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Name:Christian / RamosInstitution:Institute for Biotechnology and Bioengineering - Instituto Superior Técnico |
About my Work:
Molecular Microbiology: Generation of mutants by random plasposon mutagenesis and site-directed mutagenesis, gene cloning, His-tagging and GFP-tagging of proteins, protein expression (in vivo and in vitro), northen, southern and western-blotting, PCR optimization, Protein-DNA interaction (GMSA and DNaseI footprinting); Infection Models: Maintanence and infection of C. elegans wild-type (BN2) and mutant (DH26) strains, single worm handling, evaluation of infecting CFU's; Bioinformatics: General Clustal alignments, secondary and terciary protein analysis, 3D strutural modelling and molecular docking.
Role of small non-coding RNAs on the virulence of bacteria of the Burkholderia Cepacia Complex. Small RNAs have been implicated in the virulence of several Human pathogens. We have found that a mutation in a sRNA chaperone, Hfq, renders the bacterium B. cepacia IST408 (a CF clinical isolate) less virulent in the C. elegans infection model. Moreover, this mutant is unable to produce EPS, a major virulence determinant in the gp91phox -/- mice model. In this sense, we intend to identify sRNAs implicated in virulence of Bcc bacteria, which might then be explore to develop antibacterial agents. As suggeted by Sidney Altman, nucleic acid targets that are unique to bacteria, might be targeted for degardation by RNase P, if one can design an EGS (external guide sequence - a small oligo with a anti-sense sequence for the target sRNA, a signal sequence for activation of RNase P and a short protective peptide) that can activelly reach the bacteria growing in biofilms.
Projects:
Role of small RNAs on the virulence of bacteria of the Bcc
The Burkholderia cepacia complex (Bcc) is a group of bacterial species causing life-threatening, often lethal infections among cystic fibrosis patients.
We have recently identified a Bcc mutant with impaired virulence due to a mutation in a gene 84% similar to the Escherichia coli hfq, a chaperone of small non-coding RNAs (sRNAs).
sRNAs regulate bacterial gene expression by modulating their target mRNAs stability, playing a critical role in virulence. Almost 250 putative sRNAs were predicted by in-silico analysis of a Bcc isolate genome.
An approach involving the identification of Hfq-regulated sRNAs through transcriptomics, construction of sRNAs deletion mutants, and evaluation of sRNAs role on Bcc virulence using mice and C. elegans as infection models, will be pursued. Plasmids encoding anti-sense sRNAs will be prepared and evaluated as anti-Bcc. This program envisages contributing to unveil the molecular mechanisms of Bcc virulence involving sRNAs, exploiting them as potential targets to fight Bcc infections.
Sludge bed reactors (UASB reactors and more recently EGSB reactors) are an anaerobic technology with high potential for the treatment of various kinds of wastewaters. Yet their application for the treatment of complex fat containing effluents (e.g. dairy effluents) has some drawbacks as for instance scum forming, sludge degranulation, biomass wash-out and loss of biological activity. It is well known that this difficulties can be ascribed to the presence of complex substrates, proteins and especially fats, that accumulate in the biomass forming a film of organic matter which, besides imposing mass transfer limitations on the process kinetics, also causes sludge flotation and biomass wash-out. It is also known that the fats and long chain fatty acids (LCFA) present in dairy effluents may inhibit the anaerobic process resulting in the need of a highly adapted population for the degradation of these substrates.
The solution to overcome these problems may be an intermittent operation mode that consists in interrupting the feed to the reactor for a certain period of time (stabilisation period) allowing for the biological degradation of the accumulated organic matter having slower degrading rates. It is also known that the granular sludge is predominantly methanogenic whilst in flocculent biomass the prevailing species have higher hydrolitic and acidogenic activities. Since it is possible and quite successful to operate UASB reactors with flocculent sludge another strategy adopted in this investigation will be the use of flocculent biomass because it is more adequate for the first steps, the most problematic steps, in the degradation of complex substrates.
Finally, some published results indicate that fats and some complex substrates are more readily degraded in the termophilic temperature as compared to the rates in mesophilic temperature.
This project aims to the optimisation of the performance of UASB reactors with intermittent feed used for the treatment of complex wastewater. Therefore, several time lengths of the feed and the stabilisation periods will be studied, and also different temperature ranges in the stabilisation period as well as different recirculation ratios during the feed and/or the stabilisation periods, in order to establish the optimum operating conditions. The most important parameters to study will be the duration of the feed and the stabilisation periods in relation with the whole operating cycle (feed + stabilisation), the temperature range during the stabilisation, several recirculation flows and the applicable loads. The effect of these parameters on the biological removal, in the acidification and the conversion to methane of the removed organic matter will be used as optimisation criteria.
Simultaneously, the microbial populations developed in the intermittent UASB reactors will be studied in batch reactors to assess their capacity for the degradation of several substrates. The microbial populations will be characterized by molecular techniques based on the determination of the nucleotide sequences of genes encoding 16S RNA. Their metabolic profiles will be determined based on biochemical experiments. These approaches will allow the identification and quantification of the microbial species of relevance for the degradation of complex substrates. Results from 16S RNA-encoding genes sequencing will also be used to develop new probes which allow the application of FISH (Fluorescence in situ hybridization) techniques for controlling of this type of reactors. The obtained results will provide the establishment of correlations between the populations dynamics and reactor performance thus permitting their better optimisation and control both in start-up and in long tem operation. The results of this project will also enable the analysis of whether the improved performance of intermittent reactors as compared to continuous reactors, observed in previous studies, is due to an adaptation of the anaerobic biomass to the conditions prevailing in the stabilisation period, which if confirmed will establish intermittent operation as a way to optimise biomass adaptation.Introduction
The Burkholderia cepacia complex (BCC) is a group of opportunistic pathogens that can cause severe infections in Cystic Fibrosis patients. Aiming at the identification of new virulence determinants of the BCC, a mutant collection of B. cenocepacia K56-2 was prepared, by random plasposon mutagenesis, and screened for impaired virulence using the nematode Caenorhabditis elegans infection model.
Results and discussion
The P4A1 mutant strain was selected due its phenotype of reduced virulence to the C. elegans. Sequence analysis and homology searches revealed that B. cenocepacia P4A1 carries a Tn5 insertion in an ORF, here named pbr, encoding a putative protein 72% identical to the DNA-directed RNA polymerase subunit beta from Bacillus cereus AH1134. In silico analysis revealed that Pbr contains a HTH motif, putatively involved in nucleic acid binding. Sequence analysis of the DNA sequence upstream pbr revealed two ORFs, encoding putative proteins 97% and 91% identical, respectively, to the Pseudomonas chlororaphis phenazine biosynthesis PhzD and PhzF. The mutant was unable to produce phenazines. A putative gene encoding a protein of unknown function was also found upstream of the phzF gene. The search for homologous sequences in other BCC species with their genome sequences available, revealed that the locus here described is, most probably, unique in the BCC. Due to the low GC content of this locus, as compared to the median GC content of the genome, it is hypothesized that this could be part of a larger genomic region acquired through horizontal transfer.
Vita / Publications:
Thesis
Ramos, C.G. (2001)“Estudo, validação e implementação de 2 métodos de análise microbiológica aplicados ao controlo da qualdadeâ€Â, BSc in Biotechnology Engineering, Universidade Lusofona, Lisboa, Portugal.
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Ramos C. G. (2007), "Exploiting the Caenorhabditis elegans infection model to unveil novel virulence factors from bacteria of the Burkholderia cepacia complex ", MSc in Biotechnology (Biochemical Engineering), Instituto Superior Técnico, Lisboa, Portugal.
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Papers in international scientific periodicals with referees
Ramos, C. G., Costa, M. T., (2006) Patterns of resistance to beta-lactams and beta-lactamase inhibitors in uropathogenic Escherichia coli strains isolated from animals in Portugal. African Journal Biotechnology 5: 523-525
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Sousa, SA., Ramos, CG., Almeida, F., Meirinhos-Soares, L., Wopperer, J., Schwager, S., Eberl, L. and Leitão, JH. Burkholderia cenocepacia J2315 acyl carrier protein:a potential target for antimicrobials development ? Microbial Pathogenesis. in Press (http://dx.doi.org/10.1016/j.micpath.2008.08.002)
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Ramos, CG., Sousa, SA., Eberl, L. and Leitão, JH. Pbr: A transcriptional regulator of phenazine biosynthesis in Burkholderia cenocepacia K56-2 necessary for full virulence to the nematode Caenorhabditis elegans. In preparation
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Sousa, SA., Ramos, CG., Moreira, LM. and Leitão, JH. The Small RNA chaperone Hfq plays a role on the virulence of Burkholderia cepacia complex bacteria. In preparation
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Papers in national periodicals with referees
Ramos, C. G., Simões, S., (2006) Métodos para a identificação de Escherichia coli na Indústria Alimentar. Revista Portuguesa de Ciências Veterinárias, 101: 131-132
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Papers in conference proceedings
Ramos C.G. ,Sousa S.A. and Leitão J.H. (2008), Identification and characterization of a phenazine biosythetic locus required for virulence of Burkholderia cenocepacia K56-2 to the nematode Caenorhabditis elegans. FEBS Journal 275 (s1): 275.
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Ramos, C.G., (2001)“Caracterização fenotipica de resistência aos antibóticos de estirpes de E. coli de origem animalâ€Â, Thesis for Professional qualification level V, PRODEP III, Lisboa, Portugal. Biblioteca Nacional.
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Christian G. Ramos Silvia A. Sousa e Jorge H. Leitão (2008) "Modelos de Infecção para a identificação de factores de virulência em bactérias do complexo Burkholderia cepacia", E-learning gate e-escola (http://www.e-escola.pt/site/canal.asp?canal=5)
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S.A. Sousa, C.G. Ramos, J.H. Leitão. Nucleotide sequence of the Burkholderia cepacia IST408 hfq gene. Access number EU760353 (release date: November 2008).
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Ramos C.G., S.A. Sousa, J.H. Leitão. Nucleotide sequence of Burkholderia cenocepacia strain K56-2 locus encoding the Pbr protein required for phenazine biosynthesis. Access number EU874251 (release date: January 2009).
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Ramos C.G., J.H. Leitão (2008) "Bases de Dados Biológicos", E-learning gate e-escola (http://www.e-escola.pt/site/canal.asp?canal=5)
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Ramos C.G., J.H. Leitão (2008) "Sequenciação de Genomas", E-learning gate e-escola (http://www.e-escola.pt/site/canal.asp?canal=5)
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Ramos C.G., S.A. Sousa, I. Sá-Correia, J.H. Leitão (2008) "Sequenciação de DNA", E-learning gate e-escola (http://www.e-escola.pt/site/canal.asp?canal=5)
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Communications
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oral communications
Silvia A. Sousa, Christian G. Ramos and Jorge H. Leitão. (2008) The Acyl Carrier Protein, involved in the virulence of the Burkholderia cenocepacia J2315, is conserved within the Burkholderia genus. International Burkholderia cepacia Working Group Meeting, Treviso, Italy, 14-17 April 2008.
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Silvia A. Sousa, Christian G. Ramos and Jorge H. Leitão (2008) Burkholderia cenocepacia J2315 acyl carrier protein (ACP): a potential target for the development of antimicrobials against infections caused by B. species? IUMS Congresses 2008 (XII International Congress of Bacteriology and Applied Microbiology) 5-9 August 2008, Instambul, Turkey.
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Sousa, SA., Ramos, CG., Soares,L., Eberl, L. and Leitão, JH. Burkholderia cenocepacia J2315 acp: IDENTIFICATION OF A POTENTIAL TARGET FOR THE DEVELOPMENT OF NEW ANTIMICROBIALS. XVI Congress of Biochemistry, Azores, 22-25 October 2008, Portugal
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Posters in conferences
C. G. Ramos, S. A. Sousa, J. Wopperer, L. Eberl, J. H. Leitão, "Isochorismatase plays a role on the virulence of Burkholderia cepacia in the nematode Caenorhabditis elegans", XVth National Congress of Biochemistry, December 8th-10th, Aveiro, 2006.
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SÃlvia A. Sousa, Christian G. Ramos and Jorge H. Leitão (2007), Unveiling the role of Hfq on the virulence of Burkholderia cepacia complex, RNA 2007, Poster communication in the IV National Meeting. 8-10 November 2007, Vimeiro, Portugal
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Ramos C.G. ,Sousa S.A. and Leitão J.H. (2007), Identification of a phenazine biosynthetic locus necessary for full virulence of Burkholderia cenocepacia K56-2 to the nematode Caenorhabditis elegans, Poster communication in the MICRO’07 BIOTEC’07-XXXIII JPG congress, Lisboa, Portugal November 30th-December 2nd
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Sousa, SA., Ramos, CG., Nadais,H. and Leitão, JH. MOLECULAR CHARACTERIZATION OF MICROBIAL POPULATIONS IN SLUDGE BED REACTORS FOR DAIRY INDUSTRY WASTEWATER TREATMENT. XVI Congress of Biochemistry, Azores, 22-25 October 2008, Portugal
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Ferreira, AS., Leitão, JH., Silva, IN., Sousa, SA., Ramos, CG. and Moreira, LM. EXTENSION OF THE bce CLUSTER OF GENES INVOLVED IN THE BIOSYNTHESIS OF CEPACIAN BY THE GENUS BURKHOLDERIA. XVI Congress of Biochemistry, Azores, 22-25 October 2008, PortugalGrants and Awards:
Research Scholarship from PRODEP III (Education Ministry), 2001-2002
Research Assistant Grant, Project PTDC/AMB/65025/2006, May 2008 – present
About my Institution:
The Laboratório Associado Institute for Biotechnology and Bioengineering (IBB) is a research and development (R&D) unit, founded in October 2006 aiming to be a strategic infrastructure for the development of the Portuguese R&D and innovation policies in the areas of Biotechnology, Bioengineering, Biomaterials and Life, Biomedical and Agricultural Sciences. IBB combines its R&D activities with advanced education, technology transfer, consulting and services, with the aim of fostering the industrial, health, agriculture and environmental sectors.Additional Information:
The mission
To carry out research and education in biological sciences, focusing prokaryotic and eukaryotic microbial systems and exploring the post-genomic experimental approaches and bioinformatics.
The goals
To perform research in Molecular Microbiology focusing the yeast Saccharomyces cerevisiae and bacteria belonging to Pseudomonas-related genera, exploring the post-genomic approaches and bioinformatics.
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