Em 2015, de acordo com a Organização Mundial da Saúde, o total de mortes na população humana foi de 52,5 milhões de pessoas, em que as doenças infecciosas mais especificamente, infecções respiratórias - aparecem em terceiro lugar no ranking de causas de morte. No total foram 3,5 milhões de mortes ou 6,7% do total, demonstrando um aumento significativo quando comparado com a pesquisa anterior, realizada em 2011, na qual as doenças infecciosas foram responsáveis por 3,2 milhões ou 5,9% do total. Das doenças infecciosas, podemos destacar a Malária, HIV/AIDS, Hepatite, Tuberculose - que aparece em 8º lugar neste mesmo ranking, e a Hanseníase - que é uma importante preocupação no Brasil. A tuberculose, causada pelo Mycobacterium tuberculosis sendo uma das principais causas de morte por doenças infecciosas na população humana, já apresenta cepas resistentes ao tratamento denominadas Cepas multirresistentes (MDR) e Cepas extremamente resistentes (XDR). Enquanto que a hanseníase, causada pelo Mycobacterium leprae, apesar de não ser letal, causa a invalidez dos portadores e o Brasil possui o segundo maior número de casos desta doença. Outro patógeno de importância médica é a Pseudomonas aeruginosa que causa sérias doenças em pacientes com baixa imunidade, principalmente aqueles em hospitais em especial, pacientes com queimaduras. A via do Folato tem despertado a atenção das indústrias farmacêuticas para o desenvolvimento de novos fármacos, uma vez que está via oferece seletividade para estes novos inibidores. Isso se deve ao fato de que o tetrahidrofolato é um componente essencial para os organismos procariotos, e apenas eles, precisam realizar a produção de novo desta substância, enquanto que os eucariotos, o adquirem através da alimentação. Entretanto, o surgimento de cepas resistentes não só reforça a necessidade do desenvolvimento de novos fármacos, mas também a compreensão dos mecanismos de resistências destas cepas. O presente trabalho tem como objetivo obter informações biofísicas e estruturais de três enzimas da via do folato, a 7,8 dihidroneopterina aldolase, dihidropteroato sintase e dihidrofolato redutase. Além disso, esse trabalho teve como objetivo a compreensão do mecanismo de resistência através da indução de mutações nos códons 53 e 55 na enzima dihidropteroato sintase (DHPS), caracterização estrutural preliminar da enzima DHNA e estudos estruturais da enzima DHFR em complexo com cicloguanil. Assim, neste trabalho foi realizado a clonagem gênica da região codificando do gene para a enzima DHNA e expressão da proteína recombinante, purificada e utilizada em ensaios biofísicos. Além disso a mutagênese sítio dirigida foi feita com o intuito de se produzir DHPSs mutantes (T70S e P72R), na qual foram utilizadas nos ensaios de ITC, fluorescência, e cristalização, o que possibilitou demonstrar que a resistência de M. leprae às sulfas pode ser devido a troca de uma prolina por uma arginina na posição 72. Por fim, para DHFR, foi possível obter sua estrutura em complexo com cicloguanil que possibilitou avaliar as diferenças estruturais entre a forma aberta e fechada desta enzima. Além disso, confirmamos que o grupo nicotidamida tem essencial importância para a interação com os ligantes. / In 2015, according to the World Health Organization, the total number of deaths in the human population was 52.5 million people, in which infectious diseases - specifically, respiratory infections - appear third in the ranking of causes of death. In total, 3.5 million deaths or 6.7% of the total occurred, showing a significant increase when compared to the previous survey conducted in 2011, in which infectious diseases accounted for 3.2 million or 5.9% of the total total. Of the infectious diseases, we can highlight Malaria, HIV / AIDS, Hepatitis, Tuberculosis - which appears in 8th place in this same ranking, and Leprosy - which is an important concern in Brazil. Tuberculosis, caused by Mycobacterium tuberculosis is one of the main causes of death due to infectious diseases in the human population, and already presents resistant strains to the treatment - denominated multiresistant strains (MDR) and extremely resistant strains (XDR). While leprosy, caused by Mycobacterium leprae, despite not lethal, causes the invalidity of patients and Brazil has the second largest number of cases for this disease. Another pathogen of medical importance is Pseudomonas aeruginosa, which causes serious illness in patients with low immunity, especially those in hospitals - especially patients with burns. The Folate pathway has brought the attention of the pharmaceutical industries to the development of new drugs, since this pathway offers selectivity for these new inhibitors. This is since Tetrahydrofolate is an essential component for prokaryote organisms, and only they, need to perform the new production of this cofactor, while Eukaryotes acquire it through food. However, the emergence of resistant strains, not only reinforces the need for the development of new drugs, but also the understanding of the mechanisms of resistance of these strains. The aim of the present work was to obtain biophysical and structural information of three folate pathway enzymes, 7,8 dihydroneopterin aldolase, Dihydropteroate synthase and Dihydrofolate reductase. In addition, this work aimed to understand the mechanism of resistance through the induction of mutations at the codons 53 and 55 in the enzyme dihydropteroate synthase (DHPS), preliminary structurally characterize the DHNA enzyme and structurally study the enzyme DHFR in complex with cycloguanil. Thus, the cloning of the coding region of the gene for DHNA and the expression of the recombinant protein, purification and biophysical characterization were performed in this work. In addition, site-directed mutagenesis was performed aiming of producing mutant DHPSs (T70S and P72R), which were used in ITC, fluorescence, and crystallization tests to demonstrate that M. lepreae resistance to sulfas may be due to the exchange of a proline to an arginine at position 72. Finally, for DHFR, it was possible to obtain its structure in complex with cycloguanil which made it possible to evaluate the structural differences between the open and closed conformation of this enzyme. In addition, we have confirmed that the nicotidamide group has a crucial importance for interaction with the ligands.

Thesis submitted in fulfilment of the requirements for the degree Master of Technology: Biomedical Technology in the Faculty of Health and Wellness Sciences at the Cape Peninsula University of Technology, 2012 / In many developed countries worldwide the provision of safe, clean water is an expected commodity. In South Africa however, as in most developing countries, the access and supply of water safe for human consumption is challenged or complicated by pollution and more recently water availability. Point-source pollutants in surface- and groundwater are normally the most concentrated closest to the pollutant source (such as the end of a pipe or an underground injection system). Examples of point-source pollution are commercial and industrial businesses, that often discharge waste such as solvents and heavy metals from their operations. In contrast, non-point-source pollution occurs due to runoff moving across or through the ground and absorbing and accumulating pollutants which eventually end up in streams, rivers and dams. The lack of waste removal and adequate sanitation facilities results in the disposal of faecal matter and sewage into storm water drains which flow directly into the river systems contributing to the incidence of diseases such as gastroenteritis, diarrhoea and chronic lung ailments, caused by waterborne pathogenic bacteria, viruses and fungi. Routine water quality analysis however, does not include monitoring for viral contaminants, as this process is hampered by the lack of simple, reliable, time- and cost-effective testing methods to concentrate and detect viral pathogens. The primary aim of this study was thus to establish and optimise routine monitoring techniques for the detection of rota-, adeno- and enteroviruses in the Berg- and Plankenburg Rivers, Western Cape. Initially, various concentration and extraction methods were compared for the optimum recovery of viruses from spiked water samples. One hundred milliliter water samples were spiked with one milliliter rotavirus and two milliliters adenovirus control virions (Coris Bioconcept, Gembloux, Belgium). Optimisation testing of enterovirus was however, not completed due to the unavailability of a positive control. Four viral concentration techniques, namely the Silicon dioxide (SiO2) method, positively charged, negatively charged and the mixed-ester filters, were compared. Various nucleic acid extraction methods were also employed to establish which method would provide optimum yields for both DNA and RNA nucleic acids. The extraction techniques included the TRIzol method (Invitrogen, California, USA) for RNA extraction, the Roche High Pure PCR Template Preparation kit (Roche, Mannheim, Germany) for DNA extraction, and the QIAamp Ultrasens Virus kit (Qiagen GmbH, Hilden, Germany) for simultaneous RNA and DNA extraction. The use of virus specific primers within the PCR technique was also optimised. In addition, gene specific primers and oligo(dT)15 primers were tested and compared to establish which primers would yield the best results since gene specific primers are said to be more sensitive than oligo(dT)15 primers (van Pelt-Verkuil et al., 2008) when synthesising cDNA (rotavirus). The SiO2 concentration method yielded variable results when it was used with the various nucleic acid extraction techniques in this study, since positive PCR results were obtained when used in combination III with some techniques, while negative results were obtained with others. Similarly, variable results were also obtained when negatively charged filters were used to concentrate virus particles, and when this method was used in conjunction with various virus nucleic acid extraction techniques to identify different viruses by RT-PCR and PCR. Results for the non-charged mixed-ester filter were comparable to the positively charged filters when used in conjunction with the various nucleic acid extraction techniques in this study. Both these techniques yielded the highest viral particle concentration from the spiked water samples. Pilot study results indicated the presence of rotavirus and adenovirus detected by RT-PCR and PCR respectively, when filtering through the positively charged filter. The positively charged filter/QIAamp UltraSens virus kit combination was found to be the optimum combination when analysing the spiked water results and was then employed for the concentration of virus particles in the river water samples collected from the Plankenburg- and Berg River systems throughout the study period. The expected PCR product of 346 bp for rotavirus was absent in all 72 river water samples analysed for both river systems. In contrast to the PCR results obtained for rotavirus, the expected product of 261 bp for adenovirus was detected in 22 (30.5%) samples collected throughout the study period. Fifteen of the 22 adenovirus positive samples were found in the Plankenburg River (distributed over all sites), while seven of the 22 adenovirus positive samples were found in the Berg River (all sites). A nested PCR was used to detect enterovirus in the river water samples collected from both river systems throughout the study period. In the first round of the enterovirus PCR 15 river water samples (at various sites for both river systems) yielded a faint 513 bp product. Further amplification by nested PCR then yielded 13 (18.1%) positive nested PCR products of 297 bp. The incidence of adenovirus and enterovirus in river waters reported in the current study and the Van Heerden et al. (2003) investigation motivates for similar studies to be conducted in drinking water, dam water used for recreational purposes as well as rainwater, which is gaining popularity as a sustainable water source.

Thesis (MTech (Biomedical Technology))--Cape Peninsula University of Technology, 2012. / In many developed countries worldwide the provision of safe, clean water is an expected commodity. In South Africa however, as in most developing countries, the access and supply of water safe for human consumption is challenged or complicated by pollution and more recently water availability. Point-source pollutants in surface- and groundwater are normally the most concentrated closest to the pollutant source (such as the end of a pipe or an underground injection system). Examples of point-source pollution are commercial and industrial businesses, that often discharge waste such as solvents and heavy metals from their operations. In contrast, non-point-source pollution occurs due to runoff moving across or through the ground and absorbing and accumulating pollutants which eventually end up in streams, rivers and dams. The lack of waste removal and adequate sanitation facilities results in the disposal of faecal matter and sewage into storm water drains which flow directly into the river systems contributing to the incidence of diseases such as gastroenteritis, diarrhoea and chronic lung ailments, caused by waterborne pathogenic bacteria, viruses and fungi.