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No description available.

Human immuno deficiency virus (HIV) incorporates host cellular protein, beta-2-microglobulin (β2m), into its surface envelope during budding. β2m is a cellular protein that belongs to the major histocompatibility complex (MHC) Class I molecules. Studies have shown anti- β2m monoclonal antibodies (mAbs) has the ability of to neutralize the virus. An epitope consisting of seven amino acids of the β2m protein designated as R7V produces antibodies that protect HIV infected people from progressing to AIDS. These protective antibodies, called anti-R7V antibodies, were able to neutralize different HIV isolates, despite their genomic variations, various cellular targets and geographic origin. Anti-R7V antibodies in the format of single chain variable fragments (scFvs) were produced in our laboratory using the M13 phage display technology. These scFv antibody fragments were used during in vitro studies for the detection and neutralization of the R7V antigen by enzyme linked immune sorbent assay (ELISA). The scFv fragments produced against the R7V epitope showed interaction, however the antibody-antigen affinity was too weak for the virus neutralization assay. Hence, this project focused on the affinity maturation of the anti-R7V scFv fragments through random mutagenesis using the error prone (EP) PCR method. The EP PCR method generated two mutated anti-R7V scFvs. The mutated clones were subcloned into the pAK400 expression vector. The computer-based models, created using the Swiss PDB Deep Viewer 4.02 software, were used to predict the antigen-binding site and affinity analysis of both parent and mutated scFv’s. Mutated clone 1 failed to bind to the R7V epitope whereas mutated clone 2 had similar binding pattern as the parent scFv. Mutated clone 2 was predicted to have a higher binding affinity compared to the parent scFv. The results obtained demonstrate the efficacy of EP PCR to generate high affinity antibodies. Future experiments using high affinity anti-R7V scFv’s may lead to its potential use in diagnostics, therapeutics or vaccine development. Copyright / Dissertation (MSc)--University of Pretoria, 2012. / Biochemistry / unrestricted

This thesis describes the advances made towards a dual-species magneto-optical trap (MOT) of Li and Rb for use in photoassociation spectroscopy, Feshbach resonance studies, and, as long-term aspirations, the formation of ultracold heteronuclear polar molecules. The initial discussion will focus on a brief theoretical overview of laser cooling and trapping and the production of ultracold molecules from a cold atom source. Subsequently, details of the experimental system, including those pertaining to the required laser light, the vacuum chamber, and the computer control system will be presented. Finally, preliminary optimization and characterization measurements showing the performance of a single species Li MOT are introduced. These measurements demonstrated the loading of over 8 x 107 Li atoms directly into a MOT without the need for a Zeeman slower. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

This thesis deals with theoretical studies of electronic properties of organic conjugated molecules. The first chapter introduces different classes of organic conjugated molecules which possess high hole mobility, large quadratic non-linear response and low band gap. In this chapter, we further describe different photo-physical processes and the basic principles of various opto-electronic devices. The second chapter provides an introduction to various many-body techniques, which are employed in studying ground and excited state properties of organic conjugated systems. First, we describe the Hartree-Fock theory and the Density Functional (DFT) method. These are followed by full Configuration-Interaction (CI) methods and various semi-empirical methods (CNDO, INDO and NDDO). The INDO method is used in subsequent chapters to obtain the ground and excited state properties of organic conjugated molecules. In addition, we describe the restricted CI (SCI and SDCI) and the Density Matrix Renormalization Group (DMRG) methods. The third chapter of this thesis deals with a time evolution study to ascertain the role of the triplet state in the green emission of the ethyl-hexyl substituted poly-fluorene (PF2/6) films. To understand this phenomenon, we have modeled various non-radiative processes like (i) Inter-System Crossing (ISC), (ii) electron-hole Recombination (e-hR) and (iii) Triplet Quenching (TQ). These studies conclusively prove the contribution of triplet states to the 500 nm EL peak. In chapter four, we describe the origin of the unusual EL in tri-p-tolylamine (TTA) based hole conductors. In order to model this phenomenon, we have performed SCI calculations on TTA, its radical ions and allied hole conductors (TAPC and TPD). These calculations indicate that the unusual EL is due to low-lying charge-transfer (CT) state, which is stabilized by charge-dipole and charge-induced-dipole interactions. In chapter five, we turn our attention to the calculation of ground and excited state properties of a class of donor-acceptor (DA) system using ab-initio DFT and INDO methods. In these systems, DFT calculations along with INDO-SCI calculation, show strong intramolecular charge transfer interaction between the D and the A units. We have further calculated various properties like permanent dipole moments, oscillator strengths, Stoke’s shifts in various solvents etc. In chapter six, we focus on studying linear and non-linear optical properties of first generation nitrogen based dendrimers, using DMRG method. A novel scheme which includes the weights of the dipole allowed states in the computation of the density matrix is developed to obtain accurate dipole allowed excited states as well as the linear and nonlinear optical responses. Chapter seven deals with non-linear optical properties of weak donor-acceptor (DA) complexes formed between methyl substituted phenylenes (donor) and Chloranil or DDQ (acceptors). We have calculated the ground and the low-lying excited states of these DA complexes using INDO-SDCI method. The first hyperpolarizability (β) response coefficients are calculated using the Correction Vector (CV) technique, which are further used to obtain macroscopic depolarization ratios. By comparing the theoretical results with experimental findings, it can be shown that the slipped parallel configuration with a slight twist is the most preferred geometry of these weak DA complexes in solution.

Bestrophin-1 is a homopentameric Ca2+-gated anion channel which localises to the basolateral plasma membrane of retinal pigment epithelium (RPE) cells. Homozygous and compound heterozygous mutations in the BEST1 gene are associated with autosomal recessive bestrophinopathy (ARB), a retinopathy characterised by altered electrooculogram (EOG), deposits in the retina, and is often associated with the risk of developing angle-closure glaucoma. The mechanism by which mutations in bestrophin-1 cause disease remains unknown. Expression of four ARB-causing bestrophin-1 proteins in polarised MDCKII cells, a cell model for RPE, results in mutant proteins mislocalisation and degradation. Furthermore, when the ability of the mutant proteins to conduct Cl- ions was investigated in HEK293 cells by whole-cell patch-clamp, a reduction in the Cl- current was observed in all mutants compared to the WT.The use of a combination of the small molecules bortezomib and 4-phenylbutyrate (4PBA) successfully restored the expression and trafficking of all four ARB-causing bestrophin-1 proteins. Importantly, 4PBA was also able to restore the ability of the mutant channel to conduct Cl- ions. Biotinylation of cell surface proteins shows that the number of active channels at the plasma membrane of HEK293 cells increases following 4PBA treatment. The functional rescue achieved with 4PBA supports the hypothesis that ARB-associated missense mutations reduce the number of functional channels that reach the cell membrane rather than altering other aspects of channel function. The results presented in this thesis suggest that 4PBA may be a promising therapy for the treatment of ARB and the other bestrophinopathies resulting from missense mutations in BEST1, particularly as 4PBA is already approved for long-term use in infants and adults. These finding also pave the way for the use of small molecule therapies to treat conformational diseases caused by mutation in other protein expressed in the RPE.

It is well known that the costs associated with drug discovery are extremely high due the use of expensive in vitro methods as well as the high failure rate of drugs during clinical testing. In order to effectively fight the war against diseases such as malaria a far less expensive approach is required. Increasing the amount of in silico</i. work would decrease the amount of experiments that have to be done and so doing reduce the costs. In silico methods have the ability to predict major limiting factors in drug discovery resulting in only high quality drug molecules being subjected to clinical trials, thus increasing the probability of success. In this study a web-based chemoinformatics workspace aimed at research biologists was developed inside the FunGIMS application framework. This particular web-based chemoinformatics application was developed in the context of the FunGIMS suite of tools, specifically providing biological users with some of the most common chemoinformatics functions such as (1) the representation of chemical compounds, (2) chemical data, (3) databases and data sources, (4) structure search methods, (5) methods for calculating physical and chemical data (6) calculation of structure descriptors. The chemoinformatics module is supported by a modified version of the CHEBI database and the integration of OpenBabel and Frowns made it possible to perform analysis on molecules by means of SMILES-structures. As part of the validation of the chemoinformatics module of FunGIMS, the chemical space of the polyamine pathways in Plasmodium was explored in order to obtain a library of molecules based on similarities that could be possible lead-like compounds. This was used in a study of compounds against spermidine synthase from Plasmodium falciparum. By means of similarity and substructure searches the chemoinformatics module was able to produce molecules that are structurally similar. Additional filtering enabled a library of related molecules to be obtained, and used in a docking study. Compounds were found with high docking scores, thereby validating the effectiveness and usefulness of the chemoinformatics module of FunGIMS. Copyright / Dissertation (Msc)--University of Pretoria, 2009. / Biochemistry / unrestricted

While the human immunodeficiency virus (HIV) can be managed with antiretroviral therapy (ART), there is no cure for the disorder. If ART is discontinued, viral RNA levels rapidly increase in most individuals due to the presence of a cell-mediated hidden replication competent viral burden known as the viral reservoir. In order to successfully cure this disease, a mechanism to eliminate the viral reservoir must be developed. Preliminary research completed using a toll-like-receptor agonist 7 (TLR7) has shown favorable results supporting this goal. In a simian immunodeficiency virus (SIV) model, dosing rhesus macaques (RMs) with TLR7 agonists resulted in the development of controlled viremia. A controlled RM is a SIV positive animal that is able to maintain an undetectable viral load without continued therapeutic intervention. In cases of controlled SIV/HIV, viral RNA no longer replicates despite the discontinuation of all treatment. This implies that the viral reservoir is either completely eliminated or severely reduced. In this study, we quantified expression levels of several immune checkpoint and activation markers including CD69, CD39, CXCR5, TCF7, PD-1, PD-L1, TIGIT, CTLA-4, Tim-3, and Lag-3 on isolated peripheral blood mononuclear immune cells (PBMCs) [including CD4+ T cells, CD8+ T cells, natural killer (NK) cells, and B cells] in both controlled and non-controlled RMs. Our goal was to identify possible mechanisms by which controlled RMs are able to successfully modulate the host immune response after discontinuing TLR7 agonist treatment. The subjects each received one of two different TLR7 agonists (GS-9620 and GS-986). Isolated peripheral blood mononuclear cells (PBMCs) were obtained from two controlled RMs and two non-controlled RMs. Samples were analyzed using flow cytometry to identify and quantify levels of markers above. Expression levels of PD-1 and PD-L1 were elevated in PBMCs obtained from non-controlled RMs when compared to levels seen in controlled RMs. In contrast, levels of TIGIT and CTLA4 were downregulated in samples obtained from the controlled RMs. This suggests that immune checkpoint markers responsible for viral control and SIV/HIV pathogenesis have different functional roles. Additionally, the controlled RMs showed high expression of CD69 and CD39 on B cells and increased levels of CXCR5 on CD4+ T cells. This suggests that newly activated B cells likely contribute to the observed improvements in immune function. The results obtained provide favorable support for the potential role of immune checkpoint blockade as an HIV-specific immunotherapy that may contribute to the development of a controlled population. However, it is worthwhile to note that this study was completed using a relatively small sample size (n=4). Thus, interpretations of the findings herein must be replicated with a larger sample prior to forming any definitive conclusions.