Philosophiae Doctor - PhD / AIDS is considered a pandemic causing millions of deaths worldwide and a cure for this disease is still not available. Failure to implement early treatments due to the poor diagnostic methods and ineffective therapeutic regimens to treat HIV patients to achieve complete viral eradication from the human body has encouraged the escalation of this disease at an exponential rate. Though the current treatment regimens (High Active Antiretroviral Therapy) have aided in increasing the lifespan of HIV patients, it still suffers from some shortcomings such as adverse side effects and non-eradication of the virus. Thus, there is a need for a non-toxic therapeutic regimen to stop further infection of HIV-infected patients. Antimicrobial Peptides (AMPs) are naturally occurring peptides which are components of the first line of defence of many organisms against infections and have been proven to be promising therapeutic agents against HIV. The use of AMPs as anti-microbial agents is due to the fact that most AMPs have a net positive charge and are mostly hydrophobic molecules. These features allow AMPs to be site directed electro-statistically to the mostly negatively charged pathogens. In a previous study, a number of novel anti-HIV AMPs was identified using a predictive algorithm Profile Hidden Markov Models (HMMER). The AMP's threedimensional structures were predicted using an in-silico modelling tool I-TASSER and an insilico protein-peptide interaction study of the AMPs to HIV protein gp120 was performed using PatchDock. Five AMPs were identified to bind gp120, at the site where gp120 interacts with CD4 to prevent HIV invasion and HIV replication. Therefore, the aims of this research were to perform in-silico site-directed mutation on the parental anti-HIV AMPs to increase their binding affinity to the gp120 protein, validate the anti-HIV activity of these peptides and confirm the exclusivity of this activity by testing possible anti-bacterial and anti-cancer activities of the AMPs. Firstly, the five parental anti-HIV AMPs were used to generate mutated AMPs through insilico site-directed mutagenesis. The AMPs 3-D structures were determined using I-TASSER and the modelled AMPs were docked against the HIV protein gp120 using PatchDock. Secondly, an "in house" Lateral Flow Device (LFD) tool developed by our industrial partner, Medical Diagnostech (Pty) Ltd, was utilised to confirm the in-silico docking results. Furthermore, the ability of these AMPs to inhibit HIV-1 replication was demonstrated and additional biological activities of the peptides were shown on bacteria and cancer cell lines. In an effort to identify AMPs with increased binding affinity, the in-silico results showed that two mutated AMPs Molecule 1.1 and Molecule 8.1 bind gp120 with high affinity, at the point where gp120 bind with CD4. The molecular binding however showed that only Molecule 3 and Molecule 7 could prevent the interaction of gp120 protein and CD4 surface protein of human cells, in a competitive binding assay. Additionally, the testing of the anti-HIV activity of the AMPs showed that Molecule 7, Molecule 8 and Molecule 8.1 could inhibit HIV-1 NL4-3 with maximal effective concentration (EC₅₀) values of 37.5 μg/ml and 93.75 μg/ml respectively. The EC₅₀ of Molecule 8.1 was determined to be around 12.5 μg/ml. This result looks promising since 150 μg/ml of the AMPs could not achieve 80% toxicity of the human T cells, thus high Therapeutics Index (TI) might be obtained if 50% cytotoxic concentration (CC₅₀) is established. Further biological activity demonstrates that Molecule 3 and Molecule 7 inhibited P. aeruginosa completely after 24 hours treatment with peptide concentrations ranging from 0.5 mg/ml to 0.03125 mg/ml. Nevertheless, moderate inhibition was observed when CHO, HeLa, MCF-7 and HT-29 were treated with these peptides at peptides concentration of 100 μg/ml. The ability of these AMPs to block the entrance of HIV via the binding to CD4 of the host cells is a good concept since they pave the way for the design of anti-HIV peptide-based drugs Entry Inhibitors (FIs) or can be exploited in the production microbicide gels/films to suppress the propagation of the virus. / DST-NIC/Mintek

Masters of Science / The rapidly expanding field of nanotechnology has been the focus of many biologists with regard to drug delivery. The ability of nanoparticles to enter cellular compartments makes it possible to explore specific treatment strategies for life-threatening diseases such as AIDS. Since HIV primarily infects CD4+ cells, we aim to use CD4 as a selectable marker to deliver pro-apoptotic nano-devices to HIV infected cells. The objective is to selectively induce cell death or apoptosis in CD4+ HIV infected cells. Apoptosis is activated through a number of biochemical pathways. The apoptosis promoting protease, caspase-3 is central to the induction of apoptosis. Caspase-3 is produced as an inactive zymogen and is activated by other proteases through proteolytic cleavage. We take advantage of the fact that HIV-infected cells produce HIV-1 protease, which is responsible for the production of infectious virions through proteolytic cleavage of the HIV proteins, Gag and Pol. Our strategy was to generate a mutant form of the caspase-3 protease that is only cleavable by HIV-1 protease.

Protein sequencing has produced overwhelming amount of protein sequences, especially in the last decade. Nevertheless, the majority of the proteins' functional and structural classes are still unknown, and experimental methods currently used to determine these properties are very expensive, laborious and time consuming. Therefore, automated computational methods are urgently required to accurately and reliably predict functional and structural classes of the proteins. Several bioinformatics methods have been developed to determine such properties of the proteins directly from their sequence information. Such methods that involve signal processing methods have recently become popular in the bioinformatics area and been investigated for the analysis of DNA and protein sequences and shown to be useful and generally help better characterise the sequences. However, there are various technical issues that need to be addressed in order to overcome problems associated with the signal processing methods for the analysis of the proteins sequences. Amino acid indices that are used to transform the protein sequences into signals have various applications and can represent diverse features of the protein sequences and amino acids. As the majority of indices have similar features, this project proposes a new set of computationally derived indices that better represent the original group of indices. A study is also carried out that resulted in finding a unique and universal set of best discriminating amino acid indices for the characterisation of allergenic proteins. This analysis extracts features directly from the protein sequences by using Discrete Fourier Transform (DFT) to build a classification model based on Support Vector Machines (SVM) for the allergenic proteins. The proposed predictive model yields a higher and more reliable accuracy than those of the existing methods. A new method is proposed for performing a multiple sequence alignment. For this method, DFT-based method is used to construct a new distance matrix in combination with multiple amino acid indices that were used to encode protein sequences into numerical sequences. Additionally, a new type of substitution matrix is proposed where the physicochemical similarities between any given amino acids is calculated. These similarities were calculated based on the 25 amino acids indices selected, where each one represents a unique biological protein feature. The proposed multiple sequence alignment method yields a better and more reliable alignment than the existing methods. In order to evaluate complex information that is generated as a result of DFT, Complex Informational Spectrum Analysis (CISA) is developed and presented. As the results show, when protein classes present similarities or differences according to the Common Frequency Peak (CFP) in specific amino acid indices, then it is probable that these classes are related to the protein feature that the specific amino acid represents. By using only the absolute spectrum in the analysis of protein sequences using the informational spectrum analysis is proven to be insufficient, as biologically related features can appear individually either in the real or the imaginary spectrum. This is successfully demonstrated over the analysis of influenza neuraminidase protein sequences. Upon identification of a new protein, it is important to single out amino acid responsible for the structural and functional classification of the protein, as well as the amino acids contributing to the protein's specific biological characterisation. In this work, a novel approach is presented to identify and quantify the relationship between individual amino acids and the protein. This is successfully demonstrated over the analysis of influenza neuraminidase protein sequences. Characterisation and identification problem of the Influenza A virus protein sequences is tackled through a Subgroup Discovery (SD) algorithm, which can provide ancillary knowledge to the experts. The main objective of the case study was to derive interpretable knowledge for the influenza A virus problem and to consequently better describe the relationships between subtypes of this virus. Finally, by using DFT-based sequence-driven features a Support Vector Machine (SVM)-based classification model was built and tested, that yields higher predictive accuracy than that of SD. The methods developed and presented in this study yield promising results and can be easily applied to proteomic fields.

Actuellement le polyéthylènimine (PEI) est l’agent de transfection transitoire le plus utilisé par l’industrie pharmaceutique pour la production de protéines recombinantes à grande échelle par les cellules de mammifères. Il permet la condensation de l’ADN plasmidique (ADNp) en formant spontanément des nanoparticules positives appelées polyplexes, lui procurant la possibilité de s’attacher sur la membrane cellulaire afin d’être internalisé, ainsi qu’une protection face aux nucléases intracellulaires. Cependant, alors que les polyplexes s’attachent sur la quasi-totalité des cellules seulement 5 à 10 % de l’ADNp internalisé atteint leur noyau, ce qui indique que la majorité des polyplexes ne participent pas à l’expression du transgène. Ceci contraste avec l’efficacité des vecteurs viraux où une seule particule virale par cellule peut être suffisante. Les virus ont évolués afin d’exploiter les voies d’internalisation et de routage cellulaire pour exprimer efficacement leur matériel génétique. Nous avons donc supposé que l’exploitation des voies d’internalisation et de routage cellulaire d’un récepteur pourrait, de façon similaire à plusieurs virus, permettre d’optimiser le processus de transfection en réduisant les quantités d’ADNp et d’agent de transfection nécessaires. Une alternative au PEI pour transfecter les cellules de mammifèreest l’utilisation de protéines possédant un domaine de liaison à l’ADNp. Toutefois, leur utilisation reste marginale à cause de la grande quantité requise pour atteindre l’expression du transgène. Dans cette étude, nous avons utilisé le système E/Kcoil afin de cibler un récepteur membranaire dans le but de délivrer l’ADNp dans des cellules de mammifères. Le Ecoil et le Kcoil sont des heptapeptides répétés qui peuvent interagir ensemble avec une grande affinité et spécificité afin de former des structures coiled-coil. Nous avons fusionné le Ecoil avec des protéines capables d’interagir avec l’ADNp et le Kcoil avec un récepteur membranaire que nous avons surexprimé dans les cellules HEK293 de manière stable. Nous avons découvert que la réduction de la sulfatation de la surface cellulaire permettait l’attachement ciblé sur les cellules par l’intermédiaire du système E/Kcoil. Nous démontrons dans cette étude comment utiliser le système E/Kcoil et une protéine interagissant avec l’ADNp pour délivrer un transgène de manière ciblée. Cette nouvelle méthode de transfection permet de réduire les quantités de protéines nécessaires pour l’expression du transgène. / Pharmaceutical industry often employs polyethylenimine (PEI) for large scale protein production processes by transient transfection of mammalian cells. PEI condenses plasmid DNA (pDNA) by spontaneously forming positive nanoparticles known as polyplexes. Condensed pDNA is favoured for cell surface binding, internalization and protection from intracellular nucleases. While most of the cells efficiently uptake polyplexes, only 5 to 10% of captured pDNA reaches the nucleus for transgene expression. This suggests that polyplexes are hampered in their ability to route and to translocate to the nucleus necessitating large amounts of polyplexes to achieve high expression levels. By contrast, many viruses can efficiently transduce cells with only one or a few viral genome copies. Viruses have evolved to exploit cellular internalization and routing properties to express their own genetic material. We hypothesized that less pDNA would be used in an optimized transfection process if we exploited the internalization and routing properties that viruses use. DNA binding proteins could be used as an alternative to PEI to transfect mammalian cells. However, their usage is marginal due to the large protein quantities required to bind pDNA for transgene expression. If less pDNA is used less binding protein is needed. In this study, we used the E/Kcoil system to target a membrane receptor to deliver pDNA in mammalian cells. The Ecoil and Kcoil are two repeated heptapeptides which interact with a high affinity and specificity to form coiled-coil structures. We fused the Ecoil with a recombinant pDNA-binding protein. The Kcoil was fused to a stably-expressed membrane receptor in HEK293 cells. We discovered that low sulfation of the cell surface reduced non-specific binding of the pDNA:protein complex and permitted targeted binding via the E/Kcoil interaction. We demonstrate how to use recombinant pDNA-binding protein and the E/Kcoil system for targeted transgene delivery. This newly developed system provides a new transfection method, with reduced pDNA-binding protein quantities needed to achieve transgene expression.