Burkholderia pseudomallei is a Gram-negative intracellular bacterium that causes melioidosis, a serious disease of humans and animals in tropical countries. This pathogen can subvert the host cell actin machinery by a process known as actibased motility, for promoting its movement both within and between cells. The bacterial factor required for this process is known as BimA (Burkholderia intracellular motility A). Intracytoplasmic bacterial pathogens use distinct mechanisms for actin-based motility, hijacking host cytoskeletal proteins for their benefit. However, the molecular mechanism by which BimA subverts the cellular actin machinery is ill-defined. From an affinity approach coupled with mass spectrometry to identify cellular proteins recruited to BimA-expressing bacteria under conditions that promote actin polymerisation, a group of cellular proteins that are recruited to the B. pseudomallei surface in a BimA-dependent manner was identified. A subset of these proteins was independently validated with specific antisera including IQ motif containing GTPase activating protein 1 (IQGAP1). IQGAP1 is a ubiquitous scaffold protein that integrates several key cellular signalling pathways including those involved in actin dynamics. Previous studies demonstrated IQGAP1 was targeted by pathogens to regulate the actin cytoskeleton, for example promoting Salmonella invasion into epithelial cells or supporting cell attachment and pedestal formation of Enteropathogenic Escherichia coli. The aim of this study is to explore the roles of IQGAP1 in the intracellular life of B. pseudomallei. This present study revealed that IQGAP1 was recruited to B. pseudomallei actin tails in infected HeLa cells. This protein has not previously been associated with actin-based motility of other intracellular pathogens. To examine the effect on actibased motility of B. pseudomallei, siRNA was utilised to knockdown IQGAP1 in HeLa cells. After optimisation of siRNA transfection, IQGAP1 expression in HeLa cells was suppressed by approximately 70% as assessed by IQGAP1 immunoblotting. The siIQGAP1 knockdown cells were infected with B. pseudomallei. The bacteria could still form actin tails in the knockdown cells, however, the data showed a statistically significant increase in overall tail length with a concomitant decrease in actin density, compared with the tails formed by B. pseudomallei in control cells. Actin-based motility is essential in the life cycle of several cytoplasmic bacterial pathogens, particularly in cell-to- cell spread. After entry into the host cell cytosol, B. pseudomallei polymerises actin in a BimA-dependent manner and propels itself within and between cells. This is accompanied by cell fusion which generates multi-nucleated giant cells (MNGCs), a process mediated by a Type 6 Secretion System that is co-regulated with BimA. To gain an understanding of the impact of IQGAP1 on the intracellular life of B. pseudomallei, IQGAP1 was successfully knocked-out from HeLa cells using CRISPR-Cas9 technique. Interestingly, Burkholderia invasion was not affected in HeLa cells lacking IQGAP1. However, the bacteria showed a defect in intracellular survival in IQGAP1 knockout cells that was revealed after 6 hours post-infection. Moreover, there was no difference in the proportion of bacteria associated with actin in the control and knockout cells at 16 hours post-infection, although the bacteria formed longer actin tails in control cells with similar actin density. Consequently, the number of MNGCs decreased dramatically in the cells lacking IQGAP1, which was indicated by the absence of plaque formation. Another element of this study was to determine whether BimA and IQGAP1 are direct interacting partners. Using either an in vitro pulldown assay or in vivo yeast two-hybrid system, a direct interaction between these proteins could not be detected. It is, therefore, likely that IQGAP1 is recruited to B. pseudomallei actin tails through its intrinsic ability to interact with F-actin. Despite the lack of a direct interaction between these two proteins, an N-terminal IQGAP1 fragment significantly augmented BimA-mediated actin polymerisation in vitro. Taken together, this study provides the first evidence of the presence of IQGAP1 in B. pseudomallei actin tails and presents the importance of IQGAP1 in actin-based motility and intracellular life of this bacterium. Understanding the mechanism of B. pseudomallei actin-based motility is useful to gain insights into host cell actin dynamics and its role in pathogenesis. Targeting host cellular proteins that are required for the intracellular life of pathogens are a topical area of research, with the potential to be useful alternatives to classic antibiotic therapy. Indeed, IQGAP1 could be a potential novel therapeutic target to develop drugs for treating B. pseudomallei infection.

A thesis by Marc Ashley Bayliss entitled ‘Virus-like particles as a novel platform for delivery of protective Burkholderia antigens’ and submitted to the University of Exeter for the degree of Doctor of Philosophy. There is currently no licensed vaccine available for the global tropical pathogen Burkholderia pseudomallei which is the causative agent of melioidosis and a potential bio-threat agent. The capsule polysaccharide (CPS) expressed by B. pseudomallei has been shown to offer some protection against bacterial challenge. Polysaccharide immunogenicity can be enhanced by conjugation to a carrier protein and several licensed vaccines utilise this technology. Virus-like particles (VLPs) are non-infectious, non-replicating, viral proteins that self-assemble into viral structures and are in several licensed vaccines as primary antigens. VLPs are also effective delivery platforms for foreign antigens by genetic insertion or chemical conjugation. iQur, a collaborator on this project, has developed Tandem CoreTM that consists of two genetically linked hepatitis B core proteins that allow insertion of large proteins into each core whilst remaining assembly competent. The aim of this thesis was to assess the protective efficacy of Tandem CoreTM VLPs chemically conjugated to CPS and Tandem CoreTM Burkholderia protein fusion constructs. This involved three objectives; reduce the cost of CPS extraction; identify immunogenic Burkholderia proteins; and test candidate vaccine efficacy in an animal model of acute melioidosis against B. pseudomallei challenge. To reduce the cost of extraction, CPS was purified from B. thailandensis strain E555 and bacterial culture CPS concentration optimised which first required development of a quantitative ELISA. Immunogenic Burkholderia proteins were identified from the literature but Tandem CoreTM fusion constructs containing these proteins were not assembly competent. The Burkholderia proteins were added as co-antigens to the VLP CPS conjugate vaccine but did not improve efficacy. Tandem CoreTM VLPs conjugated to CPS were protective against B. pseudomallei challenge and were compared to CPS conjugated to Crm197: a commercially available carrier protein used in several licensed vaccines. At lower challenge doses, survival was greater in mice vaccinated with the VLP-CPS conjugate although at higher doses, Crm197-CPS efficacy was greater.

Made available in DSpace on 2016-05-11T12:56:37Z (GMT). No. of bitstreams: 2 rhaissa_vieira_ioc_mest_2016.pdf: 3046248 bytes, checksum: d61e9828a7069c1879b0fd074610f11a (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2016 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Burkholderia cenocepacia é uma bactéria gram-negativa associada a infecções pulmonares oportunistas acometendo portadores de fibrose cística, doença granulomatosa crônica ou que apresentem algum tipo de imunodeficiência, podendo resultar em declínio da função pulmonar e no quadro séptico conhecido como síndrome cepacia. Por mecanismos de escape B. cenocepacia promove atraso na maturação do fagolisossomo, enquanto fatores envolvidos na resistência a ROS desempenham papel na sobrevivência intracelular. Dados de nosso laboratório mostram que cultivos in vitro de macrófagos peritoneais de camundongos e da linhagem RAW 264.7 estimulados com IFN\03B3 e LPS, quando desafiados com B. cenocepacia, apresentam reduzidos níveis de NOx nos sobrenadantes. Assim, o objetivo do presente trabalho foi avaliar a resposta efetora de macrófagos alveolares de camundongo da linhagem AMJ2-C11 na infecção por B. cenocepacia. Além disso, avaliamos a infecção de camundongos imunocompetentes C57BL/6 ou geneticamente deficientes para a enzima óxido nítrico sintase induzível (iNOS/NOS2). Nossos resultados indicam que B. cenocepacia é capaz de desativar mecanismos efetores da resposta clássica de macrófagos, redirecionando para o perfil alternativo de ativação. Observamos em cultivos de macrófagos AMJ2-C11 expostos à B. cenocepacia (i) concentrações reduzidas de NOx e concentrações aumentadas de ureia em sobrenadantes, (ii) aumento da expressão da enzima arginase e baixa expressão de iNOS/NOS2 nestas células, (iii) menor frequência de células expressando MHC-I e moléculas coestimuladoras (CD80, CD86 e CD40), e por fim (iv) baixas concentrações de TNF nos sobrenadantes Semelhante ao encontrado nos sobrenadantes dos cultivos na presença de B. cenocepacia, os lavados pleurais de animais infectados pela bactéria apresentam aumento na produção de ureia em detrimento da produção de NOx. Em 72 horas de infecção, contagem de CFU encontra-se aumentada nos animais deficientes em iNOS/NOS2, enquanto é controlada em animais C57BL/6 imunocompetentes. Todos os camundongos infectados apresentaram perda de peso nas primeiras 72 horas de infecção. Animais C57BL/6 recuperaram peso corporal, apresentando ao término da análise ganho ponderal semelhante aos não infectados. Por outro lado, animais inos-/- infectados acumularam perda ponderal no decorrer do tempo analisado, apresentando em torno de 40% de perda de massa corpórea e apresentaram 100% de mortalidade em 13 dias pós-infecção, ao passo que não houveram mortalidade em animais controles (NI). Os pulmões dos animais inos-/- infectados apresentam alterações histológicas agudas, comparados tanto aos controles não infectados, e mais intensas que os animais C57BL/6 infectados. Em conjunto nossos dados sugerem que a infecção por B. cenocepacia interfere no balanço das vias óxido nítrico sintase induzível e arginase, favorecendo esta última, o que poderia promover a atenuação da resposta inflamatória e a persistência da infecção / Abstract: Burkholderia cenocepacia is a gram-negative bacteria associated with opportunistic lung infections affecting patients with cystic fibrosis, chronic granulomatous disease or immunodeficiencies, which can result in decreased pulmonary function and sepsis known as cepacia syndrome. By escape mechanisms B. cenocepacia promotes delay in maturation of phagolysosome, since cell factors involved in ROS resistance play a role in intracellular survival. Data from our laboratory show that cultures in vitro of mouse peritoneal macrophages and RAW 264.7 cell line stimulated with LPS and IFN\F067\F02C when challenged with B. cenocepacia present reduced NOx levels in the supernatants. The aim of the present work was to evaluate the effector response of mouse alveolar macrophages of the cell lineage AMJ2-C11 challenged by infection with B. cenocepacia. In addition, we evaluated the infection of mice immunocompetent (C57BL/6) or genetically deficient for the enzyme inducible nitric oxide synthase (iNOS/NOS2). Our results indicate that B. cenocepacia can disable effector mechanisms of classical response of macrophages, redirecting to the alternative profile of activation. We observed in AMJ2-C11 macrophage exposed to B.cenocepacia (i) reduced concentrations of NOx and increased concentrations of urea in supernatants, (ii) increased expression of arginase and low expression of iNOS/NOS2 in these cells, (iii) lower frequency of cells expressing MHC-I and costimulatory molecules (CD80, CD86, CD40), and finally (iv) lower TNF concentrations in the supernatants Akin the findings in supernatants of AMJ2-C11 macrophage exposed to B. cenocepacia, pleural washes recovered from infected mice show increased production of urea, while NOx was barely detected. At 72 hours of infection, CFU counts were increased in pleural washes of iNOS/NOS2-deficient mice, while bacteria growth was controlled in immunocompetent mice. All infected mice showed initial weight loss. After 72 hours of infection, C57BL/6 mice recovered body weight, showing similar weight gain to uninfected mice. On the other hand, B. cenocepacia-infected inos-/- mice accumulated body weight loss (~ 40%) during the course of infection, showing 100% of mortality at 13 days post-infection, whereas none of the control mice showed mortality. The lungs of B. cenocepacia-infected inos-/- mice showed acute histological alterations in comparison with uninfected controls, and more intense abnormalities when compared to infected C57BL/6 mice. Taken together our data suggest that the infection by B. cenocepacia interferes with the balance of the inducible nitric oxide synthase and arginase, favoring the latter, which could promote the attenuation of the inflammatory response and the persistence of infection / 2017-05-09

Many pathogenic microorganisms are believed to stochastically switch into low metabolic states that display resistance to supra-lethal levels of antibiotics. These so-called “persister” cells have been associated with recurrent infections and the development of antibiotic resistance. Whilst a compound that eliminates Staphylococcus aureus persister cells has been described, it is not active against Gram-negative bacteria. The aim of my PhD project was to develop a high-throughput assay for compounds that eradicate persister cells in the -proteobacterium Burkholderia thailandensis. Further to this, I aimed to develop “hit” compounds from screening into lead series through investigation of structure activity relationships and, use a chemical genetics approach to elucidate potential mechanisms of action. I developed a phenotypic assay to identify compounds that eradicate persister cells. The assay was based on the reduction of the resazurin based dye PrestoBlue. Optimization of the assay gave a Z’ prime of 0.41 when screened in high throughput at the DDU. Screening of the library of 61,250 compounds identified 2,127 compounds that gave a statistically significant reduction in persister cell numbers. Follow-up assays highlighted 29 compounds with a pIC50 greater than five. Detailed investigation allowed me to down select to six “best in class” compounds, which included the licensed drug chloroxine. A time dependent killing assay showed that chloroxine reduced levels of persister cells by three orders of magnitude over 72 hours (P = 0.01). Hit expansion around chloroxine using commercially available compounds did not identify any more potent compounds, but did highlight key features of the molecule for activity. Assay protocols were provided to collaborators at DSTL who were able to iv show that chloroxine is also active against persister cells formed by the tropical pathogen and Tier 1 biological agent Burkholderia pseudomallei. Investigations into the mechanism of action of chloroxine used Next Generation Sequencing of an over expression library, identifying two putative genes involved in inhibition of persister cells by chloroxine. My findings demonstrate a phenotypic assay against persister cells in Gram-negative bacteria, which has the power to identify potent anti-persister agents to assist in chemotherapy. Structural activity relationship and mechanism of action investigations have indicated lead series and genetic starting points for future development of this research. My PhD project has concluded with sufficient data for continuation of research following a number of leads and is at an ideal stage for instigation of a medicinal chemistry program for development of chloroxine as a clinical option for treatment of persistent melioidosis.

Submitted by BRUNO HENRIQUE DE OLIVEIRA null (bio_henri@yahoo.com.br) on 2017-04-27T19:07:13Z No. of bitstreams: 1 TESE - Bruno Henrique de Oliveira.pdf: 3915403 bytes, checksum: d2b083ce532a0ca65488edb533c50789 (MD5) / Rejected by Luiz Galeffi (luizgaleffi@gmail.com), reason: Solicitamos que realize uma nova submissão seguindo a orientação abaixo: O arquivo submetido está sem a ficha catalográfica. A versão submetida por você é considerada a versão final da dissertação/tese, portanto não poderá ocorrer qualquer alteração em seu conteúdo após a aprovação. Corrija esta informação e realize uma nova submissão com o arquivo correto. Agradecemos a compreensão. on 2017-05-03T14:48:15Z (GMT) / Submitted by BRUNO HENRIQUE DE OLIVEIRA null (bio_henri@yahoo.com.br) on 2017-05-08T12:51:56Z No. of bitstreams: 1 TESE - Bruno Henrique de Oliveira (1).pdf: 3110165 bytes, checksum: c7a30b80af11cc4e7d356a3ba35baccf (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-05-08T16:17:34Z (GMT) No. of bitstreams: 1 oliveira_bh_dr_rcla.pdf: 3110165 bytes, checksum: c7a30b80af11cc4e7d356a3ba35baccf (MD5) / Made available in DSpace on 2017-05-08T16:17:34Z (GMT). No. of bitstreams: 1 oliveira_bh_dr_rcla.pdf: 3110165 bytes, checksum: c7a30b80af11cc4e7d356a3ba35baccf (MD5) Previous issue date: 2017-02-17 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Com o desenvolvimento de uma metodologia de purificação não convencional de passo único (“MPU”) obteve-se um fator de purificação de 46,5 vezes e recuperação de 53%, observando-se banda única de 32 kDa na eletroforese SDS-PAGE. A enzima purificada apresentou atividade frente a ácidos graxos com cadeias entre 8 e 20 carbonos, destacando-se a seletividade para os ácidos palmítico (16:0) e oleico (18:1). A lipase apresentou ligeira regiosseletividade para as posições externas sn-1 e sn-3 (ésteres primários) do triacilglicerol. A cinética da reação demonstrou que a enzima segue uma cinética de Michaelis-Menten, com valor de Km de 22 mmol e Vmax de 12,7 mmol/min/mg. O valor de kcat foi de 225 s-1 e a eficiência catalítica de 104 mol-1.s-1. A temperatura para atividade máxima foi de 55 °C, sendo razoavelmente estável a 60 °C, mantendo 60% da atividade após 1 h. A atividade máxima foi obtida na faixa de pH entre 4 e 9, mantendo 100% da atividade inicial após incubação por 1 h na faixa de pH entre 2,2 e 10,0. A estabilidade em solventes orgânicos foi estudada por incubação da enzima em solventes miscíveis e imiscíveis em água. A enzima manteve 100% da atividade inicial em tolueno, n-hexano e n-heptano e apresentou estabilidade de 78%, 76% e 90% quando incubada com 50% (v/v) de metanol, etanol e isopropanol. Com relação às aplicações em reações de interesse industrial, a lipase de B. lata apresentou comportamento predominantemente de hidrolase (90%) em detrimento a ação de aciltransferase. Para a síntese de ésteres de aroma produz o éster caprilato de isoamila com 45% de rendimento (24 h e 50 °C em n-hexano). Para a síntese de um éster de cera (oleil oleato, C36), apresentou 87% de rendimento (30 h, 55 °C). A obtenção de ácidos graxos epoxidados alcançou rendimento de 44,4% em reação quimio-enzimática de epoxidação do oleato de metila (48 h, 40 °C e 10% (v/v) de H2O2). A atividade frente a galactolipídeos de espinafre in situ atingiu 2700 U/mg. Em ambientes gástricos simulados apresentou alta atividade e estabilidade em presença de proteases (pepsina e tripsina) em condições de baixo pH, além de demonstrar surpreendente ativação na presença de altas concentrações de sais biliares (NaTDC). / With the development of a non-conventional one-step purification methodology, was obtained a purification factor of 46.5 and recovery of 53%, noting single band of 32 kDa in SDS-PAGE. The purified enzyme showed activity against fatty acids with chains from 8 to 20 carbons, highlighting the preference for palmitic (16:0) and oleic acids (18:1). The lipase showed slight regioselectivity for external positions sn-1 and sn-3 (primary esters). The reaction kinetics showed that the enzyme follows a Michaelis-Menten model with Km value of 22 mmol and Vmax of 12.7 mmol/min/mg. The kcat value was 225 s-1 and the catalytic efficiency was 104 mol-1.s-1. The temperature for maximum activity was 55 °C, and reasonably stable at 60 °C, keeping 60% of activity after 1 h. The maximum activity was obtained at pH values between 4 and 9, maintaining 100% of the initial activity after 1 h incubation in a pH range from 2.2 to 10.0. Stability in organic solvents was studied by incubating the enzyme in miscible and immiscible solvents. The enzyme retained 100% activity in toluene, n-hexane and n-heptane and was 78%, 76% and 90% stable when incubated with 50% (v/v) methanol, ethanol and isopropanol, respectively. Regarding the applications in industrial interest reactions, B. lata lipase presented predominantly hydrolase behavior (90%) over acyltransferase action. For the flavor esters synthesis, the isoamyl caprylate ester is produced with 45% yield (24 h and 50 °C). For a wax ester synthesis (oleyl oleate, C36) showed 87% yield (30 h, 55 °C). The epoxidized fatty acids obtainment reached 44.4% yield in methyl oleate chemo-enzymatic epoxidation (48h, 40 °C and 10% (v/v) H2O2). The activity against spinach galactolipids in situ reached 2700 U/mg. In simulated gastric environment showed high activity and stability in the presence of proteases (pepsin and trypsin) in low pH conditions, besides demonstrating surprising activation in the presence of high concentrations of bile salts (NaTDC). / CNPq: 140129/2013-8

Submitted by (lucia.rodrigues@ufrpe.br) on 2016-06-08T14:30:49Z No. of bitstreams: 1 Jose Andreey Almeida Teles.pdf: 1056555 bytes, checksum: 1cd9d8325b9ce830ca450c9fea318bad (MD5) / Made available in DSpace on 2016-06-08T14:30:49Z (GMT). No. of bitstreams: 1 Jose Andreey Almeida Teles.pdf: 1056555 bytes, checksum: 1cd9d8325b9ce830ca450c9fea318bad (MD5) Previous issue date: 2012-02-13 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Glanders is an infectious-contagious disease of acute or chronic character which principally affects horses, causing enormous losses in the productive chain of this animal. To control the disease, the Ministry of Agriculture, Husbandry and Supply instituted mandatory sanitation measures in the entire national territory which include an official diagnosis through the complement fixation (CF) test, maleinization and sacrifice of the animals that are positive. Nowadays the kits used for the diagnosis of the disease are imported, making their routine application difficult and more expensive. The objective of this study was to standardize an indirect ELISA test, using the proteic extract of Burkholderia mallei isolated from a carrier horse in the state of Pernambuco. The samples were cultivated in 10% blood agar and incubated for 48h at 37ºC; later, one of the isolated colonies was characterized phenotypically and genotypically and immediately cultivated in brain heart infusion (BHI) for enrichment; then it was peaked (repicada) for the Dor-set Henley medium which was incubated at 37ºC under 60rpm for eight weeks. To standardize the test the Protein G Peroxidase Sigma Conjugate was used in the dilution of 1:90.000, with serums diluted in 1:100 and the antigen in 1:400. Sixty serums were used as negative controls, tested before the CF to determine the cutting point which was 0.042nm. After establishing the standardization, 300 samples were tested, of which 99% (297) were in agreement with the results obtained in the CF. At the end, of assay presented 100% sensibility and 98.2% specificity, with predictive (preditivo) positive and negative values of 97.7% and 100% respectively. The Kappa concordance test was 0.98 and the intra and interplac repeatability were 8.8% and 10.3% respectively. From the results obtained, it is possible to affirm that the indirect ELISA test can be used as an efficient diagnosis tool. However, more essays must be carried out to consolidate the reliability of this test. / O mormo é uma enfermidade infecto-contagiosa de caráter agudo ou crônico que acomete principalmente os equídeos, causando enormes prejuízos na cadeia produtiva do cavalo. Para controlar a enfermidade, o Ministério da Agricultura, Pecuária e Abastecimento instituiu medidas sanitárias obrigatórias em todo território nacional que incluem o diagnóstico oficial pela fixação do complemento (FC), maleinização e sacrifício dos animais positivos. Os kits atuais utilizados no diagnóstico da doença são importados, dificultando e encarecendo sua aplicação na rotina. Objetivou-se com este estudo padronizar um teste de ELISA indireto utilizando o extrato protéico de Burkholderia mallei isolada a partir de equídeo portador no estado de Pernambuco. As amostras foram cultivadas em ágar sangue 10%, incubada por 48h a 37ºC; posteriormente caracterizou-se fenotípica e genotipicamente uma das colônias isoladas, e em seguida a cultivou em BHI para enriquecimento; logo após, esta foi repicada para o meio Dor-set Henley o qual foi incubado a 37ºC sob 60rpm por oito semanas. Para padronização do teste utilizouse o conjugado Proteína G Peroxidase Sigma na diluição de 1:90.000, com soros diluídos em 1:100 e o antígeno em 1:400. Utilizou-se 60 soros como controle negativo testados frente à FC para determinação do ponto de corte o qual ficou em 0,042nm. Feitas as padronizações, foram testadas 300 amostras, onde 99% (297) foram concordantes com os resultados obtidos na FC. Ao final, o ensaio apresentou 100% de sensibilidade e 98,2% de especificidade, com valores preditivo positivo e negativo de 97,7% e 100% respectivamente. O teste de concordância kappa foi 0,98 e a repetibilidade intra e interplaca ficaram em 8,8% e 10,3% respectivamente. Diante dos resultados obtidos durante os ensaios, atesta-se que o teste de ELISA indireto pode ser utilizado como uma ferramenta de diagnóstico eficiente. Entretanto, mais ensaios devem ser realizados visando consolidar a confiabilidade do referido teste.

Submitted by Pedro Barros (pedro.silvabarros@ufpe.br) on 2018-08-21T20:32:01Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) DISSERTAÇÃO Josineide Neri Monteiro.pdf: 2021284 bytes, checksum: 33052b504f3c70e6a33eb5267d37322b (MD5) / Approved for entry into archive by Alice Araujo (alice.caraujo@ufpe.br) on 2018-08-29T17:42:19Z (GMT) No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) DISSERTAÇÃO Josineide Neri Monteiro.pdf: 2021284 bytes, checksum: 33052b504f3c70e6a33eb5267d37322b (MD5) / Made available in DSpace on 2018-08-29T17:42:19Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) DISSERTAÇÃO Josineide Neri Monteiro.pdf: 2021284 bytes, checksum: 33052b504f3c70e6a33eb5267d37322b (MD5) Previous issue date: 2016-09-08 / CAPES / O gênero Burkholderia compreende bactérias gran-negativas, aeróbicas pertencentesà classe β-proteobacteria. Estudos de 16S rDNA revelaram que o gênero Burkholderia é composto por bactérias que, apesar de intimamente relacionadas e fenotipicamente muito similares, possuem múltiplas diferenças genéticas, suficientes para permitir subdivisões em espécies ou variantes genômicas, que formam o complexo B. cepacia. Dados biológicos, especialmente os de sequenciamento genômico, vêm sendo gerados em ritmo acelerado nas últimas décadas. Com o surgimento da Bioinformática, podemos aplicar técnicas computacionais para manipular dados biológicos. O alinhamento múltiplo de sequências (MAS) é um conjunto de técnicas utilizadas para entender informações biológicas de um conjunto de sequências sendo considerada a tarefa mais comum e mais importante da bioinformática, visto que pode fornecer consideráveis informações sobre estrutura e função de genes. Os algoritmos genéticos (AGs) permitem uma simplificação na formulação e solução de problemas de otimização visto que incorporam uma solução potencial para um problema específico numa estrutura semelhante à de um cromossomo e aplicam operadores de seleção e cruzamento a essas estruturas de forma a preservar informações críticas relativas à solução do problema. O presente trabalho objetivou aplicar técnicas computacionais visando solucionar o problema de alinhamento genético de sequências biológicas de DNA de bactérias do complexo Burkholderia cepacia. As sequências analisadas (586) foram obtidas através do banco de dados GenBank do National Center for Biotechnology Information (NCBI). Para alinhamento das sequências, utilizou-se as seguintes ferramentas: Clustal ômega e Kalign. Das ferramentas utilizadas, nenhuma conseguiu gerar dados de boa acurácia. Desse modo, conclui-se que existe a necessidade de desenvolvimento de novos algoritmos/ferramentas de alinhamento genético visando trabalhar com grande quantidade de dados para obtenção de uma otimização. Para o caso de várias sequências, o problema do alinhamento múltiplo é considerado NP-difícil. Desse modo, foi observado que é necessário desenvolver novos algoritmos, para sua resolução em tempo hábil buscando sempre soluções bem aproximadas da solução ótima. / The genus Burkholderia comprises gran-negative bacteria, aerobic belonging to β-proteobacteria class. 16S rDNA analyzes have revealed that the genus Burkholderia is composed of bacteria which, although closely related and phenotypically very similar, have multiple genetic enough differences to allow subdivisions species or genomic variants that constitute the B. cepacia complex. Biological data, especially the genomic sequencing, are being generated at a rapid pace in recent decades. With the emergence of bioinformatics, we can apply computational techniques to manipulate biological data. The multiple sequence alignment (MAS) is a set of techniques used to understand biological information from a set of sequences is considered the most common and most important task of bioinformatics, since it can provide considerable information about the structure and function of genes. AGs allow a simplification in the design and optimization of troubleshooting as incorporate a potential solution to a specific problem in a structure similar to a chromosome and apply selection and crossover operators such critical information to preserve the form of structures for the solution problem. This study aimed to apply computational techniques aimed at solving the genetic alignment problem of biological DNA sequences of bacteria Burkholderia cepacia complex. The sequences analyzed (586) were obtained from the GenBank database of the National Center for Biotechnology Information (NCBI). For aligning the sequences, the following tools were used: Clustal omega and Kalign. The tools used, none was able to generate good data accuracy. Thus, it is concluded that there is a need to develop new algorithms / alignment tools genetic targeting working with large amounts of data to obtain an optimization. In the case of multiple sequences, the problem of multiple alignment is considered to be NP-hard. Thus, it was observed that it is necessary to develop new algorithms for its resolution in a timely manner and always seeking approximate solutions of the optimal solution.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2018-06-12T14:14:59Z No. of bitstreams: 1 Cecilia Maria de Souza Leao e Silva.pdf: 1493945 bytes, checksum: 8bcc1ab9e28f749b73d9d7ff2cbaa285 (MD5) / Made available in DSpace on 2018-06-12T14:16:15Z (GMT). No. of bitstreams: 1 Cecilia Maria de Souza Leao e Silva.pdf: 1493945 bytes, checksum: 8bcc1ab9e28f749b73d9d7ff2cbaa285 (MD5) Previous issue date: 2018-02-28 / Glanders is a highly contagious disease caused by Burkholderia mallei solipeds, a Gram - negative bacterium, not motility and aerobic coccobacillus, primarily infecting horses, donkeys and mules, though humans are considered accidental hosts . The Burkholderia mallei is listed in the list of the World Organisation for Animal Health (OIE) as an important public health disease, and due to its high potential for infection is referred to as a bioterrorism agent. According to the OIE comprises the serological diagnosis, allergy testing and bacterial isolation, and complement fixation, the official test to be performed for trade of animals. This method of diagnosis is recommended in Brazil by Instruction Nº 22, 16 of march of 2018, Ministry of Agriculture, Livestock and Food Supply by its high sensitivity and specificity. According to the OIE comprises the serological diagnosis, allergy testing and bacterial isolation, and complement fixation, the official test to be performed for trade of animals and bacterial isolation is considered gold test for identification of the agent. From the bacterial isolation the presence of Burkholderia was identified in 11 samples from epidemiological surveillance in the disease outbreaks. Sequencing demonstrated the circulation of the strains Burkholderia pseudomallei and Burkholderia mallei in Brazilian territory where Mormo is present. The results show the differentiation of the circulating strains in the national territory and provides the knowledge of their epidemiology and virulence, later assisting in formulations of disease control and eradication measures / O Mormo constitui-se em uma doença altamente contagiosa dos solípedes causada pela Burkholderia mallei, uma bactéria Gram-negativa, não móvel e cocobacilo aeróbio, infectando primariamente equínos, asininos e muares, entretanto humanos são considerados hospedeiros acidentais. Burkholderia mallei está relacionada na lista de doenças da Organização Mundial para a Saúde Animal (OIE) como doença de importância de saúde pública, e devido ao seu alto potencial de infecção é referenciada como agente de bioterrorismo. De acordo com a OIE o diagnóstico compreende o teste sorológico, alérgico, isolamento bacteriano e testes de biologia molecular, sendo a fixação do complemento, o teste oficial a ser realizado para trânsito de animais. O Ministério da Agricultura, Pecuária e Abastecimento, através da Portaria Nº 22, de 16 de março de 2018 do Ministério, utiliza o teste da Fixação do Complemento e Elisa para triagem dos animais, sendo aqueles reagentes, testados posteriormente com o Western Blotting para confirmação. Neste trabalho, temos como objetivo identificar filogeneticamente as cepas circulantes em território nacional para os animais suspeitos de Mormo. A partir do isolamento bacteriano foram identificados 08 resultados compatíveis com a Burkholderia em amostras oriundas de vigilância epidemiológica em áreas focos da doença. O sequenciamento demonstrou a circulação das cepas Burkholderia pseudomallei e Burkholdeia mallei, em território brasileiro onde há presença do Mormo. Os resultados mostram a diferenciação das estirpes circulantes em território nacional e proporciona o conhecimento de sua epidemiologia e virulência, auxiliando posteriormente em formulações de medidas para controle e erradicação da enfermidade.

The trimethoprim and sulfamethoxazole combination, co-trimoxazole, plays a vital role in the treatment of Burkholderia pseudomallei infections. Previous studies demonstrated that the B. pseudomallei BpeEF-OprC efflux pump confers widespread trimethoprim resistance in clinical and environmental isolates, but this is not accompanied by significant resistance to co-trimoxazole. Using the excluded select-agent strain B. pseudomallei Bp82, we now show that in vitro acquired trimethoprim versus cotrimoxazole resistance is mainly mediated by constitutive BpeEF-OprC expression due to bpeT mutations or by BpeEF-OprC overexpression due to bpeS mutations. Mutations in bpeT affect the carboxy-terminal effector-binding domain of the BpeT LysR-type activator protein. Trimethoprim resistance can also be mediated by dihydrofolate reductase (FolA) target mutations, but this occurs rarely unless BpeEF-OprC is absent. BpeS is a transcriptional regulator that is 62% identical to BpeT. Mutations affecting the BpeS DNA-binding or carboxy-terminal effector-binding domains result in constitutive BpeEF-OprC overexpression, leading to trimethoprim and sulfamethoxazole efflux and thus to cotrimoxazole resistance. The majority of laboratory-selected co-trimoxazole-resistant mutants often also contain mutations in folM, encoding a pterin reductase. Genetic analyses of these mutants established that both bpeS mutations and folM mutations contribute to co-trimoxazole resistance, although the exact role of folM remains to be determined. Mutations affecting bpeT, bpeS, and folM are common in co-trimoxazole-resistant clinical isolates, indicating that mutations affecting these genes are clinically significant. Cotrimoxazole resistance in B. pseudomallei is a complex phenomenon, which may explain why resistance to this drug is rare in this bacterium. IMPORTANCE Burkholderia pseudomallei causes melioidosis, a tropical disease that is difficult to treat. The bacterium's resistance to antibiotics limits therapeutic options. The paucity of orally available drugs further complicates therapy. The oral drug of choice is co-trimoxazole, a combination of trimethoprim and sulfamethoxazole. These antibiotics target two distinct enzymes, FolA (dihydrofolate reductase) and FolP (dihydropteroate synthase), in the bacterial tetrahydrofolate biosynthetic pathway. Although co-trimoxazole resistance is minimized due to two-target inhibition, bacterial resistance due to folA and folP mutations does occur. Co-trimoxazole resistance in B. pseudomallei is rare and has not yet been studied. Co-trimoxazole resistance in this bacterium employs a novel strategy involving differential regulation of BpeEF-OprC efflux pump expression that determines the drug resistance profile. Contributing are mutations affecting folA, but not folP, and folM, a folate pathway-associated gene whose function is not yet well understood and which has not been previously implicated in folate inhibitor resistance in clinical isolates.

Melioidosis is an important cause of community-acquired sepsis, endemic to Southeast Asia and Northern Australia. Melioidosis is caused by the soil saprophyte, Burkholderia pseudomallei, a motile Gram-negative bacillus, and is associated with a mortality rate that approaches 50% in Northeast Thailand. The most important risk factor for melioidosis is diabetes mellitus, and two-thirds of all adult patients with melioidosis have diabetes as a risk factor. It has been noted previously, however, that patients with diabetes have lower mortality than patients without diabetes. In this dissertation, we look at a cohort of 1160 consecutive adult melioidosis patients presenting to Sappasithiprasong Hospital in Ubon Ratchathani, Thailand, 410 (35%) of whom were diagnosed with diabetes prior to admission. We confirmed previous findings that diabetes protected from mortality in melioidosis, but also found that this protective effect was confined to a smaller subset of patients (208 patients) who were treated with glibenclamide prior to admission. Patients with hyperglycaemia (but no diagnosis of diabetes prior to admission) had the same mortality rate as patients without diabetes. In vitro experiments found no inhibitory effect of glibenclamide on bacterial growth, and we therefore looked for evidence of an effect of glibenclamide on the host. We conducted a gene expression study of circulating blood leukocytes in melioidosis patients and compared them to uninfected controls. In this study, we found that glibenclamide was associated with an anti-inflammatory effect on the host response to melioidosis. To further elucidate a mechanism for the action of glibenclamide, we studied the effect of glibenclamide therapy in a mouse model of melioidosis and found that the effect of glibenclamide was specific to interleukin-1β secretion. This reduction in interleukin-1β secretion was associated with reduced cellular influx into the lungs as well as lower bacterial loads in blood, liver and spleen.