Made available in DSpace on 2015-04-11T13:38:40Z (GMT). No. of bitstreams: 1 Dissertacao_Kariza_PDF.pdf: 850701 bytes, checksum: b37877619512bd71829e75c0c32ae9fd (MD5) Previous issue date: 2006-08-28 / Fundação de Amparo à Pesquisa do Estado do Amazonas / Endophytic microorganisms can be utilized in distinct ways in Biotechnology science. Among them, one of most interesting uses is as heterologue gene carriers into plants, allowing the development of new Biotechnologic processes, which makes relevant the development of vectors from native plasmids from the endophytic bacterias itselves for genetic transformation of this kind of bacteria. Studies involving Enterobacter agglomerans, a endophytic bacteria isolated from Copaifera multijuga (copaiba tree), demonstrated the presence of a small, cryptic plasmid named pEA1. Based on this plasmid, the pEA1.0 and pEA2.4 plasmids were developed. pEA1.0 was built from a fragment of PstI (1000 bp), cloned in pUC18. pEA2.4 was developed from an amplified fragment (2415 bp), cloned in the vector pCR2.1TOPO (Invitrogen), which allowed, by primer walking, the determination of the complete sequence of the original plasmid (2545 bp), which had been previously recorded in GenBank (access DQ659147). The sequence analysis showed a GC level of 34% and an AT level of 66%. The restriction map was determined using NEBCutter2.0. Comparison between pEA1 sequence and the data bank revealed high similarity (62%) with the sequence of the pIGMS31 plasmid (2520 bp) from Klebsiella pneumoniae. Using the BlastX and ORF finder softwares, the result demonstrated the presence of two ORFs, one of them similar (E value=-98) to ORF2 of pIGMS31 (AY543072.1) isolated from K. pneumoniae. The pEA2.4 plasmid was used to genetically transform Escherichia coli and E. agglomerans by the Tris-calcium/thermal shock method. Besides the capability of pEA2.4 to genetically transform E. coli cells, it has showed itself capable of transforming E. agglomerans as well, which can actually acquire resistance to kanamicine. This reflects the bifunctional chacter of pEA2.4. The transformation efficacy of E. agglomerans using pEA2.4 extracted from E. coli was about 5 x 104 T/μg, while the same process with pEA2.4 extracted from E. agglomerans itself showed a ten times higher efficacy (5,1 x 105 T/μg), probably because of the avoidance of host restriction. The pEA2.4 plasmid will be used as foundation for the development of heterologue gene expression vectors in E. agglomerans. / Microrganismos endofíticos podem ser utilizados de diversas formas em biotecnologia. Dentre estas, se destaca o uso como carreadores de genes heterólogos para o interior de plantas possibilitando o desenvolvimento de novos processos biotecnológicos, o que torna relevante o desenvolvimento de vetores a partir de plasmídeos nativos das próprias bactérias endofíticas para transformação genética desse tipo de bactérias. Estudos envolvendo a Enterobacter agglomerans, uma bactéria endofítica isolada de Copaifera multijuga (copaíba) demonstraram a presença de um pequeno plasmídeo críptico denominado pEA1. Com base neste plasmídeo foram desenvolvidos os plasmídeos pEA1.0 e pEA2.4. O pEA1.0 foi construído a partir do fragmento de PstI (1000 pb) clonado em pUC18. O plasmídeo pEA2.4 foi desenvolvido a partir de um fragmento amplificado (2415 pb) clonado no vetor pCR2.1TOPO (INVITROGEN), o qual por primer walking permitiu a determinação da seqüência completa do plasmídeo original (2545 pb), que foi depositada no GenBank (acesso DQ659147). A análise da seqüência mostrou um índice GC de 34% e AT de 66%, e o mapa de restrição foi determinado utilizando a ferramenta NEBCutter2.0. Comparando a seqüência do pEA1 com o banco de dados, observou-se alta similaridade (62%) com a seqüência do plasmídeo pIGMS31 (2520 pb) de Klebsiella pneumoniae. Utilizando as ferramentas BlastX e ORF finder, o resultado demonstrou a presença de duas ORFs, sendo uma delas similar (E value = -98) à ORF2 do plasmídeo pIGMS31 (AY543072.1) isolado de K. pneumoniae. O plasmídeo pEA2.4 foi utilizado para transformar geneticamente bactérias Escherichia coli e E. agglomerans pelo método Tris-Cálcio/choque térmico. O pEA2.4 além de ser capaz de transformar geneticamente células de E. coli, mostrou-se também capaz de transformar geneticamente a E. agglomerans tornando-a resistente a canamicina, evidenciando seu caráter bifuncional. A eficiência de transformação da E. agglomerans com pEA2.4 extraído de E. coli foi de 5 x 104 T/μg, enquanto que a transformação desta bactéria com o pEA2.4 extraído da própria E. agglomerans, apresentou eficiência 1 ordem de grandeza maior (5,1 x 105 T/μg) provavelmente por ter sido, desta forma, evitado o processo de restrição da hospedeira. O plasmídeo pEA2.4 será utilizado como base para o desenvolvimento de vetores de expressão de genes heterólogos em E. agglomerans.

A expressão de proteínas na forma de corpos de inclusão em bactérias é uma alternativa muito interessante para obtenção de proteínas recombinantes. No entanto, a agregação é uma dificuldade frequentemente encontrada durante a renaturação dessas proteínas. Altas pressões hidrostáticas são capazes de solubilizar os corpos de inclusão na presença de baixas concentrações de reagentes desnaturantes, favorecendo a renaturação protéica com alto rendimento e redução de custos. O presente trabalho tem como objetivo a renaturação de proteínas recombinantes expressas em Escherichia coli sob a forma de corpos de inclusão usando altas pressões hidrostáticas. Três toxinas, todas apresentando cinco ou mais pontes dissulfídicas foram estudadas: NXH8, Naterina 2 e Bothropstoxina 1. Suspensões dos corpos de inclusão das três proteínas foram pressurizadas em 2000 bares de pressão durante 16 horas. Os tampões de renaturação foram otimizados para as três proteínas. O tampão utilizado no processo de renaturação da NXH8 foi Tris HCl 50 mM, pH 9,0 com proporção de 1GSH:4GSSG em concentração de 6 mM e 2 M GdnHCl. Foram utilizados corpos de inclusão em D.O.(A600nm) de 0,5. Após o processo de renaturação foi realizada diálise em pH 7,0. O rendimento final de recuperação de NXH8 solúvel foi de 40%, sendo obtidos 28,6 mg/L de meio de cultura. A renaturação de Bothropstoxina 1 foi obtida em tampão de renaturação Tris HCl 50 mM pH 7,5 na proporção de 2 GSH:3 GSSG em concentração de 3 mM e 1 M GdnHCl. Utilizamos uma suspensão com D.O.(A600nm) de 0,5. O rendimento final de recuperação de Bothropstoxina 1 renaturada foi de 32 %, obtendo-se 9,2 mg/L de meio de cultura. A renaturação de Naterina 2 foi obtida em tampão de renaturação com 20 mM de Tris HCl pH 9,0 na proporção de 2 GSH:3 GSSG e concentração de 10 mM e 1 M GdnHCl e corpos de inclusão na D.O. (A600nm) de 6,0. Foram obtidas 3,7 mg de Nateria 2 renaturada /L de meio de cultura (20% de recuperação a partir dos corpos de inclusão). O rendimento da Naterina 2 renaturada foi de 20 %. Para a análise e a comprovação da eficácia do processo de renaturação sob pressão foram utilizadas as técnicas de SDS-PAGE, western blot, microscopia eletrônica de varredura, ensaios biológicos in vivo e in vitro e estruturais. As análises físicoquímicas realizadas em NXH8 não mostraram nenhuma comprovação da sua renaturação. O ensaio in vivo realizado com a Naterina 2 mostrou uma leve atividade de contração de vênulas, indicando que ela esteja em sua conformação correta. Os ensaios in vitro com a Bothropstoxina 1 mostraram uma atividade citotóxica dose-dependente em células musculares. / The expression of proteins as inclusion bodies in bacteria is a widely used alternative for production of recombinante protein. However, the aggregation is a problem often encountered during refolding of these proteins. High hydrostatic pressure are able to solubilize the inclusion bodies in the presence of low concentrations of denaturant reagents, encouraging refolding protein with high efficiency and reduce costs. This work aims to refolding of recombinant proteins expressed in Escherichia coli from inclusion bodies using high hydrostatic pressure. Three toxins, all featuring five or more disulfide bonds were studied: NXH8, Natterin 2 and Bothropstoxin 1. Suspensions of inclusion bodies of the three proteins were pressurized to 2000 bars for 16 hours. The buffers were optimized for refolding of the three proteins. The buffer used in the refolding of NXH8 was 50 mM Tris HCl, pH 9.0 with proportion of 1GSH: 4GSSG at a concentration of 6 mM and 2 M GdnHCl. Inclusion bodies were used in O.D. (A600nm) of 0.5. After refolding process, dialysis was performed at pH 7.0. The final yield of obtaining soluble NXH8 was 40% (28,6 mg of soluble NXH8/L of culture medium). The refolding of Bothropstoxin 1 was obtained in refolding buffer of Tris HCl 50 mM, pH 7,5 with proportion of 2 GSH: GSSG 3 and concentration of 3 mM and 1 M GdnHCl. Use with a suspension of O.D. (A600nm) of 0.5. The final yield of recovery of Bothropstoxin 1 refolded was 32% (9,2 mg of refolded Bothropstoxin 1/L of culture medium). The refolding of Natterin 2 was performed in the refolding buffer: 20 mM Tris HCl pH 9.0 at a ratio of 2 GSH: 3GSSG and concentration of 10 mM and 1 M GdnHCl and inclusion bodies O.D. (A600nm) of 6.0. The yield of Natterin 2 refolded was 20% (3,7 mg/L of culture medium). Physico-chemical and biological analysis were performed by SDS-PAGE, western blot, scanning electron microscopy, biological tests in vivo and in vitro and structural. The analysis conducted in NXH8 did not show any evidence of refolding. An activity of contraction of venules was show by the in vivo test indicating a correct conformation of Natterin 2. Tests in vitro with Bothropstoxin 1 showed a dosedependent cytotoxic activity in muscle cells.

A expressão de proteínas na forma de corpos de inclusão em bactérias é uma alternativa muito interessante para obtenção de proteínas recombinantes. No entanto, a agregação é uma dificuldade frequentemente encontrada durante a renaturação dessas proteínas. Altas pressões hidrostáticas são capazes de solubilizar os corpos de inclusão na presença de baixas concentrações de reagentes desnaturantes, favorecendo a renaturação protéica com alto rendimento e redução de custos. O presente trabalho tem como objetivo a renaturação de proteínas recombinantes expressas em Escherichia coli sob a forma de corpos de inclusão usando altas pressões hidrostáticas. Três toxinas, todas apresentando cinco ou mais pontes dissulfídicas foram estudadas: NXH8, Naterina 2 e Bothropstoxina 1. Suspensões dos corpos de inclusão das três proteínas foram pressurizadas em 2000 bares de pressão durante 16 horas. Os tampões de renaturação foram otimizados para as três proteínas. O tampão utilizado no processo de renaturação da NXH8 foi Tris HCl 50 mM, pH 9,0 com proporção de 1GSH:4GSSG em concentração de 6 mM e 2 M GdnHCl. Foram utilizados corpos de inclusão em D.O.(A600nm) de 0,5. Após o processo de renaturação foi realizada diálise em pH 7,0. O rendimento final de recuperação de NXH8 solúvel foi de 40%, sendo obtidos 28,6 mg/L de meio de cultura. A renaturação de Bothropstoxina 1 foi obtida em tampão de renaturação Tris HCl 50 mM pH 7,5 na proporção de 2 GSH:3 GSSG em concentração de 3 mM e 1 M GdnHCl. Utilizamos uma suspensão com D.O.(A600nm) de 0,5. O rendimento final de recuperação de Bothropstoxina 1 renaturada foi de 32 %, obtendo-se 9,2 mg/L de meio de cultura. A renaturação de Naterina 2 foi obtida em tampão de renaturação com 20 mM de Tris HCl pH 9,0 na proporção de 2 GSH:3 GSSG e concentração de 10 mM e 1 M GdnHCl e corpos de inclusão na D.O. (A600nm) de 6,0. Foram obtidas 3,7 mg de Nateria 2 renaturada /L de meio de cultura (20% de recuperação a partir dos corpos de inclusão). O rendimento da Naterina 2 renaturada foi de 20 %. Para a análise e a comprovação da eficácia do processo de renaturação sob pressão foram utilizadas as técnicas de SDS-PAGE, western blot, microscopia eletrônica de varredura, ensaios biológicos in vivo e in vitro e estruturais. As análises físicoquímicas realizadas em NXH8 não mostraram nenhuma comprovação da sua renaturação. O ensaio in vivo realizado com a Naterina 2 mostrou uma leve atividade de contração de vênulas, indicando que ela esteja em sua conformação correta. Os ensaios in vitro com a Bothropstoxina 1 mostraram uma atividade citotóxica dose-dependente em células musculares. / The expression of proteins as inclusion bodies in bacteria is a widely used alternative for production of recombinante protein. However, the aggregation is a problem often encountered during refolding of these proteins. High hydrostatic pressure are able to solubilize the inclusion bodies in the presence of low concentrations of denaturant reagents, encouraging refolding protein with high efficiency and reduce costs. This work aims to refolding of recombinant proteins expressed in Escherichia coli from inclusion bodies using high hydrostatic pressure. Three toxins, all featuring five or more disulfide bonds were studied: NXH8, Natterin 2 and Bothropstoxin 1. Suspensions of inclusion bodies of the three proteins were pressurized to 2000 bars for 16 hours. The buffers were optimized for refolding of the three proteins. The buffer used in the refolding of NXH8 was 50 mM Tris HCl, pH 9.0 with proportion of 1GSH: 4GSSG at a concentration of 6 mM and 2 M GdnHCl. Inclusion bodies were used in O.D. (A600nm) of 0.5. After refolding process, dialysis was performed at pH 7.0. The final yield of obtaining soluble NXH8 was 40% (28,6 mg of soluble NXH8/L of culture medium). The refolding of Bothropstoxin 1 was obtained in refolding buffer of Tris HCl 50 mM, pH 7,5 with proportion of 2 GSH: GSSG 3 and concentration of 3 mM and 1 M GdnHCl. Use with a suspension of O.D. (A600nm) of 0.5. The final yield of recovery of Bothropstoxin 1 refolded was 32% (9,2 mg of refolded Bothropstoxin 1/L of culture medium). The refolding of Natterin 2 was performed in the refolding buffer: 20 mM Tris HCl pH 9.0 at a ratio of 2 GSH: 3GSSG and concentration of 10 mM and 1 M GdnHCl and inclusion bodies O.D. (A600nm) of 6.0. The yield of Natterin 2 refolded was 20% (3,7 mg/L of culture medium). Physico-chemical and biological analysis were performed by SDS-PAGE, western blot, scanning electron microscopy, biological tests in vivo and in vitro and structural. The analysis conducted in NXH8 did not show any evidence of refolding. An activity of contraction of venules was show by the in vivo test indicating a correct conformation of Natterin 2. Tests in vitro with Bothropstoxin 1 showed a dosedependent cytotoxic activity in muscle cells.

La maladie de Crohn (MC) est une maladie inflammatoire chronique de l’intestin (MICI), dont la physiopathologie résulterait d’une interaction anormale entre le microbiote intestinal et le système immunitaire de l’hôte sous l’influence de facteurs génétiques et environnementaux. Au sein de ce microbiote, les E. coli adhérents et invasifs (AIEC) colonisent la muqueuse iléale des patients atteints de la MC et sont capables de survivre et se multiplier à l’intérieur des macrophages. Par ailleurs, les objectifs thérapeutiques de la MC et notamment la cicatrisation muqueuse endoscopique nécessitent des endoscopies répétées, peu acceptables du point de vue des patients. Parmi les moyens alternatifs, la calprotectine fécale est le marqueur fécal de référence même si ses performances semblent diminuées dans certaines situations comme la maladie iléale pure. Le premier objectif de ces travaux étaient de comparer la capacité des macrophages dérivés de monocytes (MDM) issus de patients atteints de MC, de rectocolite hémorragique (RCH) ou de sujets sains à contrôler l’infection par les AIEC et d’identifier les facteurs associés à cette multiplication des AIEC et notamment le rôle des polymorphismes génétiques associés à la MC en lien avec l’autophagie. Les AIEC se multipliaient de manière plus importante que la souche non pathogène K12 dans les macrophages quel que soit leur origine. L’entrée des AIEC (1h post- infection) ne variait pas en fonction de la provenance des macrophages. La survie des AIEC était augmentée dans les MDM issus de patients MC comparés à ceux issus de RCH ou de sujets contrôles. En analyse multivariée, cette survie était positivement corrélée à la sécrétion d’IL1β mais était diminuée en présence des variants à risque pour ULK1 (p=0,046) et XBP1 (p=0,014). Les MDM issus de patients MC étaient incapable de contrôler la multiplication des AIEC contrairement à ceux issus de RCH ou de sujets contrôles d’autant plus en présence du variant à risque pour IRGM (p=0,045). L’infection des MDM de patients MC par les bactéries AIEC induit un profil de sécrétion cytokinique pro-inflammatoire. La deuxième partie de ces travaux avait pour but de comparer les performances de la chitinase 3-like 1 fécale (CHI3L1), une protéine de l’hôte interagissant avec un facteur de virulence des AIEC, et la métalloprotéase matricielle 9 (MMP-9) pour détecter l’activité inflammatoire endoscopique de la MC en comparaison du marqueur fécal de référence, la calprotectine. Les taux de CHI3L1, de MMP-9 et de calprotectine fécales étaient corrélés au ‘Crohn’s Disease Endoscopic Index of Severity’ (CDEIS) et étaient significativement augmentés en présence d’ulcérations endoscopiques. En cas d’atteinte iléale pure, la CHI3L1 fécale semblait mieux corrélée au CDEIS que la calprotectine fécale. Le seuil de CHI3L1 fécale de 15 ng/g présentait de meilleures performances que la calprotectine fécale pour détecter la présence d’ulcérations endoscopiques. La MMP-9 étaient un marqueur performant pour détecter la présence de lésions endoscopiques dans les MICI. En conclusion, nous avons montré qu’il existe un défaut des macrophages à contrôler l’infection par les bactéries AIEC chez les patients atteints de MC en rapport avec les variants à risque impliqués dans l’autophagie conduisant à un phénotype de macrophages pro-inflammatoires. La CHI3L1 fécale, connue comme une protéine de l’hôte interagissant avec un facteur de virulence des AIEC, tout comme la MMP-9 semblent être de bons marqueurs d’activité endoscopique dans les MICI. / Crohn's disease (CD) is a chronic inflammatory bowel disease (IBD) whose pathophysiology results from an abnormal interaction between the gut microbiota and the host's immune system under the influence of genetic and environmental factors. . Within this microbiota, adherent and invasive E. coli (AIEC) colonize the ileal mucosa of patients with CD and are able to survive and multiply within macrophages. Moreover, the therapeutic objectives of CD, and especially endoscopic mucosal healing, require repeated endoscopies, which are not acceptable from the patients' point of view. Among alternative means, fecal calprotectin is the fecal marker of reference even if its performance seems to be diminished in certain situations like pure ileal disease. The primary objective of this work was to compare the ability of monocyte-derived macrophages (MDM) from patients with CD, ulcerative colitis (UC) or healthy subjects to control AIEC infection and to identify associated with this multiplication of AIEC and in particular the role of genetic polymorphisms associated with CD in connection with autophagy. AIEC multiplied more than non-pathogenic strain K12 in macrophages irrespective of their origin. The entry of the AIEC (1h post-infection) did not vary according to the origin of the macrophages. The survival of AIEC was increased in MDM from MC patients compared to those from HCR or control subjects. In multivariate analysis, this survival was positively correlated with the secretion of IL1β but was decreased in the presence of the variants at risk for ULK1 (p = 0.046) and XBP1 (p = 0.014). MDM from MC patients were unable to control the multiplication of AIEC, unlike those from HCR or control subjects, especially in the presence of the variant at risk for IRGM (p = 0.045). Infection of MDM from MC patients by AIEC bacteria induces a pro-inflammatory cytokine secretion pattern. The second part of this work aimed to compare the performance of faecal chitinase 3-like 1 (CHI3L1), a host protein interacting with AIEC virulence factor, and matrix metalloprotease 9 (MMP-9). to detect the endoscopic inflammatory activity of MC in comparison with the standard fecal marker, calprotectin. Fecal CHI3L1, MMP-9 and calprotectin levels were correlated with Crohn's Disease Endoscopic Index of Severity (CDEIS) and were significantly increased in the presence of endoscopic ulcerations. In case of pure ileal involvement, fecal CHI3L1 seemed better correlated with CDEIS than fecal calprotectin. The fecal CHI3L1 threshold of 15 ng / g showed better performance than faecal calprotectin in detecting the presence of endoscopic ulcerations. MMP-9 was a powerful marker for detecting the presence of endoscopic lesions in IBD. In conclusion, we have shown that there is a macrophage defect to control infection by AIEC bacteria in patients with CD related to atopic risk variants involved in autophagy leading to a pro-inflammatory macrophage phenotype . Fecal CHI3L1, known as a host protein interacting with AIEC virulence factor, as well as MMP-9 appear to be good markers of endoscopic activity in IBD.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (p. 98-113).

Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / "May 2003." Typescript. Vita. Includes bibliographical references (leaves 152-162).

Not available / text

Escherichia coli, one of the most common pathogens causing urinary tract infections, has shown increased resistance to commonly used antibiotics. In this study we analyzed the β-lactamase profiles of 38 inhibitor-resistant E. coli isolates obtained from public hospitals at three different levels of healthcare in KwaZulu-Natal, selected on the basis of their resistance profiles to the three antibiotic/inhibitor combinations, viz., amoxicillin/clavulanate, ampicillin/ sulbactam and piperacillin/ tazobactam. The isolates were subjected to MIC determinations, IEF analysis, plasmid profile analysis, PCR of the different β-lactamase genes and sequencing thereof to detect the possible mechanism/s of resistance. A range of β-lactamases including two novel inhibitor-resistant TEM β-lactamases, TEM-145 and TEM-146 were detected in two isolates whilst a novel plasmid-mediated AmpC-type β-lactamase, CMY-20 was detected in three isolates. Other β-lactamases included OXA-1, TEM-55, SHV-2, CTX-M-l and TEM-1. Changes were detected in the chromosomal AmpC promoter/attenuator regions in one isolate. Diverse β-lactamase genes and plasmid profiles inferred extensive mobilization of β-lactamase genes causing the concern of limited therapeutic options in the face of increasing resistance. / Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2008.

Diss. (sammanfattning) Stockholm : Univ., 2005. / Härtill 3 uppsatser.

Thesis (Ph.D.)--McMaster University, 2006. / Supervisor: Paul J. Berti. Includes bibliographical references.