The recent advent of bioinformatics has given rise to the central and recurrent problem of optimally aligning biological sequences. Many techniques have been proposed in an attempt to solve this complex problem with varying degrees of success. This thesis investigates the application of a computational intelligence technique known as particle swarm optimization (PSO) to the multiple sequence alignment (MSA) problem. Firstly, the performance of the standard PSO (S-PSO) and its characteristics are fully analyzed. Secondly, a scalability study is conducted that aims at expanding the S-PSO’s application to complex MSAs, as well as studying the behaviour of three other kinds of PSOs on the same problems. Experimental results show that the PSO is efficient in solving the MSA problem and compares positively with well-known CLUSTAL X and T-COFFEE. / Dissertation (MSc)--University of Pretoria, 2009. / Computer Science / Unrestricted

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Orientador: Lucimara Gaziola de la Torre / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-26T17:50:46Z (GMT). No. of bitstreams: 1 Sipoli_CarolineCasagrande_D.pdf: 4303538 bytes, checksum: 416499c84401c29f761041b6fe23813d (MD5) Previous issue date: 2014 / Resumo: O estudo de processos de produção de nanopartículas poliméricas, mais especificamente a quitosana (CHI), para aplicações em vacinação/terapia gênica é um desafio. Este trabalho teve como objetivo o desenvolvimento do processo de produção de nanopartículas de CHI com o agente reticulante tripolifosfato de sódio (TPP), sendo dividido em 3 etapas. A primeira etapa consistiu na produção destas partículas, avaliando a influência do pH da solução inicial de CHI (pH 4; 5 e 5,5). Para isso, utilizou-se um sistema de reator com chicanas e agitação mecânica com impelidor do tipo cowles facilmente escalonável. A caracterização físico-química das nanopartículas permitiu verificar as diferenças das partículas produzidas nos diferentes pH¿s. Um estudo de estabilidade simulando o ambiente de aplicação biológica foi realizado para avaliar as nanopartículas produzidas em pH¿s diferentes e verificou-se que as partículas produzidas nos pH¿s menores apresentaram maior estabilidade no período estudado (4 horas). A avaliaçãp biológica dos complexos preparados (DNA-CHI/TPP), em termos de transfecção in vitro em células HeLa indicou que não houve diferença entre as partículas produzidas nos diferentes pHs. A segunda, etapa do trabalho avaliou a influência da temperatura durante o processo de produção de nanopartículas de CHI/TPP utilizando o mesmo sistema anteriormente descrito. Foram avaliadas três diferentes condições isotérmicas e também a variação de temperatura ao longo da gelificação ionotrópica. Para este último caso, obteve-se nanopartículas com índice de polidispersidade significativamente menor do que os processos isotérmicos. 2 porcentagens de DNA (10 e 40% m/m) foram incoporadas às nanopartículas e a diferença em termos de pDNA incoporado apresentou diferentes eficiências de transfecção em células A293 ao longo do tempo, indicando uma liberação sustentada. Em termos de citotoxicidade, observou-se queda da viabilidade celular após 120 horas de incubação, sem ocorrer dependência da dose. Na terceira etapa realizou-se o uma análise exploratória para o desenvolvimento de um processo microfluídico para produção de partículas de CHI/TPP. Dois métodos foram estudados: focalização hidrodinâmica e emulsão temporária. O processo de emulsão temporária não foi reprodutível visto que a finalização desta etapa foi comprometida devido a precipitação da quitosana nos microcanais. As condições avaliadas para o método de focalização hidrodinâmica geraram nanopartículas de CHI/TPP com alto índice de polidispersidade. Este trabalho apresenta uma nova área de desenvolvimento de processos de produção de partículas poliméricas / Abstract: The study of polymeric nanoparticle production process, specifically chitosan (CHI), for gene vaccine/therapy application is a challenge. This work aims to the development of production process of chitosan (CHI) with the crosslinking agent pentasodium triphosphate (TPP), and it was divided into three steps. The first step was the production of these nanoparticles evaluating the influence of pH of chitosan solution (pH 4; 5; 5.5). For this purpose, the system used was a reactor with baffles, mechanical stirring with cowles impeller, easy to scale up. Physico-chemical characterization reveals the difference between the produced nanoparticle in the different pH¿s. One stability tests simulating the biological application environment was carried out to evaluate the nanoparticle produced in different pH¿s and it was possible to verify that the nanoparticles produced in lower pH¿s presented higher stability at the evaluated period (4 hours). The biological study of the prepared complexes (DNA-CHI/TPP), in terms of in vitro tranfection in in HeLa cells indicated no difference between between the nanoparticles produced in the different pHs. The second step of this work studied the influence of temperature the CHI/TPP nanoparticles production using the same system as previously reported. Three isothermal conditions and also temperature variation during the ionic gelation were tested. For this last condition, nanoparticles produced were significantly smaller in terms of PDI if compared to the isothermal process. 2 percentage of pDNA were incorporated (10 and 40% w/w) to the nanoparticles and the difference in terms of amount of pDNA showed different transfection efficiency in A293 cells over the time, suggesting sustained release capability. In terms of cytotoxicity, it was noticed that the cell viability decreased after 120 hours of incubation, and it is no dose-dependent. The third step was the exploratory investigation of microfluidic processes to obtain CHI/TPP nanoparticles. Two methods were studied: (i) temporary emulsion and (ii) hydrodynamic flow focusing. The temporary emulsion process was not reproducible since this step was not concluded due to the chitosan precipitation inside the microchannels. In hydrodynamic flow focusing, CHI/TPP nanoparticles presented high values of polydispersity index at the studied conditions. This work presents new area in the development of polymeric nanoparticles production processes. Keywords: chitosan nanoparticles, sodium triphsosphate, pH, temperature, plasmidial DNA, microfluidic, HeLa cells, A293 cells / Doutorado / Engenharia Química / Doutora em Engenharia Quimica

Feline babesiosis, first described in domestic cats in South Africa in 1937, is regarded to be of great importance in the coastal regions although isolated cases also occur on the eastern highlands of Mpumalanga Province. Babesia felis (described from domestic cats) and B. leo (described from lions) are the two best characterised Babesia species in felids. These two parasites are morphologically similar when examined under a light microscope, but are serologically and genetically distinct. In this study the prevalence of these two Babesia species in various wild and domestic felid species was determined. A total of 358 samples were tested using the reverse line blot hybridization (RLB) assay. This assay makes it possible to simultaneously detect and differentiate between blood parasites using DNA probes. The RLB consists of three basic steps, the first being amplification of the variable region (V4) in the 18S rRNA gene using genus-specific primers where one is labelled with biotin. This is followed by a blotting step, where the amplicons are hybridized to oligonucleotides bound to a nitrocellulose membrane. The third and last step is the detection of the hybridized amplicons by using chemiluminescence reagents. This assay is a screening tool utilizing the variable (V4) region in the 18S rRNA gene to detect and differentiate between blood parasites. A new B. felis-specific DNA probe was developed to use in the RLB assay. Results demonstrated that these two parasites not only occur in the felid species from which they have been described, but also in other felid species. Babesia microti was also detected in various felid species, while B. rossi was detected in 1 of the lion samples. Two hundred and twelve samples tested positive for Babesia spp., of which only 54.24% of the samples reacted with the genus-specific probe. This indicates the presence of a novel Babesia or Theileria species or variant of a species. / Dissertation (MSc)--University of Pretoria, 2010. / Veterinary Tropical Diseases / unrestricted

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The fact that loosely bonded DNA bases could tolerate large structural fluctuations, form a dissipative environment for a charge traveling through the DNA. Nonlinear stochastic nature of structural fluctuations facilitates rich charge dynamics in DNA. We study the complex charge dynamics by solving a nonlinear, stochastic, coupled system of differential equations. Charge transfer between donor and acceptor in DNA occurs via different mechanisms depending on the distance between donor and acceptor. It changes from tunneling regime to a polaron assisted hopping regime depending on the donor-acceptor separation. Also we found that charge transport strongly depends on the feasibility of polaron formation. Hence it has complex dependence on temperature and charge-vibrations coupling strength. Mismatched base pairs, such as different conformations of the G・A mispair, cause only minor structural changes in the host DNA molecule, thereby making mispair recognition an arduous task. Electron transport in DNA that depends strongly on the hopping transfer integrals between the nearest base pairs, which in turn are affected by the presence of a mispair, might be an attractive approach in this regard. I report here on our investigations, via the I –V characteristics, of the effect of a mispair on the electrical properties of homogeneous and generic DNA molecules. The I –V characteristics of DNA were studied numerically within the double-stranded tight-binding model. The parameters of the tight-binding model, such as the transfer integrals and on-site energies, are determined from first-principles calculations. The changes in electrical current through the DNA chain due to the presence of a mispair depend on the conformation of the G・A mispair and are appreciable for DNA consisting of up to 90 base pairs. For homogeneous DNA sequences the current through DNA is suppressed and the strongest suppression is realized for the G(anti)・A(syn) conformation of the G・A mispair. For inhomogeneous (generic) DNA molecules, the mispair result can be either suppression or an enhancement of the current, depending on the type of mispairs and actual DNA sequence.

Software to display and analyse DNA sequences is a crucial tool for bioinformatics research. The data of a DNA sequence has a relatively simple format but the length and sheer volume of data can create difficulties in navigation while maintaining overall context. This is one reason that current bioinformatics applications can be difficult to use. This research examines techniques for navigating through large single DNA sequences and their annotations. Navigation in DNA sequences is considered here in terms of the navigational activities: exploration, wayfinding and identifying objects. A process incorporating user-centred design was used to create prototypes involving panning and zooming of DNA sequences. This approach included a questionnaire to define the target users and their goals, an examination of existing bioinformatics applications to identify navigation designs, a heuristic evaluation of those designs, and a usability study of prototypes. Three designs for panning and five designs for zooming were selected for development. During usability testing, users were asked to perform common navigational activities using each of the designs. The “Connected View” design was found to be the most usable for panning while the “Zoom Slider” design was best for zooming and most useful zooming tool for tasks involving browsing. For some tasks the ability to zoom was unnecessary. The research provides important insights into the expectations that researchers have of bioinformatics applications and suitable methods for designing for that audience. The outcomes of this type of research can be used to help improve bioinformatics applications so that they will be truly usable by researchers.

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Organometallic chemotherapeutic agents, many of which target DNA, have been shown to be effective in the treatment of cancer. With that said though, these compounds have a number of side affects such as nephrotoxicity. Two novel compounds, Ferrocene [ferrocenoyltrichloroacetone] and Rhodium-ferrocene [(1.5 cyclooctadiene)(1-ferrocenyl- 4,4,4-trichloro-1,3-butanedionate], synthesised by the research group of J Swarts (University of the Free State) were evaluated to determine their mechanism of action and their potential use as novel therapeutic agents. It is hypothesized, by merit of their chemical structures, that these compounds’ anti-cancer activity is due to their interaction with DNA. Both drugs were evaluated from a cellular to a molecular level, in vitro, to validate this hypothesis. Linearised DNA was exposed to both drugs and digested with a variety of restriction enzymes. It was found that the compounds bind to the PstI restriction site; thereby inhibiting the enzyme’s restriction activity. From this point it was necessary to show that the compounds are able to interact with DNA in a cellular system. By exposing a transformed breast epithelial cell line (MCF-12A) and a cancerous breast epithelial cell line (MCF-7) to the compounds, for various times, followed by flow cytometric analyses, it was found that both affect progression through the cell cycle. Cells accumulated at various phases of the cell cycle, as a result of checkpoint gene activation. Further flow cytometric analyses showed that both drugs induce necrosis in MCF-7 cells. The “normal” cell line however did not show this response as it is believed that cell cycle arrest and repair mechanisms were initiated, which would delay cell death. Gene expression analyses were performed by reverse transcriptase real-time PCR in which panels of cell cycle related genes as well as DNA damage associated genes were probed in two separate array formats. These studies revealed that a number of DNA damage and repair genes are activated; specifically those associated with excision repair and free-radical induced DNA damage. Members of the RAD family as well as the genes GADD45A, XPC and OGG1 were found to be upregulated as a result of Ferrocene treatment. This could be expected as it was shown that ferrocene binds to DNA, and it logically then follows that this would lead to excision repair being attempted by the cell. Similar gene expression patterns were found following Rhodium-ferrocene treatment with the up-regulation of genes such as OGG1, ATM and GADD45G, albeit to a lesser extent. It is hypothesised that the larger molecule may not interact as effectively with DNA, due to steric hinderance. Arrest mechanisms, for both drugs, were more pronounced in the “normal” cell line and it is believed that this is due to the fact that many of these genes have been inactivated in the cancerous cell line. We have shown, on multiple levels, that both compounds’ therapeutic action is as a result of their interaction with the cell’s DNA. This interaction leads to cell death in both the transformed and the cancerous cell line. In order to clarify these mechanisms it is suggested that proteomic and metabolomic studies should be performed. / Dissertation (MSc)--University of Pretoria, 2012. / Genetics / unrestricted

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