Succinic Acid (SA) was continuously produced using glucose and a Mg2CO3(OH)2 slurry as feed. Glucose feed concentrations of 20 and 40 g ℓ-1 were employed with corresponding Mg2CO3(OH)2 slurry concentrations of 60 and 120 g ℓ-1. The reactor pH was passively maintained between 6,4 and 6,8 by the buffer properties of the slurry in conjunction with the pH adjusted glucose feed. The suspended cell (SC) reactor was operated at 37°C with dilution rates varying between 0,04 h-1and 0,6 h-1. Groperl® particles were used as inert supports in the biofilm reactor; dilution rates of 0,11 h-1 to 1 h-1 were investigated. Two SC fermentations were conducted for the 20 g ℓ-1 glucose feed concentration and one for the 40 g ℓ-1. All SC fermentation runs were operated in excess of 12 days, while the biofilm run lasted 6,5 days. Fermentations were terminated only after contamination by lactic acid bacteria was observed. SC fermentations with the glucose feed concentration of 20 g ℓ-1 achieved a maximum SA productivity of 5,2 g ℓ-1h-1 at 0,6 h-1 with a corresponding SA yield of 0,65 g g-1. SC fermentations with the glucose feed concentration of 40 g ℓ-1 achieved a maximum SA productivity of 3,76 g ℓ-1h-1 at 0,4 h-1 with a SA yield of 0,82 g g-1. The results were comparable to the other continuous studies with Actinobacillus succinogenes, despite the fact that either biofilms or membranes were employed in these studies. The preliminary biofilm study demonstrated the capability of A. succinogenes to produce SA in high productivities and yields. SA productivities and yields for the dilution rates of 0,33 h-1 and 1,0 h-1, were 5,72 g ℓ-1h-1 (0,95 g g-1) and 12 g ℓ-1h-1 (1,0 g g-1), respectively. The biofilm reactor at 0,33 h-1 achieved twice the SA productivity of the SC reactor at 0,3 h-1 with a 42 % increase in SA yield. Copyright / Dissertation (MEng)--University of Pretoria, 2012. / Chemical Engineering / unrestricted

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2016-12-23T12:26:14Z No. of bitstreams: 1 texto completo.pdf: 716349 bytes, checksum: 491af184849bdcb2477fa46e3081a75f (MD5) / Made available in DSpace on 2016-12-23T12:26:14Z (GMT). No. of bitstreams: 1 texto completo.pdf: 716349 bytes, checksum: 491af184849bdcb2477fa46e3081a75f (MD5) Previous issue date: 2016-07-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os insetos respondem à infecção através da montagem de reações imunes do tipo celular e humoral. Os reguladores primários dessas respostas são células chamadas hemócitos, os quais medeiam importantes respostas celulares, incluindo a fagocitose, encapsulamento, nodulação, e também segregam fatores humorais, tais como opsoninas, fatores de melanização e peptídeos antimicrobianos. Os hemócitos circulam ao longo da hemocele (cavidade corporal do inseto) pelo fluxo rápido da hemolinfa (sangue), além disso, partes desses hemócitos também existem como células sésseis que estão associados aos tecidos. As larvas de Galleria mellonella são uma alternativa viável para os modelos tradicionais dos mamíferos para estudar a eficácia de drogas antimicrobianas e a patogênese de microrganismos in vivo. No entanto, apesar da sua importância como um modelo de infecção, aspectos biológicos sobre as células do sistema imunológico, tais como a densidade e dinâmica dos hemócitos das larvas são mal compreendidos. No presente trabalho, investigamos a resposta imune celular dos hemócitos circulantes das larvas de G. mellonella contra diferentes cepas da bactéria Gram-negativa Actinobacillus pleuropneumoniae: baixa virulência (780), alta virulência (1022), e cepas de referência do sorotipo 8 (R8). Os hemócitos foram classificados com base no seu tamanho, morfologia, coloração e seus papeis na resposta imune, incluindo cinco tipos: prohemócitos, plasmatócitos, granulócitos, esferulócitos, e oenocitóides. Contagem total de hemócitos, contagem diferencial de hemócitos, atividade dos fagolisossomos, resposta autofágica, viabilidade celular, e a ativação da caspase-3 (como indicador de apoptose) foram determinados em hemócitos circulantes provenientes de larvas desafiadas e controle. Demostramos pela primeira vez no modelo de G. mellonella que os plasmatócitos e granulócitos ativam suas respostas autofágicas através da formação dos autofagossomos após o contato com A. pleuropneumoniae. Além disso, nossos dados demonstram que a imunidade celular do presente modelo de infecção muda dependendo do grau de virulência das cepas bacterianas. / Insects respond to infection by mounting cellular and humoral immune reactions. The primary regulators of these immune responses are cells called hemocytes, which mediate important cellular immune responses including phagocytosis, encapsulation, nodulation and also secrete immune factors such as opsonins, melanization factors and antimicrobial peptides. Hemocytes circulate through the hemocoel (body cavity) by the swift flow of hemolymph (blood), and part of these hemocytes population are sessile and are attached to tissues. Larvae of Galleria mellonella is a widely used factitious host as a viable alternative to traditional mammalian models to study the efficacy of antimicrobial drugs and the microbial pathogenesis in vivo. However, despite their importance as an infection model, biological aspects about the immune cells, such as density and hemocyte dynamic of larvae are poorly understood. In the present study, we investigated the cellular immune response of hemocytes from G. mellonella larvae against three strains of the gram-negative bacterium Actinobacillus pleuropneumoniae: low virulent (780), high virulent (1022), and the serotype 8 reference strain (R8). Five types of larval hemocytes, prohemocytes, plasmatocytes, granulocytes, oenocytoids, and spherulocytes, were distinguished according to size, morphology, detection by molecular probes, dye-staining properties, and their role in the immune response. Total hemocyte count, differential hemocyte count, lysosome activity, autophagic response, cell viability, and caspase-3 activation were determined in circulating hemocytes of naïve and infected larvae. Granulocytes and plasmatocytes were the major hemocyte types involved in the cellular defense against A. pleuropneumoniae; these hemocytes activated phagolysosome activities associated with an autophagic response against the bacteria. Moreover, our results showed that apoptosis in circulating hemocytes after exposure to virulent bacterial strains was related to an excessive autophagic cell death response induced by stress and subsequent caspase-3 activation.

Carbon dioxide serves as co-substrate in the production of succinic acid by Actinobacillus succinogenes. The transient concentration of dissolved CO2 in the broth (CCO2) controls the uptake of CO2 in the cell. Based on CCO2 , three distinct regimes could be identified in which the behaviour of the organism differed with CCO2 availability. When CCO2 was higher than 8.4 mM (44.4% saturated at an atmospheric pressure of 86 kPa), there was no evidence of CO2 limiting succinic acid productivity and flux to succinic acid remained constant. When CCO2 decreased below 8.4 mM a decrease in the succinic acid production and glucose consumption rates was observed to 28.01% and 19.89% of their original value respectively, at the lowest CCO2 value investigated. Below a CCO2 of 4 mM (21.16% saturated at an atmospheric pressure of 86 kPa), the productivity continued to decrease along with a shift in the total carbon flux from the succinic acid-producing pathway (C4-pathway) to the by-product-producing pathway (C3-pathway). The fraction of total carbon flux directed to the C4-pathway decreased from 0.48 to 0.33 at the lowest CCO2 value investigated. Although the by-product acetic acid concentration decreased to 88% of the original value, formic acid remained relatively stable and the ethanol concentration increased from an average of 0.26 g.L-1 to 1 g.L-1. The organism starts producing ethanol in order to satisfy the redox balance when the C4-pathway becomes less active. It was calculated that the flux shift to the C3-pathway does not favour ATP production. The organism is, however, still viable at the very low ATP production rates found at very low values of CCO2. Since succinic acid production is not limited at relatively low values of CCO2 (44.4% saturation), adequate CO2 supply to the fermenter can be achieved without major CO2 sparging which is beneficial from an industrial processing perspective. / Dissertation (MEng)--University of Pretoria, 2016. / Chemical Engineering / MEng / Unrestricted

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Actinobacillus pleuropneumoniae et Streptococcus suis sont des bactéries pathogènes entraînant des maladies porcines mortelles et des pertes économiques importantes dans l’industrie porcine mondiale. Ces bactéries sont notamment reconnues pour leur implication dans certaines maladies pulmonaires porcines, dont la pneumonie, et leurs mécanismes de pathogénicité ne sont pas parfaitement élucidés. De ce fait, nous nous sommes intéressés à l’activité de ces deux bactéries sur la barrière épithéliale de la trachée du porc, soit un modèle représentatif de la première ligne de défense des voies respiratoires supérieures du porc. Plus précisément, nous avons étudié la capacité d’A. pleuropneumoniae et de S. suis à induire une perte d’intégrité de la barrière épithéliale, à la traverser, et à induire une réponse inflammatoire. En considérant les cytokines pro-inflammatoires sécrétées par les cellules épithéliales en présence de ces bactéries pathogènes, les effets du TNF-α sur la barrière épithéliale ont également été analysés. Nous avons démontré qu’A. pleuropneumoniae, S. suis et le TNF-α sont aptes à induire des dommages importants à la barrière épithéliale et à permettre la translocation de ces bactéries pathogènes à travers cette barrière. Ces résultats suggèrent que la barrière épithéliale puisse représenter une cible thérapeutique contre les infections engendrées par les bactéries pathogènes environnantes afin de limiter leur capacité d’invasion. Pour ce faire, il serait nécessaire d’investiguer davantage les facteurs de virulence de ces bactéries contribuant à la perte d’intégrité de la barrière épithéliale des voies respiratoires supérieures du porc afin d’élaborer de potentiels thérapies ciblées inhibant leur pathogénicité

Submitted by Simone Souza (simonecgsouza@hotmail.com) on 2017-10-18T12:24:30Z No. of bitstreams: 1 DISS_2014_Laila Natasha Santos Brandão.pdf: 1739750 bytes, checksum: 16eb165dd6c3a1476424ddf9bd18fe73 (MD5) / Approved for entry into archive by Jordan (jordanbiblio@gmail.com) on 2017-11-07T15:29:45Z (GMT) No. of bitstreams: 1 DISS_2014_Laila Natasha Santos Brandão.pdf: 1739750 bytes, checksum: 16eb165dd6c3a1476424ddf9bd18fe73 (MD5) / Made available in DSpace on 2017-11-07T15:29:45Z (GMT). No. of bitstreams: 1 DISS_2014_Laila Natasha Santos Brandão.pdf: 1739750 bytes, checksum: 16eb165dd6c3a1476424ddf9bd18fe73 (MD5) Previous issue date: 2014-03-05 / CAPES / A possibilidade de detecção de agentes patogênicos representou um grande passo para a ciência. O desenvolvimento e melhorias nas técnicas de diagnóstico como a técnica de Reação em Cadeia da Polimerase (PCR), ao longo dos anos ainda requer infraestrutura laboratorial, apesar de sua sensibilidade e custo decrescente, investimentos em equipamentos e cuidados com fontes de contaminação externa. Técnicas que possam ser executas com facilidade e pouca mão de obra ou exigência de pessoas capacitadas, têm grande possibilidades de aplicabilidade. Neste estudo desenvolveu-se uma técnica de rápida execução e baixo custo, para detecção de um dos principais agentes causadores de pneumonias em granjas de suínos o Actinobaccilus pleuropneumoniae, com sensibilidade 93,8% e especificidade de 84,6% em amostras de pulmões com e sem lesão. O teste Kappa entre a PCR e nanopartícula de ouro foi 0,684 representando boa concordância. A técnica pode ser utilizada como alternativa aos testes convencionais, já que é de fácil e rápida execução e não exige infraestrututra e mão de obra especializada. / The possibility of detection of pathogens was a major step for science as a whole, developing and improving these techniques over the years to increase visibly. The development of the technique of Polymerase Chain Reaction ( PCR ) , although its sensitivity and decreasing cost over the years, it's still a handy little technique that requires laboratory infrastructure, high investments in equipment and care with sources of external contamination. Techniques that can be performed through the ease and little manpower or requirement of skilled people, always have great scope of applicability. This study develops a technique for quick and low- cost detection of a major causative agent of pneumonia in swine herds the Actinobaccilus pleuropneumoniae , with 93.8 % sensitivity and 84.6% specificity in samples of lungs with and without injury, the Kappa test between PCR and gold nanoparticle was 0,684 representing good agreement . The technique can be used as an alternative to conventional tests, since it is easy and quick to implement and does not require infrastructure and skilled labor.

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A Diabetes mellitus do tipo I (IDDM), doença metabólica caracterizada pela deficiência na produção de insulina, causa profundas alterações sistêmicas, incluindo a cavidade bucal. A doença periodontal, que se apresenta mais agressiva em pacientes diabéticos, até mesmo na infância, tem como agentes etiológicos microrganismos denominados periodontopatógenos, dentre eles o Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), anteriormente chamado Actinobacillus actinomycetemcomitans, que em associação às alterações na resposta imune e inflamatória decorrentes da diabetes, poderá causar maior reabsorção do osso alveolar precocemente em crianças. A identificação precoce destes agressores poderá ser extremamente útil para a instituição de medidas preventivas e terapêuticas que possam inibir ou minimizar os efeitos nocivos da IDDM e suas conseqüências para a cavidade bucal. Para este fim, métodos moleculares de investigação estão sendo cada vez mais utilizados, destacando-se a Reação em Cadeia da Polimerase em Tempo Real (Real-Time PCR) com alta especificidade e sensibilidade para detectar e quantificar microrganismos, mesmo em pequeno número e em meios, até então, pouco explorados como a saliva. Por fim, a utilização de um antimicrobiano seguro, que possa ser aplicado topicamente por meio da escovação dentária, como o metronidazol 25% na forma de gel, poderia reduzir ou eliminar o A. actinomycetemcomitans evitando a progressão da doença periodontal. Assim, este estudo foi realizado para observar a presença do A. actinomycetemcomitans antes e após o uso do gel de metronidazol 25% uma vez ao dia, em substituição a uma das escovações diárias. Avaliou também a influência deste protocolo de tratamento nos índices de placa, gengival e profundidade de sondagem em 32 crianças, na faixa etária de 3 a 12 anos de idade, divididas em dois grupos: diabéticos tipo I (D) e não diabéticos (ND). Para avaliação da presença do A. actinomycetemcomitans foi utilizado o método da Real-Time PCR. Os resultados obtidos sugerem que o gel de metronidazol 25%, aplicado segundo o protocolo estabelecido neste trabalho foi efetivo para a redução dos índices de placa, gengival e do A. actinomycetemcomitans, no entanto, pareceu não atuar sobre a profundidade de sondagem. Os dados obtidos sugerem que, ainda que a ocorrência da bactéria nos indivíduos avaliados tenha sido baixa e a diferença entre o grupo diabéticos e não diabéticos tenha restringido-se apenas à profundidade de sondagem, o gel estudado demonstrou ser promissor na prevenção da doença periodontal em crianças diabéticas, havendo necessidade da realização de mais estudos clínicos randomizados. / Type I Diabetes mellitus (IDDM) is a metabolic disease characterized by deficiency on the insulin production, causing great systemic alterations, including at the oral cavity. Periodontal disease is more aggressive in diabetic patients, even during the childhood, and presents as etiological microorganisms the Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), formerly Actinobacillus actinomycetemcomitans. This microorganisms in association to alterations in the immune and inflammatory answers caused by diabetes, can promote a larger and early alveolar bone resorption even in children. So, the early identification of these aggressors could be extremely useful for the institution of preventive and therapeutic measures in order to inhibit or minimize the noxious effects of IDDM and their consequences in the oral cavity. Aiming this early identification, molecular biology methods of investigation are being used more frequently, pointing out the Real Time Polymerase Chain Reaction (Real-Time PCR), which presents high specificity and sensibility to detect and to quantify microorganisms, even in small number and in different media little explored like saliva. The use of a safe topical antibiotic, such as metronidazole 25% gel, which used in association with dental brushing, could reduce or eliminate A. actinomycetemcomitans, avoiding the progression of the periodontal disease. This study evaluated the presence of A. actinomycetemcomitans before and after the use of metronidazole 25% gel once a day, in substitution to one of the daily teeth brushing. It also evaluated the influence of this treatment protocol on the plaque, gingival and probing depth indexes. Thirty-two children, aging from 3 to 12 years were included in this study and they were divided into two groups: type I diabetic (D) and no diabetic (ND). Real-Time PCR methods were used to evaluate the presence of A. actinomycetemcomitans. The obtained data suggest that metronidazole 25% gel, applied according to the established protocol in this study, effectively reduced plaque and gingival indexes and the number of A. actinomycetemcomitans. However, no influence was observed regarding the probing depth. The obtained data suggest that, although the number of bacteria has been small and the difference between the D and ND groups was not observed only regarding probing depth, the studied antibiotic gel demonstrated to be promising in preventing periodontal disease in diabetic children. More randomized clinical trials are necessary for a final conclusion.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal