32 resultados para Cloning, Organism
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RESUMO: O Cell Fusing Agent Vrus (CFAV), considerado como o primeiro flavivrus especficos de insectos (ISF), parece estar exclusivamente adaptado aos seus hospedeiros, no replicando em clulas de vertebrados. Apesar de ter sido identificado h mais de trs dcadas (1975), a verdade que muito pouco se conhece sobre a sua biologia. Dado o seu parentesco filogentico com alguns outros flavivrus encontrados naturalmente em mosquitos de diferentes gneros colhidos em diferentes regies do globo, este vrus poder ser usado como modelo para o estudo de ISF. No entanto, necessitam do desenvolvimento de ferramentas bsicas, tais como clones moleculares ou baterias de soros contendo anticorpos que reconheam uma ou mais protenas codificadas pelo genoma viral, produzidas, por exemplo, a partir de antignios virais produzidos de forma recombinante. Com este trabalho pretendeu-se a optimizao de protocolos que permitiram a expresso e purificao parcial de quatro protenas [duas protenas estruturais (C e E) e duas no estruturais (NS3hel e NS5B)] do CFAV em E. coli, todas elas produzidas como protenas de fuso com caudas (tags) de hexahistidina nos seus extremos carboxilo. Para a expanso do CFAV foram utilizadas clulas Aedes albopictus (C6/36). Aps a realizao da extraco do RNA viral e a obteno de cDNA, procedeu-se amplificao, por RT-PCR, das regies codificantes das protenas C, E, NS3hel e NS5B, utilizando primers especficos. Os quatro fragmentos de DNA foram independentemente inseridos no vector pJTE1.2/blunt usando E. coli NovaBlue como hospedeira de clonagem e, posteriormente, inseridos em vectores de expresso pET-28b e pET-29b usando E. coli BL21(DE3)pLysS e Rosetta(DE3)pLysS como hospedeiras de expresso. Aps da induo, expresso e purificao das protenas recombinantes C, E, NS3hel e NS5B, foi confirmada a autenticidade destas protenas produzidas atravs do mtodo Western Blot com um anticorpo anti-histidina. --------- ABSTRACT: The Cell Fusing Agent virus (CFAV) considered as the first "insect- specific flavivirus" (ISF) and seems to be uniquely adapted to their hosts, not replicating in vertebrate cells. Although it has been known for more than three decades (1975), the truth is very little is known about its biology. Given its close phylogenetic relationship with other flavivirus naturally circulating in various genera of mosquitoes collected from different regions of the globe, this virus could be used as a model for the study of ISF. However, such studies require the development of experimental basic tools, such as molecular clones or serum batteries containing antibodies that recognize one or more proteins encoded by the viral genome, produced, for example, from viral antigens recombinant produced. In this work, we carried out the optimization of protocols that allowed the expression and partial purification of four proteins [two structural proteins (C and E) and two nonstructural proteins (NS3hel and NS5B)] CFAV in E. coli as fusion protein for c-terminal hexahistidine tags. For the expansion of the CFAV we used Aedes albopictus (C6/36) cells. After completion of the viral RNA extraction and cDNA obtained, amplification of the coding regions of the C, E, NS5B and NS3hel proteins was carried out by RT-PCR using specific primers. The four DNA fragments were independently inserted into the vector pJTE1.2/blunt using E. coli NovaBlue as cloning host and then inserted into expression vectors pET-28b and pET-29b using E. coli BL21(DE3)pLysS and Rosetta(DE3)pLysS as expression host. After induction, expression and purification of recombinant C, E, NS3hel and NS5B proteins Western Blot analyses with an anti-histidine antibody confirmed the authenticity of these proteins produced.
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Dissertation presented to obtain the Ph.D degree in Evolutionary Biology
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J Biol Inorg Chem (2007) 12:691698 DOI 10.1007/s00775-007-0219-9
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J Biol Inorg Chem (2006) 11: 433444 DOI 10.1007/s00775-006-0090-0
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J Biol Inorg Chem (2004) 9: 839849 DOI 10.1007/s00775-004-0584-6
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J Biol Inorg Chem (2004) 9: 145151 DOI 10.1007/s00775-003-0506-z
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Chlamydia trachomatis has a unique obligate intracellular developmental cycle that ends by the lysis of the cell and/or the extrusion of the bacteria in order to allow for re-infections. While Chlamydia trachomatis infections are often asymptomatic the diagnosis of Chlamydia trachomatis is usually late, occurring after manifestation of persistency. Investigations on the consequences of long-term infections and the molecular mechanisms behind it will reveal light to what extent bacteria can modulate host cell function and what the ultimate fate of host cells after clearance of an infection is. Such studies on the host cell fate could be greatly facilitated if the infected cells become permanently marked during and after the infection. Therefore, this project intends to develop a new genetic tool that would allow permanently labeling of Chlamydia trachomatis host cells. The plan was to generate a Chlamydia trachomatis strain that encodes a recombinant CRE recombinase, fused to a secretory effector function of the Chlamydia type 3 secretion system (T3SS). Upon translocation into the host cell, this recombinant CRE enzyme could then, owing to its site-specific recombination function, switch a reporter gene contained in the host cell genome. To this end, the reporter line carried a membrane-tagged tdTomato (mT) gene flanked by two LoxP sequences followed by a GFP gene. The translocation of the recombinant CRE recombinase into this cell line was designed to trigger the recombination of the LoxP sites whereby the cells would turn from red fluorescence to green as an irreversible label of the infected cells. Successful execution of this mechanism would allow to draw a direct link between Chlamydia trachomatis infection and the subsequent fate of the infected cell.
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The work presented in this thesis describes the functional characterization of hydrogenases in the overall energy metabolism of the sulfate reducing bacterium Desulfovibrio gigas. With the complete annotation of the D. gigas genome, we were able to verify that only the two previously described hydrogenases are present in this organism, the periplasmic [NiFe] HynAB and the cytoplasmic membrane-bound [NiFe] Ech.(...)
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Dissertation presented to obtain the Ph.D degree in Computational Biology
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Functional regeneration of organs upon injury is a key process for animals survival. Contrary to humans, some vertebrates are remarkably competent in regenerating after acute organ or appendage lesions. This advantageous skill allows overcoming limitations in repair even in adult stages, when tissues are fully developed, via a process of epimorphic regeneration. One such organism is the zebrafish, which can regenerate several organs, namely its heart, retina, spinal cord and fins. (...)
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Mannans (linear mannan, glucomannan, galactomannan and galactoglucomannan) are the major constituents of the hemicellulose fraction in softwoods and show great importance as a renewable resource for fuel or feedstock applications. As complex polysaccharides, mannans can only be degraded through a synergistic action of different mannan-degrading enzymes, mannanases. Microbial mannanases are mainly extracellular enzymes that can act in wide range of pH and temperature, contributing to pulp and paper, pharmaceutical, food and feed, oil and textile successful industrial applications. Knowing and controlling these microbial mannan-degrading enzymes are essential to take advantage of their great biotechnological potential. The genome of the laboratory 168 strain of Bacillus subtilis carries genes gmuA-G dedicated to the degradation and utilization of glucomannan, including an extracellular -mannanase. Recently, the genome sequence of an undomesticated strain of B. subtilis, BSP1, was determined. In BSP1, the gmuA-G operon is maintained, interestingly, however, a second cluster of genes was found (gam cluster), which comprise a second putative extracellular -mannanase, and most likely specify a system for the degradation and utilization of a different mannan polymer, galactoglucomannan. The genetic organization and function of the gam cluster, and whether its presence in BSP1 strain results in new hemicellulolytic capabilities, compared to those of the laboratory strain, was address in this work. In silico and in vivo mRNA analyses performed in this study revealed that the gam cluster, comprising nine genes, is organized and expressed in at least six different transcriptional units. Furthermore, cloning, expression, and production of Bbsp2923 in Escherichia coli was achieved and preliminary characterization shows that the enzyme is indeed a -mannanase. Finally, the high hemicellulolytic capacity of the undomesticated B. subtilis BSP1, demonstrated in this work by qualitative analyses, suggests potential to be used in the food and feed industries.
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Os resultados apresentados no captulo 2 foram includos no artigo Dantas JM, Campelo LM, Duke NEC, Salgueiro CA, Pokkuluri PR (2015) "The structure of PccH from Geobacter sulfurreducens a novel low reduction potential monoheme cytochrome essential for accepting electrons from an electrode", FEBS Journal, 282, 2215-2231.
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RESUMO: Clostridium difficile presentemente a principal causa de doena gastrointestinal associada utilizao de antibiticos em adultos. C. difficile uma bactria Gram-positiva, obrigatoriamente anaerbica, capaz de formar endsporos. Tem-se verificado um aumento dos casos de doena associada a C. difficile com sintomas mais severos, elevadas taxas de morbilidade, mortalidade e recorrncia, em parte, devido emergncia de estirpes mais virulentas, mas tambm devido m gesto do uso de antibiticos. C. difficile produz duas toxinas, TcdA e TcdB, que so os principais fatores de virulncia e responsveis pelos sintomas da doena. Estas so codificadas a partir do Locus de Patogenicidade (PaLoc) que codifica ainda para um regulador positivo, TcdR, uma holina, TcdE, e um regulador negativo, TcdC. Os esporos resistentes ao oxignio so essenciais para a transmisso do organismo e recorrncia da doena. A expresso dos genes do PaLoc ocorre em clulas vegetativas, no final da fase de crescimento exponencial, e em clulas em esporulao. Neste trabalho construmos dois mutantes de eliminao em fase dos genes tcdR e tcdE. Mostrmos que a auto-regulao do gene tcdR no significativa. No entanto, tcdR sempre necessrio para a expresso dos genes presentes no PaLoc. Trabalho anterior mostrou que, com a exceo de tcdC, os demais genes do PaLoc so expressos no pr-esporo. Mostrmos aqui que TcdA detectada superfcie do esporo maduro e que a eliminao do tcdE no influencia a acumulao de TcdA no meio de cultura ou em associao s clulas ou ao esporo. Estas observaes tm consequncias para o nosso entendimento do processo infecioso: sugeremque o esporo possa ser tambm um veculo para a entrega da toxina nos estgios iniciais da infeco, que TcdA possa ser libertada durante a germinao do esporo, e que o esporo possa utilizar o mesmo receptor reconhecido por TcdA para a ligao mucosa do clon.---------------------------ABSTRACT: Clostridium difficile is currently the major cause of antibiotic-associated gastrointestinal diseases in adults. This is a Gram-positive bacterium, endospore-forming and an obligate anaerobe that colonizes the gastrointestinal tract. Recent years have seen a rise in C. difficile associated disease (CDAD) cases, associated with more severe disease symptoms, higher rates of morbidity, mortality and recurrence, which were mostly caused due to the emergence of hypervirulent strains but also due to changing patterns of antibiotics use. C. difficile produces two potent toxins, TcdA and TcdB, which are the main virulence factors and the responsible for the disease symptoms. These are codified from a Pathogenicity Locus (PaLoc), composed also by the positive regulator, TcdR, the holin-like protein, TcdE, and a negative regulator, TcdC. Besides the toxins, the oxygen-resistant spores are also essential for transmission of the organism through diarrhea; moreover, spores can accumulate in the environment or in the host, which will cause disease recurrence. The expression of the PaLoc genes occurs in vegetative cells, at the end of the exponential growth phase, and in sporulating cells. In this work, we constructed two in-frame deletion mutants of tcdR and tcdE. We showed that the positive auto regulation of tcdR is not significant. However, tcdR is always necessary for the expression of the PaLoc genes. A previous work showed that, except tcdC, all the PaLoc genes are expressed in the forespore. Here, we detected TcdA at the spore surface. Furthermore, we showed that the in-frame deletion of tcdE does not affect the accumulation of TcdA in the culture medium or in association with cells or spores. This data was important for us to conclude about the infeccious process: it suggests that the spore may be the vehicle for the delivery of TcdA in early stages of infection, that TcdA may be released during spores germination and that this spore may use the same receptor recognized by TcdA to bind to the colonic mucosa.
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RESUMO: As clulas eucariticas evoluram um sistema de sinalizao complexo que lhes permite responder aos sinais extracelulares e intracelulares. Desta forma, as vias de sinalizao so essenciais para a sobrevivncia da clula e do organismo, uma vez que regulam processos fundamentais, tais como o desenvolvimento, o crescimento, a imunidade, e a homeostase dos tecidos. A via de transduo de sinal Hedgehog (Hh) envolve o receptor Patched1 (Ptch1), que tem um efeito inibidor sobre a protena Smoothened (Smo) na ausncia dos seus ligandos, as protenas Sonic hedgehog (Shh). Estas protenas so reguladores fundamentais do desenvolvimento embrionrio, como ilustrado pelas malformaes drsticas observadas em embries humanos e de murganho com perturbaes da transduo de sinal da via Hh e que incluem polidactilia, defeitos craniofaciais e malformaes sseas. Igualmente importantes so as consequncias da ativao inapropriada da via de sinalizao Hh na formao de tumores. Curiosamente, os componentes desta via localizam-se nos clios primrios. Alm disso, demonstrou-se que esta localizao crucial para a sinalizao atravs da via Hh. Na presena dos ligandos, Ptch1 internalizado e destinado a degradao ou sequestrado num compartimento da clula de onde no pode desempenhar o seu papel inibitrio. A protena Arl13b uma pequena GTPase pertencente famlia Arf/Arl da superfamlia Ras de pequenas GTPases e foi implicada no sndrome de Joubert, uma ciliopatia caracterizada por ataxia congnita cerebelar, hipotonia, atrso mental e cardiopatia congnita. Murganhos deficientes para Arl13b, chamado hennin (hnn) morrem morrem prematuramente ao dia 13,5 de gestao (E13,5) e exibem anomalias morfolgicas nos clios que levam interrupo da sinalizao Hh. Alm disso, a Arl13b est diretamente envolvida na regulao da via Hh, controlando a localizao de vrios componentes desta via nos clios primrios. Neste trabalho, mostramos que a Arl13b se localiza em circular dorsal ruffles (CDRs), que so estruturas de actina envolvidas em macropinocitose e internalizao de recetores, e que regula a sua formao. Alm disso, aprofundmos o conhecimento do processo de ativao da via de sinalizao Hh, mostrando que as CDRs sequestram seletivamente e internalizam o recetor Ptch1. As CDRs formam-se minutos aps ativao da via por ligandos Shh ou pelo agonista de Smo SAG e continuam a ser formadas a partir da, sugerindo uma induo contnua da reorganizao do citoesqueleto de actina quando a via est ativada. Observmos ainda que a inibio da formao de CDRs atravs do silenciamento de WAVE1, uma protena necessria para a formao destas estruturas, resulta na diminuio da ativao da via de sinalizao Hh. Alm disso, o bloqueio da macropinocitose, que se segue ao fecho das CDRs, atravs do silenciamento de uma protena necessria para a ciso de macropinossomas, nomeadamente a protena BARS, tem um efeito semelhante. Estes resultados sugerem que as CDRs e a macropinocitose so necessrias para a ativao da via de sinalizao Hh e indicam que esta via de internalizao controla os nveis de sinal Hh. Durante o desenvolvimento, as clulas proliferativas dependem do clio primrio para a transduo de vrias vias de sinalizao. A via Hh induz a diferenciao do msculo cardaco. Por conseguinte, os murganhos deficientes na via de sinalizao Hh exibem uma variedade de defeitos de lateralidade, incluindo alterao do looping do corao, como pode ser visto em murganhos deficientes para Arl13b. Por conseguinte, investigmos o papel da Arl13b no desenvolvimento do corao. Mostramos que a Arl13b altamente expressa no corao de embries de murganho e de murganhos adultos ao nvel do mRNA e da protena. Alm disso, o perfil de distribuio da Arl13b no corao segue o dos clios primrios, que so essenciais para o desenvolvimento cardaco. Coraes de murganhos hnn no estadio E12,5 mostram um canal trio-ventricular aberto, espessamento da camada compacta ventricular e aumento do ndice mittico no ventrculo esquerdo. Alm disso, um atraso de 1 a 2 dias no desenvolvimento observado em coraes de murganhos hnn, quando comparados com controlos selvagens no estadio E13,5. Assim, estes resultados sugerem que a Arl13b necessria para o desenvolvimento embrionrio do corao e que defeitos cardacos podem contribuir para a letalidade embrionria de murganhos hnn. Em suma, foi estabelecido um novo mecanismo para a regulao dos nveis de superfcie do recetor Ptch1, que envolve a remodelao do citoesqueleto de actina e a formao de CDRs aps a ativao da via de sinalizao Hh. Este mecanismo permite um feedback negativo que evita a represso excessiva da via atravs da remoo de Ptch1 da superfcie da clula. Alm disso, determinou-se que uma mutao de perda de funo na Arl13b causa defeitos cardacos durante o desenvolvimento, possivelmente relacionados com a associao dos defeitos em clios primrios e na sinalizao Hh, existentes em murganhos deficientes para Arl13b. A via de sinalizao Hh tem tido um papel central entre as vias de sinalizao, uma vez que a sua regulao crucial para o funcionamento apropriada da clula. Assim, a descoberta de um novo mecanismo de trfego atravs de macropinocitose e CDRs que controla a ativao e represso da via de sinalizao Hh traz novas perspetivas de como esta via pode ser regulada e pode ainda conduzir identificao de novos alvos e estratgias teraputicas. --------------------ABSTRACT: Eukaryotic cells have evolved a complex signaling system that allows them to respond to extracellular and intracellular cues. Signaling pathways are essential for cell and organism survival, since they regulate fundamental processes such as development, growth, immunity, and tissue homeostasis. The Hedgehog (Hh) pathway of signal transduction involves the receptor Patched1 (Ptch1), which has an inhibitory effect on Smoothened (Smo) in the absence of its ligands, the Sonic hedgehog (Shh) proteins. These proteins are fundamental regulators of embryonic development, as illustrated by the dramatic malformations seen in human and mouse embryos with perturbed Hh signal transduction that include polydactyly, craniofacial defects and skeletal malformations. Equally important are the consequences of inappropriate activation of the Hh signaling response in tumor formation. Interestingly, the components of this pathway localize to primary cilia. Moreover, it has been shown that this localization is crucial for Hh signaling. However, in the presence of the ligands, Ptch1 is internalized and destined for degradation or sequestered in a cell compartment where it no longer can play its inhibitory role. ADP-ribosylation factor-like (Arl) 13b, a small GTPase belonging to Arf/Arl family of the Ras superfamily of small GTPases has been implicated in Joubert syndrome, a ciliopathy characterized by congenital cerebellar ataxia, hypotonia, intellectual disability and congenital heart disease. Arl13b-deficient mice, called hennin (hnn) die at embryonic day 13.5 (E13.5) and display morphological abnormalities in primary cilia that lead to the disruption of Hh signaling. Furthermore, Arl13b is directly involved in the regulation of Hh signaling by controlling the localization of several components of this pathway to primary cilia. Here, we show that Arl13b localizes to and regulates the formation of circular dorsal rufles (CDRs), which are actin-basedstructures known to be involved in macropinocytosis and receptor internalization. Additionally, we extended the knowledge of the Hh signaling activation process by showing that CDRs selectively sequester and internalize Ptch1 receptors. CDRs are formed minutes after Hh activation by Shh ligands or the Smo agonist SAG and keep being formed thereafter, suggesting a continuous induction of actin reorganization when the pathway is switched on. Importantly, we observed that disruption of CDRs by silencing WAVE1, a protein required for CDR formation, results in down-regulation of Hh signaling activation. Moreover, the blockade of macropinocytosis, which follows CDR closure, through silencing of a protein necessary for the fission of macropinosomes, namely BARS has a similar effect. These results suggest that CDRs and macropinocytosis are necessary for activation of Hh signaling and indicate that this pathway of internalization controls Hh signal levels. During development, proliferating cells rely on the primary cilium for the transduction of several signaling pathways. Hh induces the differentiation of cardiac muscle. Accordingly, Hh-deficient mice display a variety of laterality defects, including alteration of heart looping, as seen in Arl13b-deficient mice. Therefore, we investigated the role of Arl13b in heart development. We show that Arl13b is highly expressed in the heart of both embryonic and adult mice at mRNA and protein levels. Also, Arl13b localization profile mimics that of primary cilia, which have been shown to be essential to early heart development. E12.5 hnn hearts show an open atrioventricular channel, increased thickening of the ventricular compact layer and increased mitotic index in the left ventricle. Moreover, a delay of 1 to 2 days in development is observed in hnn hearts, when compared to wild-type controls at E13.5. Hence, these results suggest that Arl13b is necessary for embryonic heart development and that cardiac defects might contribute to the embryonic lethality of hnn mice. Altogether, we established a novel mechanism for the regulation of Ptch1 surface levels, involving cytoskeleton remodeling and CDR formation upon Hh signaling activation. This mechanism allows a negative feedback loop that prevents excessive repression of the pathway by removing Ptch1 from the cell surface. Additionally, we determined that the Arl13b loss-offunction mutation causes cardiac defects during development, possibly related to the associated ciliary and Hh signaling defects found in Arl13b-deficient mice. Hh signaling has taken a center stage among the signaling pathways since its regulation is crucial for the appropriate output and function of the cell. Hence, the finding of a novel trafficking mechanism through CDRs and macropinocytosis that controls Hh signaling activation and repression brings new insights to how this pathway can be regulated and can lead to the discovery of novel therapeutic targets and strategies.
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Polysaccharides are gaining increasing attention as potential environmental friendly and sustainable building blocks in many fields of the (bio)chemical industry. The microbial production of polysaccharides is envisioned as a promising path, since higher biomass growth rates are possible and therefore higher productivities may be achieved compared to vegetable or animal polysaccharides sources. This Ph.D. thesis focuses on the modeling and optimization of a particular microbial polysaccharide, namely the production of extracellular polysaccharides (EPS) by the bacterial strain Enterobacter A47. Enterobacter A47 was found to be a metabolically versatile organism in terms of its adaptability to complex media, notably capable of achieving high growth rates in media containing glycerol byproduct from the biodiesel industry. However, the industrial implementation of this production process is still hampered due to a largely unoptimized process. Kinetic rates from the bioreactor operation are heavily dependent on operational parameters such as temperature, pH, stirring and aeration rate. The increase of culture broth viscosity is a common feature of this culture and has a major impact on the overall performance. This fact complicates the mathematical modeling of the process, limiting the possibility to understand, control and optimize productivity. In order to tackle this difficulty, data-driven mathematical methodologies such as Artificial Neural Networks can be employed to incorporate additional process data to complement the known mathematical description of the fermentation kinetics. In this Ph.D. thesis, we have adopted such an hybrid modeling framework that enabled the incorporation of temperature, pH and viscosity effects on the fermentation kinetics in order to improve the dynamical modeling and optimization of the process. A model-based optimization method was implemented that enabled to design bioreactor optimal control strategies in the sense of EPS productivity maximization. It is also critical to understand EPS synthesis at the level of the bacterial metabolism, since the production of EPS is a tightly regulated process. Methods of pathway analysis provide a means to unravel the fundamental pathways and their controls in bioprocesses. In the present Ph.D. thesis, a novel methodology called Principal Elementary Mode Analysis (PEMA) was developed and implemented that enabled to identify which cellular fluxes are activated under different conditions of temperature and pH. It is shown that differences in these two parameters affect the chemical composition of EPS, hence they are critical for the regulation of the product synthesis. In future studies, the knowledge provided by PEMA could foster the development of metabolically meaningful control strategies that target the EPS sugar content and oder product quality parameters.
