<p> Proteins vary tremendously in many of their physical and chemical properties. In order to perform in vitro application or analysis, one protein must be separated from other cellular components. This process is called protein purification. With the advances of modem science and technology, many protein purification schemes have been developed. Among them, the ELP-intein protein purification system has recently attracted an increasing amount of attention because of its positive characteristics: it is simple, inexpensive, scalable, with a high throughput, protease-free, etc. However, although the scientific literature reports all those good aspects of the system, several bad responses to it still exist. In this thesis, through comparing expression and purification of four different self-cleavage ELP fusion proteins, we propose a general solution to these problems for the first time. This makes a significant contribution to increased utility of the method of protein purification using self-cleavable stimulus responsive tags. </p> <p> When ELP-intein fusion proteins are expressed in bacteria, formation of non-native cytoplasmic aggregates (inclusion bodies) is a common problem which affects the yield of target protein. Inclusion bodies are generally assumed to contain misfolded or partially folded protein through exposure of hydrophobic patches and the consequent intermolecular interactions. Despite a loss of total expression yield and the need for more time, culturing at a lower temperature was reported to promote the expression of genes into soluble proteins and alleviate IB formation. Directly motivated by previous reports, we have applied a low-temperature expression strategy to solve the problems in this research. As expected, most of the T4-ELP inclusion bodies disappeared, and were transformed into a soluble expression, when culturing at lower temperatures. </p> <p> Inverse transition cycling (lTC), as the core method for the system we investigated has proved successful in the past with proteins that were expressed to high levels. However poor level ELP-intein tagged protein expression happens from time to time. It is hypothesized that if an ELP tagged molecule is present in a solution at a very low concentration, adding an excess amount of free ELP to the sample would form hybrid aggregates via the interaction of ELP moieties of the two molecules. We used this efficient and reversible capture system for low yield recombinant protein purification, and found it is perform very well. </p> / Thesis / Master of Applied Science (MASc)

Orientador: Sérgio Luís de Carvalho / Banca: Edson Pereira Tangerino / Banca: Beatriz Susana Ovruski de Ceballos / Resumo: O aumento na contaminação dos mananciais destinados ao abastecimento público por fármacos e sua freqüente ocorrência no ambiente aquático e na água potável tem levantado a questão sobre o seu impacto no ambiente e na saúde pública. Estes compostos constituem uma classe de micropoluentes ambientais de elevada persistência na água e de difícil remoção em sistemas convencionais de tratamento de água. Possuem alto potencial para bioacumulação e baixa biodegradabilidade, podendo ter efeitos adversos. O uso de filtro lento de areia com ação ecológica, melhor denominado como "Sistema de Purificação Ecológica", representa uma promissora tecnologia de tratamento, em razão desta não necessitar da aplicação de produtos químicos, bem como sua constatada capacidade em remoção de diversos compostos. Através deste processo pode-se oferecer água de baixo custo, insípida, inodora, incolor e segura. Dentro do tanque de filtração ecológica estabelece-se entre os seres vivos, a relação de cadeia alimentar. O uso de filtros de carvão biologicamente ativado para remoção de fármacos vem sendo estudado, e mostrando grande eficiência. Nessa pesquisa foram aplicados quatro compostos farmacológicos diferentes (Diclofenaco, Naproxeno, Ibuprofeno e Paracetamol), e houve remoção de todos eles, tanto no FEco como no FCB durante as etapas 1, 2 e 3. Outros parâmetros analisados, como turbidez, coliformes totais e termotolerantes, também foram removidos. Foi identificada grande variedade de algas e cianobactérias no Filtro Ecológico, e os resultados obtidos permitem supor que tiveram importantíssima ação nesse filtro, sendo um dos compartimentos biológicos mais importantes, representando a base de toda a cadeia trófica deste sistema. As porcentagens de remoção dos fármacos foram: diclofenaco: 97,43%, ibuprofeno: 85,03%, naproxeno: 94,11% e paracetamol: 84,07% / Abstract: The increase in the contamination of water sources for public supply by drugs and the frequent occurrence in the aquatic environment and drinking water has raised the question of its impact on the environment and public health. These compounds are a class of highly persistent environmental micropollutants in water and difficult to remove in conventional water treatment. Have high potential for bioaccumulation and low biodegradability, which can have adverse effects. The use of slow sand filter with eco- action, better known as "Ecological Purification System", represents a promising treatment technology, because this does not require the application of chemicals, as well as its observed ability to remove various compounds. Through this process we can offer low-cost water, tasteless, odorless, colorless and safe.Within the ecological filtration tank is between living beings, the relationship of the food chain. The use of biologically activated carbon filters for removal of drugs has been studied, and showing great efficiency. In this research were applied four different pharmacological compounds (Diclofenac, Naproxen, Ibuprofen and Paracetamol) and there was removal of all of them, both as in FEco FCB during steps 1, 2 and 3. Other parameters, turbidity, total and thermotolerant coliforms were also removed. Was identified wide variety of algae and cyanobacteria in ecological filter, and the results obtained allow to suppose that they had important work in this filter, one of the most important biological compartments, representing the basis of the whole food chain of this system. The percentages of removal of drugs were: diclofenac: 97.43%, ibuprofen: 85.03%, naproxen, acetaminophen and 94.11%, 84.07% / Mestre

DNA in cells is constantly damaged by both endogenous and exogenous genotoxic agents. DNA repair is a cellular machinery that counters DNA damage and thus preserve genome integrity. Nucleotide excision repair (NER) in Escherichia coli (E. coli) is one of the DNA repair systems that recognizes and removes a variety of DNA damage such as pyrimidine dimers, bulky chemical adducts, DNA intrastrand cross-links, etc. The genes responsible for E. coli NER incisions are UvrA, UvrB, and UvrC. As the first step of E. coli NER, DNA damage recognition is achieved through the UvrA2B complex. Purification of UvrA, UvrB, and UvrC is essential for research to understand the molecular mechanisms of NER and carcinogenesis. Although UvrA, a 115 kDa protein, has been successfully purified in our lab in the past, the experimental procedures were very time-consuming and technically challenging. In this study we employed IMPACT (Intein Mediated Purification with an Affinity Chitin-binding Tag) system to subclone the cDNA of UvrA and express and purify the recombinant UvrA protein by a single-column step using the cloned expression construct. Furthermore, the purified protein was found to be fully functional in the UvrABC incision assay in which the DNA adduct of FABP [N-(20-deoxyguanosin-8-yl)-4-fluoro-4-aminobiphenyl] was efficiently cleaved in a time course-dependent manner.

Made available in DSpace on 2014-06-11T19:29:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-08Bitstream added on 2014-06-13T20:38:37Z : No. of bitstreams: 1 erba_cm_me_ilha.pdf: 2751709 bytes, checksum: f13703e351afb548144d878131c5c8a2 (MD5) / O aumento na contaminação dos mananciais destinados ao abastecimento público por fármacos e sua freqüente ocorrência no ambiente aquático e na água potável tem levantado a questão sobre o seu impacto no ambiente e na saúde pública. Estes compostos constituem uma classe de micropoluentes ambientais de elevada persistência na água e de difícil remoção em sistemas convencionais de tratamento de água. Possuem alto potencial para bioacumulação e baixa biodegradabilidade, podendo ter efeitos adversos. O uso de filtro lento de areia com ação ecológica, melhor denominado como “Sistema de Purificação Ecológica”, representa uma promissora tecnologia de tratamento, em razão desta não necessitar da aplicação de produtos químicos, bem como sua constatada capacidade em remoção de diversos compostos. Através deste processo pode-se oferecer água de baixo custo, insípida, inodora, incolor e segura. Dentro do tanque de filtração ecológica estabelece-se entre os seres vivos, a relação de cadeia alimentar. O uso de filtros de carvão biologicamente ativado para remoção de fármacos vem sendo estudado, e mostrando grande eficiência. Nessa pesquisa foram aplicados quatro compostos farmacológicos diferentes (Diclofenaco, Naproxeno, Ibuprofeno e Paracetamol), e houve remoção de todos eles, tanto no FEco como no FCB durante as etapas 1, 2 e 3. Outros parâmetros analisados, como turbidez, coliformes totais e termotolerantes, também foram removidos. Foi identificada grande variedade de algas e cianobactérias no Filtro Ecológico, e os resultados obtidos permitem supor que tiveram importantíssima ação nesse filtro, sendo um dos compartimentos biológicos mais importantes, representando a base de toda a cadeia trófica deste sistema. As porcentagens de remoção dos fármacos foram: diclofenaco: 97,43%, ibuprofeno: 85,03%, naproxeno: 94,11% e paracetamol: 84,07% / The increase in the contamination of water sources for public supply by drugs and the frequent occurrence in the aquatic environment and drinking water has raised the question of its impact on the environment and public health. These compounds are a class of highly persistent environmental micropollutants in water and difficult to remove in conventional water treatment. Have high potential for bioaccumulation and low biodegradability, which can have adverse effects. The use of slow sand filter with eco- action, better known as Ecological Purification System, represents a promising treatment technology, because this does not require the application of chemicals, as well as its observed ability to remove various compounds. Through this process we can offer low-cost water, tasteless, odorless, colorless and safe.Within the ecological filtration tank is between living beings, the relationship of the food chain. The use of biologically activated carbon filters for removal of drugs has been studied, and showing great efficiency. In this research were applied four different pharmacological compounds (Diclofenac, Naproxen, Ibuprofen and Paracetamol) and there was removal of all of them, both as in FEco FCB during steps 1, 2 and 3. Other parameters, turbidity, total and thermotolerant coliforms were also removed. Was identified wide variety of algae and cyanobacteria in ecological filter, and the results obtained allow to suppose that they had important work in this filter, one of the most important biological compartments, representing the basis of the whole food chain of this system. The percentages of removal of drugs were: diclofenac: 97.43%, ibuprofen: 85.03%, naproxen, acetaminophen and 94.11%, 84.07%

Devido ao número crescente de surtos de infecção hospitalar, torna-se proeminente o estabelecimento de um programa de sanitização que liste os agentes químicos a serem empregados e o modo de aplicação mais efetivo. Processos de desinfecção também são relevantes em sistemas de tratamento de água (em indústrias farmacêuticas e centros de saúde) para que a qualidade da água seja assegurada e atenda os parâmetros estabelecidos, evitando proliferação microbiana. Validação da eficácia de descontaminação é uma tarefa ao mesmo tempo importante e desafiadora. Indicadores biológicos são sistemas ou moléculas que detectam atividade biológica, permitindo a validação de processos de descontaminação ou desinfecção. O indicador biológico pode ser uma suspensão de microrganismos específicos (sistema biológico) com resistência definida a um determinado processo de descontaminação. Enzimas e proteínas também têm sido empregadas como indicadores biológicos para avaliar a eficácia de processos industriais. A proteína verde fluorescente (GFP) tem sido sugerida como potencial indicador biológico para tratamentos de desinfecção, devido facilidade de sua detecção por espectrofluorimetria ou por inspeção visual. Para estudar e comparar o comportamento dos microrganismos selecionados e da GFP foram realizados ensaios de concentração inibitória mínima (CIM) e tempo de redução decimal (valor D). A CIM capaz de reduzir o bioburden inicial (>8log10) foi: 59 - 156 mg/L- 3250 mg/mL de glutaraldeído, 39 - 246 mg/L de formaldeído, 43750 - 87500 mg/L de álcool etílico 1250 - 6250 mg/L de polivinilpirrolidona iodo, 150 - 4491 mg/L de compostos liberadores de cloro, 469 -2500 mg/L de peróxido de hidrogênio e 2310 - 18500 mg/L de ácido peracético. A. calcoaceticus apresentou resistência à maioria dos agentes químicos testados, seguido de E. cloacae e S. marcescens. No sistema de purificação de água os resultados para Pseudomonas aeruginosa foram: (i) 0,5% de ácido cítrico, D = 3,8 min; (ii) 0,5% de ácido clorídrico, D = 6,9 min; (iii) 70% álcool etílico, D = 9,7 min; (iv) 0,5% bissulfito de sódio, D = 5,3 min; (v) 0,4% de hidróxido de sódio, D = 14,2 min; (vi) 0,5% de hipoclorito de sódio, D = 7,9 min; (vii) mistura de peróxido de hidrogênio (2,2%) e ácido peracético (0,45%), D = 5,5 min. GFP testada frente a soluções cloradas (com diferentes valores de pH e concentração) resultou em diminuição da fluorescência, sendo mais evidente em concentrações de cloro maiores que 150 ppm, com valores D entre 1,3 - 1,7 min. Em soluções de cloro em tampão fosfato (pH=7,15±0,08), a proteína manteve sua estrutura em contato com soluções 52-94 ppm de cloro. Frente a 110 ppm de cloro a estabilidade da proteína foi reduzida 10 vezes. A proteína GFP se mostrou um marcador fluorescente apropriado para monitorar eficácia de desinfecção. Para ser empregada como indicador biológico a proteína deve ser purificada através de um método de fácil ampliação de escala e com boa relação custo benefício, com este intuito o sistema micelar de duas fases aquosas foi estudado, e a proteína GFP foi parcialmente recuperada do homogeneizado celular de E. coli, outros contaminantes presentes no meio foram removidos na mesma etapa. A demonstração de que é possível se extrair uma biomolécula alvo utilizando ligantes de afinidade representa um passo importante no desenvolvimento de um método eficaz de separação que poderá ser utilizado na purificação de outras biomoléculas. / Due to the growing number of outbreaks of infection in hospital and nurseries, it becomes essential to set up a sanitation program that indicates that the appropriate chemical agent was chosen for application in the most effective way. Disinfection processes are also relevant in water purification systems (in pharmaceutical industries and in health environments) to assure water quality, meeting ionic and organic chemical standards, and avoiding microbial proliferation. Validating the effectiveness of decontamination and disinfection is an important and often challenging task. A biological indicator is a system or a molecule that enables the detection of biological activity, and as such, permits the validation of decontamination or disinfection treatments. The biological indicator can be a specific microorganism suspension (microbiological test system) with a defined resistance to a particular decontamination treatment. Enzymes and proteins have also been used as biological indicators to evaluate the immediate efficacy of industrial procedures, such as blanching, pasteurization, and disinfection treatments, as well as to monitor the satisfactory preservation of a product subjected to disinfection or sterilization. Green fluorescent protein (GFP) has been proposed as an ideal choice for a protein-based biological indicator for use in the validation of decontamination or disinfection treatments. In order to study and compare microorganism (in hospital infections outbreaks and isolated from the water purification system) and protein behavior, microorganisms were submitted to minimal inhibitory concentration (CIM) and decimal reduction time determination (D value). The CIM intervals, which reduced bacteria populations over 8log10, were: 59 to 156 mg/L of quaternarium ammonium compounds (QACs); 63 to 10000 mg/L of chlorhexidine; 1375 to 3250 mg/L of glutaraldehyde; 39 to 246 mg/L of formaldehyde; 43750 to 87500 mg/L of ethanol; 1250 to 6250 mg/L of iodine in polyvinyl-pyrolidone complexes, 150 to 4491 mg/L of chlorine-releasing-agents (CRAs); 469 to 2500 mg/L of hydrogen peroxide; and, 2310 to 18500 mg/L of peracetic acid. Chlorhexidine showed non inhibitory activity over germinating spores. A. calcoaceticus showed resistance to the majority of the agents tested, followed by E. cloacae and S. marcescens. In the water purification system the results were for P. aeruginosa into: (i) 0.5% citric acid, D = 3.8 min; (ii) 0.5% hydrochloric acid, D = 6.9 min; (iii) 70% ethanol, D = 9.7 min; (iv) 0.5% sodium bisulfite, D = 5.3 min; (v) 0.4% sodium hydroxide, D = 14.2 min; (vi) 0.5% sodium hypochlorite, D = 7.9 min; (vii) mixture of hydrogen peroxide (2.2%) plus peracetic acid (0.45%), D = 5.5 min. GFP was challenged with chlorine solutions (different pH and chlorine concentrations) and its fluorescence decreased abruptly on contact with chlorine in concentrations greater than 150 ppm, with D-values between 1.3 min (147 ppm chlorine) and 1.7 min (183 ppm chlorine). In phosphate buffered chlorine solutions (pH=7.15±0.08), GFP maintained its structure between 52-94 ppm of chlorine, but protein stability decreased 10-fold when exposed to 110 ppm chlorine. GFP performed as a suitable fluorescent marker for monitoring disinfection effectiveness. To be used as a biological indicator GFP has to be purified through a potentially scalable and cost-effective way to purify the recombinant protein, produced by E. coli. The method studied was two-phase aqueous micellar system, and GFP was partially recovered from a clarified E. coli cell lysate. Other contaminating proteins were simultaneously removed. The demonstration of proof-ofprinciple of the direct affinity-enhanced extraction of CBM9-GFP from the cell lysate represents an important first step towards developing a cost-effective separation method for GFP, and more generally, for other proteins of interest.

Devido ao número crescente de surtos de infecção hospitalar, torna-se proeminente o estabelecimento de um programa de sanitização que liste os agentes químicos a serem empregados e o modo de aplicação mais efetivo. Processos de desinfecção também são relevantes em sistemas de tratamento de água (em indústrias farmacêuticas e centros de saúde) para que a qualidade da água seja assegurada e atenda os parâmetros estabelecidos, evitando proliferação microbiana. Validação da eficácia de descontaminação é uma tarefa ao mesmo tempo importante e desafiadora. Indicadores biológicos são sistemas ou moléculas que detectam atividade biológica, permitindo a validação de processos de descontaminação ou desinfecção. O indicador biológico pode ser uma suspensão de microrganismos específicos (sistema biológico) com resistência definida a um determinado processo de descontaminação. Enzimas e proteínas também têm sido empregadas como indicadores biológicos para avaliar a eficácia de processos industriais. A proteína verde fluorescente (GFP) tem sido sugerida como potencial indicador biológico para tratamentos de desinfecção, devido facilidade de sua detecção por espectrofluorimetria ou por inspeção visual. Para estudar e comparar o comportamento dos microrganismos selecionados e da GFP foram realizados ensaios de concentração inibitória mínima (CIM) e tempo de redução decimal (valor D). A CIM capaz de reduzir o bioburden inicial (>8log10) foi: 59 - 156 mg/L- 3250 mg/mL de glutaraldeído, 39 - 246 mg/L de formaldeído, 43750 - 87500 mg/L de álcool etílico 1250 - 6250 mg/L de polivinilpirrolidona iodo, 150 - 4491 mg/L de compostos liberadores de cloro, 469 -2500 mg/L de peróxido de hidrogênio e 2310 - 18500 mg/L de ácido peracético. A. calcoaceticus apresentou resistência à maioria dos agentes químicos testados, seguido de E. cloacae e S. marcescens. No sistema de purificação de água os resultados para Pseudomonas aeruginosa foram: (i) 0,5% de ácido cítrico, D = 3,8 min; (ii) 0,5% de ácido clorídrico, D = 6,9 min; (iii) 70% álcool etílico, D = 9,7 min; (iv) 0,5% bissulfito de sódio, D = 5,3 min; (v) 0,4% de hidróxido de sódio, D = 14,2 min; (vi) 0,5% de hipoclorito de sódio, D = 7,9 min; (vii) mistura de peróxido de hidrogênio (2,2%) e ácido peracético (0,45%), D = 5,5 min. GFP testada frente a soluções cloradas (com diferentes valores de pH e concentração) resultou em diminuição da fluorescência, sendo mais evidente em concentrações de cloro maiores que 150 ppm, com valores D entre 1,3 - 1,7 min. Em soluções de cloro em tampão fosfato (pH=7,15±0,08), a proteína manteve sua estrutura em contato com soluções 52-94 ppm de cloro. Frente a 110 ppm de cloro a estabilidade da proteína foi reduzida 10 vezes. A proteína GFP se mostrou um marcador fluorescente apropriado para monitorar eficácia de desinfecção. Para ser empregada como indicador biológico a proteína deve ser purificada através de um método de fácil ampliação de escala e com boa relação custo benefício, com este intuito o sistema micelar de duas fases aquosas foi estudado, e a proteína GFP foi parcialmente recuperada do homogeneizado celular de E. coli, outros contaminantes presentes no meio foram removidos na mesma etapa. A demonstração de que é possível se extrair uma biomolécula alvo utilizando ligantes de afinidade representa um passo importante no desenvolvimento de um método eficaz de separação que poderá ser utilizado na purificação de outras biomoléculas. / Due to the growing number of outbreaks of infection in hospital and nurseries, it becomes essential to set up a sanitation program that indicates that the appropriate chemical agent was chosen for application in the most effective way. Disinfection processes are also relevant in water purification systems (in pharmaceutical industries and in health environments) to assure water quality, meeting ionic and organic chemical standards, and avoiding microbial proliferation. Validating the effectiveness of decontamination and disinfection is an important and often challenging task. A biological indicator is a system or a molecule that enables the detection of biological activity, and as such, permits the validation of decontamination or disinfection treatments. The biological indicator can be a specific microorganism suspension (microbiological test system) with a defined resistance to a particular decontamination treatment. Enzymes and proteins have also been used as biological indicators to evaluate the immediate efficacy of industrial procedures, such as blanching, pasteurization, and disinfection treatments, as well as to monitor the satisfactory preservation of a product subjected to disinfection or sterilization. Green fluorescent protein (GFP) has been proposed as an ideal choice for a protein-based biological indicator for use in the validation of decontamination or disinfection treatments. In order to study and compare microorganism (in hospital infections outbreaks and isolated from the water purification system) and protein behavior, microorganisms were submitted to minimal inhibitory concentration (CIM) and decimal reduction time determination (D value). The CIM intervals, which reduced bacteria populations over 8log10, were: 59 to 156 mg/L of quaternarium ammonium compounds (QACs); 63 to 10000 mg/L of chlorhexidine; 1375 to 3250 mg/L of glutaraldehyde; 39 to 246 mg/L of formaldehyde; 43750 to 87500 mg/L of ethanol; 1250 to 6250 mg/L of iodine in polyvinyl-pyrolidone complexes, 150 to 4491 mg/L of chlorine-releasing-agents (CRAs); 469 to 2500 mg/L of hydrogen peroxide; and, 2310 to 18500 mg/L of peracetic acid. Chlorhexidine showed non inhibitory activity over germinating spores. A. calcoaceticus showed resistance to the majority of the agents tested, followed by E. cloacae and S. marcescens. In the water purification system the results were for P. aeruginosa into: (i) 0.5% citric acid, D = 3.8 min; (ii) 0.5% hydrochloric acid, D = 6.9 min; (iii) 70% ethanol, D = 9.7 min; (iv) 0.5% sodium bisulfite, D = 5.3 min; (v) 0.4% sodium hydroxide, D = 14.2 min; (vi) 0.5% sodium hypochlorite, D = 7.9 min; (vii) mixture of hydrogen peroxide (2.2%) plus peracetic acid (0.45%), D = 5.5 min. GFP was challenged with chlorine solutions (different pH and chlorine concentrations) and its fluorescence decreased abruptly on contact with chlorine in concentrations greater than 150 ppm, with D-values between 1.3 min (147 ppm chlorine) and 1.7 min (183 ppm chlorine). In phosphate buffered chlorine solutions (pH=7.15±0.08), GFP maintained its structure between 52-94 ppm of chlorine, but protein stability decreased 10-fold when exposed to 110 ppm chlorine. GFP performed as a suitable fluorescent marker for monitoring disinfection effectiveness. To be used as a biological indicator GFP has to be purified through a potentially scalable and cost-effective way to purify the recombinant protein, produced by E. coli. The method studied was two-phase aqueous micellar system, and GFP was partially recovered from a clarified E. coli cell lysate. Other contaminating proteins were simultaneously removed. The demonstration of proof-ofprinciple of the direct affinity-enhanced extraction of CBM9-GFP from the cell lysate represents an important first step towards developing a cost-effective separation method for GFP, and more generally, for other proteins of interest.

<p>Human Coronaviruses (HCoVs) are found within the family Coronaviridae (genus, Coronavirus) and are enveloped, single-stranded, positive-sense RNA viruses. Infections of humans by&nbsp / coronaviruses are not normally associated with severe diseases. However, the identification of the coronavirus responsible for the outbreak of severe acute respiratory syndrome (SARS-CoV)&nbsp / showed that highly pathogenic coronaviruses can enter the human population. The SARS-CoV epidemic resulted in 8 422 cases with 916 deaths globally (case fatality rate: 10.9%). In 2004 a&nbsp / group 1 Coronavirus, designated Human Coronavirus NL63 (HCoV-NL63), was isolated from a 7 month old Dutch child suffering from bronchiolitis. In addition, HCoV-NL63 causes disease in&nbsp / children (detected in approximately 10% of respiratory tract infections), the elderly and the immunocompromised. This study was designed to express the full length nucleocapsid (N) proteins of&nbsp / HCoV-NL63 and SARS-CoV for antibody production in an animal model. The NL63-N/pFN2A and SARSN/ pFN2A plasmid constructs were used for this study. The presence of the insert on the Flexi &reg / vector was confirmed by restriction endonuclease digest and sequence verification. The sequenced chromatographs obtained from Inqaba Biotec were consistent with sequences from&nbsp / the NCBI Gen_Bank. Proteins were expressed in a KRX Escherichia coli bacterial system and analysed using 15% SDS-PAGE and Western Blotting. Thereafter, GST-tagged proteins were purified&nbsp / ith an affinity column purification system. Purified fusion proteins were subsequently cleaved with Pro-TEV Plus protease, separated on 15% SDS-PAGE gel and stained with Coomassie&nbsp / Brilliant Blue R250. The viral fusion proteins were subsequently used to immunize Balbc mice in order to produce polyclonal antibodies. A direct ELISA was used to analyze and validate the&nbsp / production of polyclonal antibodies by the individual mice. This is a preliminary study for development of diagnostic tools for the detection of HCoV-NL63 from patient samples collected in the&nbsp / Western Cape.</p>

<p>Human Coronaviruses (HCoVs) are found within the family Coronaviridae (genus, Coronavirus) and are enveloped, single-stranded, positive-sense RNA viruses. Infections of humans by&nbsp / coronaviruses are not normally associated with severe diseases. However, the identification of the coronavirus responsible for the outbreak of severe acute respiratory syndrome (SARS-CoV)&nbsp / showed that highly pathogenic coronaviruses can enter the human population. The SARS-CoV epidemic resulted in 8 422 cases with 916 deaths globally (case fatality rate: 10.9%). In 2004 a&nbsp / group 1 Coronavirus, designated Human Coronavirus NL63 (HCoV-NL63), was isolated from a 7 month old Dutch child suffering from bronchiolitis. In addition, HCoV-NL63 causes disease in&nbsp / children (detected in approximately 10% of respiratory tract infections), the elderly and the immunocompromised. This study was designed to express the full length nucleocapsid (N) proteins of&nbsp / HCoV-NL63 and SARS-CoV for antibody production in an animal model. The NL63-N/pFN2A and SARSN/ pFN2A plasmid constructs were used for this study. The presence of the insert on the Flexi &reg / vector was confirmed by restriction endonuclease digest and sequence verification. The sequenced chromatographs obtained from Inqaba Biotec were consistent with sequences from&nbsp / the NCBI Gen_Bank. Proteins were expressed in a KRX Escherichia coli bacterial system and analysed using 15% SDS-PAGE and Western Blotting. Thereafter, GST-tagged proteins were purified&nbsp / ith an affinity column purification system. Purified fusion proteins were subsequently cleaved with Pro-TEV Plus protease, separated on 15% SDS-PAGE gel and stained with Coomassie&nbsp / Brilliant Blue R250. The viral fusion proteins were subsequently used to immunize Balbc mice in order to produce polyclonal antibodies. A direct ELISA was used to analyze and validate the&nbsp / production of polyclonal antibodies by the individual mice. This is a preliminary study for development of diagnostic tools for the detection of HCoV-NL63 from patient samples collected in the&nbsp / Western Cape.</p>

>Magister Scientiae - MSc / Human Coronaviruses (HCoVs) are found within the family Coronaviridae (genus, Coronavirus) and are enveloped, single-stranded, positive-sense RNA viruses. Infections of humans by coronaviruses are not normally associated with severe diseases. However, the identification of the coronavirus responsible for the outbreak of severe acute respiratory syndrome (SARS-CoV) showed that highly pathogenic coronaviruses can enter the human population. The SARS-CoV epidemic resulted in 8 422 cases with 916 deaths globally (case fatality rate: 10.9%). In 2004 a group 1 Coronavirus, designated Human Coronavirus NL63 (HCoV-NL63), was isolated from a 7 month old Dutch child suffering from bronchiolitis. In addition, HCoV-NL63 causes disease in children (detected in approximately 10% of respiratory tract infections), the elderly and the immunocompromised. This study was designed to express the full length nucleocapsid (N) proteins of HCoV-NL63 and SARS-CoV for antibody production in an animal model. The NL63-N/pFN2A and SARSN/ pFN2A plasmid constructs were used for this study. The presence of the insert on the Flexi ® vector was confirmed by restriction endonuclease digest and sequence verification. The sequenced chromatographs obtained from Inqaba Biotec were consistent with sequences from the NCBI Gen_Bank. Proteins were expressed in a KRX Escherichia coli bacterial system and analysed using 15% SDS-PAGE and Western Blotting. Thereafter, GST-tagged proteins were purified ith an affinity column purification system. Purified fusion proteins were subsequently cleaved with Pro-TEV Plus protease, separated on 15% SDS-PAGE gel and stained with Coomassie Brilliant Blue R250. The viral fusion proteins were subsequently used to immunize Balbc mice in order to produce polyclonal antibodies. A direct ELISA was used to analyze and validate the production of polyclonal antibodies by the individual mice. This is a preliminary study for development of diagnostic tools for the detection of HCoV-NL63 from patient samples collected in the Western Cape. / South Africa

Água purificada para uso em ambientes de saúde e preparação industrial de produtos médico-odonto-hospitalar deve ter uma carga reduzida de contaminação microbiológica e pirogênio. As amostras analisadas foram coletadas em 13 (treze) diferentes estágios de um sistema de purificação de água, sendo isoladas 78 colônias. Para a identificação dos microrganismos foram utilizados: (i) identification system for non-enteric gram-negative rods (api 20 NE, bio Mérieux) e (ii) identification system for enteric and nonfermenter (BBL crystal, Becton Dickinson). De acordo com os resultados pode-se observar uma maior prevalência para Pseudomonas aeruginosa 32,05% (25 colônias dentre as 78 isoladas); Pseudomonas picketti 23,08% (18); Pseudomonas vesiculares 12,82% (10); Pseudomonas diminuta 11,54% (09); Flavobacterium aureum 6,42% (05); Pseudomonas fluorescences 5,13% (04); Acinetobacter Iwoffi 2,56% (02); Pseudomonas putida 2,56% (02); Pseudomonas alcaligenes 1,28% (01); Pseudomonas paucimobilis 1,28% (01), Flavobacterium multivorum 1,28% (01). A eficácia dos agentes sanitizantes utilizados para higienização dos diferentes pontos foi determinada pela concentração inibitória mínima (CIM) e tempo de redução decimal (valor D). Como o hipoclorito de sódio é largamente utilizado na higienização do tanque de água de alimentação, tanque de estocagem da água purificada e nas linhas de distribuição aos pontos de uso, este foi testado frente a todos os microrganismos sendo observado menor resistência para Escherichia coli ATCC 25922 (0.06%=600mg/L), quando comparada a P. aeruginosa isolada e identificada (O,25%=2500mg/L). O tempo de redução decimal (Valor D) da Escherichia coli ATCC 25922 foi de 4 minutos e o valor D da P. aeruginosa isolada e identificada e isolada in house foi de 9 minutos. / The samples of water, which were taken directly from the public distribution water tank and at twelve different stages of a typical purification system, were analyzed for the identification of isolated bacteria. The efficacy of the chemical sanitizers used in the stages of the system, over the isolated and identified bacteria in the sampling water, was valuated by the minimum inhibitory concentration (MIC and decimal reduction time (D-values). According to the miniature kits used in the identification, there was a prevalence of isolation by P. aeruginosa 32,05 % , P. picketti (Ralstonia picketti) 23,08%, P. vesiculares 12,82%, P. diminuta 11.54%, F.aureum 6,42%, P.fluorescens 5,13%, A.lowffi 2,56%, P.putida 2,56%, P. alcaligenes 1,28%, P.paucimobilis 1,28%, and F. multivorum 1,28%. The efficacy of the agents varied with the concentration and time of contact to reduce a decimal logarithmic (loglO) population (n cycles): (i) 0.5% citric acid (0= 4 min) for 30 min reduced n=7 cycles; (ii) 0.5% chloridric acid (0= 6 min) for 30 min reduced n= 4 cycles; (iii) 70% alcohol (0= 9 min) for 1.0 min (n=0.2 cycles); (iv) 0.5% sodium bisulphite (0= 6 min) for 90 min (n=14 cycles); (v) 0.4% sodium hydroxide (0= 8 min) for 30 min (n=3.0 cycles); (vi) 0.5% sodium hypochlorite (0=9 min) for 180 min (n = 19 cycles); (vii) mixture of hydrogen peroxide (2,2%) plus peracetic acid (0.45%), 0= 6 min, contact time of 180 min, reduced n = 32 cycles of the population. The sterility assurance level (SAL) of 10-6 was attained for the 0.5% sodium hypochlorite solution applied in the water purified storage tank and distribution loop; and for the mixture of H2O2 plus peracetic acid, used in the reverse osmose and in the deionizator systems.