cell cycle and c-Haras( V12) transfection may be studied. Transfection of this cell line with the c-Haras( V12) oncogene gave rise to the invasive MCF10AneoT premalignant derivate, in which distribution of cathepsin B (CB), cathepsin L (CL) and cathepsin D (CD), membrane-type 1 matrix metalloprotease (MT1-MMP), a membrane-bound collagenase, is altered. The possible role of these proteases in the premalignant invasive phenotype, as well as the role of the V12 mutation and the effect of p53 on vesicle trafficking, was explored. In the MCF10AneoT cell line lack of negative feedback by p53 and other Ha-Ras effectors such as Rac, Rho and CDC42, seems to result in lack of control over the cytoskeleton and thus cell polarity during growth stimulus-related migration. Luminal alkalinization, especially of vesicles distant from the perinuclear region, as well as degradative efficiency seem affected, possibly as functional assembly of the acidifying vacuolar-ATPase proton pump on these vesicles is compromized. In normal cells CB and CD seem discretely located, while a spread of proteases was noted in transfected cells, from a perinuclear position to along the basal plane. Increased association of CB with lysosome-associated membrane protein-2 (LAMP- 2), and of CD with an acidic juxta-nuclear structure (JNS) was also noted, while this structure was observed in two sites in transfected cells, compared to only one in normal cells. In invasive cancers increased levels of both CB and MT1-MMP have been found to correlate with accelerated pathological degradation and invasion of the underlying basement membrane (BM) barrier and extracellular matrix (ECM). MT1- MMP is known to regulate BM turnover, while the manner in which the association of CB with the plasma membrane (PM) supports such turnover, ECM degradation and migration, is not yet clear. The current investigation showed altered distribution of PM-associated CB and MT1-MMP in transformed cells, compared to normal. This phenotype seems explained in terms of the effects of the mutationally activated c-Ha- Ras(V12) on its downstream effectors, Rac and PI3K and their effectors, on cytoskeletal organization and vesicle trafficking, increased calcium and, via Rho, cytoplasmic alkalinization due to proton extrusion by an activated NHE-1 membraneassociated proton pump. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.

Thesis (Ph. D.)--University of Adelaide, Dept. of Paediatrics, 1985. / Offprints of several author's journal articles inserted. Includes bibliographical references.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 4 uppsatser.

Thesis (M.Sc. (Biochemistry))--Mahidol University, 1979.

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / "May 1998." Typescript. Vita. Includes bibliographical references (leaves 126-138). Also available on the Internet.

Thesis (Ph. D.)--Ohio State University, 2005. / Document formatted into pages; contains 215 p. Includes bibliographical references. Abstract available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 March 3.