Developing a model for cystic fibrosis sociomicrobiology based on antibiotic and environmental stress

Cystic fibrosis (CF) infections are invariably biofilm-mediated and polymicrobial, being safe to assume that a myriad of factors affects the sociomicrobiology within the CF infection site and modulate the CF community dynamics, by shaping their social activities, overall functions, virulence, ultimately affecting disease outcome. This work aimed to assess changes in the dynamics (particularly on the microbial composition) of dual-/three-species biofilms involving CF-classical (Pseudomonas aeruginosa) and unusual species (Inquilinus limosus and Dolosigranulum pigrum), according to variable oxygen conditions and antibiotic exposure. Low fluctuations in biofilm compositions were observed across distinct oxygen environments, with dual-species biofilms exhibiting similar relative proportions and P. aeruginosa and/or D. pigrum populations dominating three-species consortia. Once exposed to antibiotics, biofilms displayed high resistance profiles, and microbial compositions, distributions, and microbial interactions significantly challenged. The antibiotic/oxygen environment supported such fluctuations, which enhanced for three-species communities. In conclusion, antibiotic therapy hugely disturbed CF communities dynamics, inducing significant compositional changes on multispecies consortia. Clearly, multiple perturbations may disturb this dynamic, giving rise to various microbiological scenarios in vivo, and affecting disease phenotype. Therefore, an appreciation of the ecological/evolutionary nature within CF communities will be useful for the optimal use of current therapies and for newer breakthroughs on CF antibiotherapy.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/ BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER006684). The authors also acknowledge the financial support provided by FCT through the projects: PTDC/SAU-ESA/646091/2006/ FCOMP01-0124-FEDER-007480FCT; strategic project PEst-OE/EQB/LA0023/2013; “BioHealth – Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124-FEDER-000027, cofunded by the Programa Operacional Regional do Norte (ON.2–O Novo Norte), QREN, FEDER; RECI/BBB-EBI/0179/2012 – Consolidating Research Expertise and Resources on Cellular and Molecular Biotechnology at CEB/IBB, FCOMP-01-0124-FEDER-027462, FEDER; and the DNA mimics project PIC/IC/82815/2007. The FCT BPD fellowship of Susana P. Lopes SFRH/BPD/95616/2013 and the support of the COST-Action TD1004:Theragnostics for imaging and therapy is also acknowledged.info:eu-repo/semantics/publishedVersio

Service and auto-configuration framework for secure Ad-hoc environments in Android and Linux

Dissertação de mestrado em Engenharia de InformáticaAd-hoc networks can be useful in many contexts because they can be spontaneously created and do not require any sort of infrastructure. They can be useful for small groups when no other network is accessible. They can also be used in wider areas as a low cost replacement for wireless infrastructure networks with multiple dedicated access points. Despite this, ad-hoc networks are not a very popular option for most users. Unfortunately, ad-hoc networks are not as user friendly as infrastructure networks. The latter ones usually provide standardized mechanisms that perform the essential configurations for the correct functioning of the network. Ad-hoc networks do not have standardized mechanisms adapted to them. Each wireless network manager supports a different set of configuration mechanisms. There is usually no problem when every machine uses the same operating system but when different ones are used, users may need to manually perform the required configurations. Another cause for this low popularity is the lack of useful and easy to use applications. These applications are usually hosted on the Internet, as it provides a larger variety of business models. To tackle these problems, new forms of automatically configuring machines and providing services should be explored. These services must be easy to develop, in order to attract the developers that would develop them. The designed solutions must also be adapted to ad-hoc environments. Another important aspect that must be addressed is security. In some contexts, such as public and corporate environments, security can be essential to provide authentication and even to allow the correct functioning of the network.As redes ad-hoc podem ser uteis em muitos contextos visto poderem ser criadas espontaneamente e não necessitarem de qualquer tipo de infraestrutura. Elas podem ser uteis para grupos pequenos quando nenhuma outra rede pode ser acedida. Tamb em podem ser usadas em areas amplas como uma alternativa de baixo custo para redes sem os infraestruturadas com vários pontos de acesso dedicados. Ainda assim, as redes ad-hoc não são uma opção muito popular para a maioria dos utilizadores. Infelizmente, as redes ad-hoc não são tão simples de utilizar como as redes de infraestrutura. Estas ultimas normalmente utilizam mecanismos standardizados para efectuar as configurações que são essenciais para o correcto funcionamento da rede. As redes ad-hoc não têm mecanismos standardizados adaptados a elas. Cada aplicação gestora de redes sem os suporta um conjunto diferente de mecanismos de confi guração. Normalmente não há problemas quando todas as máquinas usam o mesmo sistema operativo mas quando são usados diversos, os utilizadores podem ter que con- figurar manualmente as máquinas. Outra causa para esta baixa popularidade e a falta de aplicações uteis e de fácil utilização. Estas aplicações são normalmente utilizadas na Internet, visto que esta fornece uma maior variedade de modelos de negócio. Para abordar estes problemas, novas formas de configurar máquinas automaticamente e de fornecer serviços devem ser exploradas. Estes serviços devem ser fáceis de desenvolver, de forma a atrair os developers que os irão desenvolver. As solu ções desenhadas também devem estar adaptadas a ambientes ad-hoc. Outro aspecto importante que tem de ser focado e a segurança. Em alguns contextos, tais como ambientes públicos e empresariais, a segurança pode ser essencial para fornecer autenticação e mesmo permitir o correcto funcionamento da rede. Neste trabalho, uma framework funcional que trata estes problemas e desenhada e implementada. A framework e capaz de efectuar automaticamente as configurações necessárias a criação de um ambiente ad-hoc funcional e seguro. E também capaz de fornecer serviços especializados e de fácil desenvolvimento, sobre o ambiente seguro criado de forma espontânea. A framework desenvolvida e fácil de utilizar e foi exaustivamente testada em Android e Linux, embora possa ser facilmente estendida de forma a funcionar em muitos outros sistemas operativos

Cf multispecies dynamics: role of oxygen milieu and antibiotic therapy

EUROBIOFILMS 2017 - 5th European Congress on Microbial BiofilmsPortuguese Foundation for Science and Technology (FCT), under the scope of the strategic funding UID/BIO/04469/2013 and COMPETE 2020 (POCI-01-0145-FEDER -006684). This study was also supported by FCT and European Community fund FEDER, through Program COMPETE, and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020 -Programa Operacional Regional do Norte. FTC is also acknowledged for S.Lopes Post-Doc Grant (SFRH/BPD/95616/2013)info:eu-repo/semantics/publishedVersio

Quantitative assessment of individual populations within polymicrobial biofilms

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Selecting appropriate tools providing reliable quantitative measures of individual populations in biofilms is critical as we now recognize their true polymicrobial and heterogeneous nature. Here, plate count, quantitative real-time polymerase chain reaction (q-PCR) and peptide nucleic acid probe-fluorescence in situ hybridization (PNA-FISH) were employed to quantitate cystic fibrosis multispecies biofilms. Growth of Pseudomonas aeruginosa, Inquilinus limosus and Dolosigranulum pigrum was assessed in dual- and triple-species consortia under oxygen and antibiotic stress. Quantification methods, that were previously optimized and validated in planktonic consortia, were not always in agreement when applied in multispecies biofilms. Discrepancies in culture and molecular outcomes were observed, particularly for triple-species consortia and antibiotic-stressed biofilms. Some differences were observed, such as the higher bacterial counts obtained by q-PCR and/or PNA-FISH (?4 log10 cells/cm2) compared to culture. But the discrepancies between PNA-FISH and q-PCR data (eg D. pigrum limited assessment by q-PCR) demonstrate the effect of biofilm heterogeneity in method's reliability. As the heterogeneity in biofilms is a reflection of a myriad of variables, tailoring an accurate picture of communities? changes is crucial. This work demonstrates that at least two, but preferentially three, quantification techniques are required to obtain reliable measures and take comprehensive analysis of polymicrobial biofilm-associated infections.The authors thank the financial support from the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI-01–0145-FEDER-006684) and BioTecNorte operation (NORTE01-0145-FEDER-000004) funded by the European Regional Development Fund (ERDF) under the scope of Norte2020 - Programa Operacional Regional do Norte. This work was also the result of the projects: (i) POCI01-0145-FEDER-006939 (Laboratory for Processing Engineering, Environment, Biotechnology and Energy – UID/EQU/00511/2013) funded by the ERDF, through COMPETE2020 and by national funds, through FCT; (ii) NORTE-01-0145-FEDER-000005 – LEPABE-2-ECO-INNOVATION, supported by North Portugal Regional Operational Programme (NORTE2020), under the Portugal2020 Partnership Agreement, through the ERDF; (iii) Coded-FISH PTDC/DTP-PIC/4562/2014/16678; (iv) POCI-01-0145-FEDER-029841, through COMPETE2020 - Programa Operacional Competitividade e Internacionalização and by national funds, through FCT). Also, the fellowship of Susana P. Lopes SFRH/BPD/95616/2013 is acknowledged. The authors would also like to thanks to Dr Michael Surette (Farncombe Family Digestive Health Research Institute, McMaster University, Hamilton, ON, Canada) for kindly providing the I. limosus strain used in this study.info:eu-repo/semantics/publishedVersio

Development of an aptamer-based biosensor for the detection of food toxins

According to the last EFSA/ECDC report, bacterial toxins are the second leading causative agent (17.0%) of foodborne outbreaks in Europe [1]. Aptasensors, as biosensors that use aptamers are called, are seen as a simple, rapid and cost-effective assay format with high suitability for point-of-care testing, allowing a sensitive and mostly qualitative detection of analytes [2]. In this work, DNA aptamers previously selected by our group (Apt1, Apt2, Apt3, Apt4 and Apt5) for staphylococcal enterotoxin A (SEA), one of the most reported bacterial toxins, were applied as recognition molecules in a lateral flow assay. For this, lateral flow strips consisting of a sample pad with glass fibre, test zone with nitrocellulose membrane and absorbent pad with cellulose membrane were assembled. Gold nanoparticles (AuNPs) covalently attached with Apt5 were synthesized. Biotinylated aptamers (140 pmol of Apt1, Apt2, Apt3 and Apt4) were immobilized in the test zone using streptavidin as an anchor. A DNA probe (140 pmol) complementary to Apt5 was also immobilized as a test control. Then, SEA solutions (0.3 ng/L) as well as negative samples were prepared and incubated with Apt5- AuNPs (OD3) in binding buffer for 10 min. Different assay combinations (Apt5-AuNPs + Apt1/Apt2/Apt3/Apt4) were tested. The samples (60 L) were applied to the sample pad, allowing the solution to flow on the strip until the test lines were visualized by the accumulation of AuNPs. SEA samples were positively detected, with the combination of Apt5-AuNPs with Apt3, providing the best result, followed by Apt4, Apt2 and Apt1. Negative controls were validated by the control line. Further tests to determine the detection limit and improve the noise ratio are being carried out. These results show that aptasensors can be a simple and rapid alternative for the detection of SEA. Furthermore, this format assay can be easily adapted to any food toxin.This work was financially supported by: LA/P/0045/2020 (ALiCE), UIDB/00511/2020 and UIDP/00511/2020 (LEPABE), funded by national funds through FCT/MCTES (PIDDAC); project POCI01-0145- FEDER-028659, funded by FEDER funds through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI). The authors also thank FCT for the PhD Fellowship SFRH/BD/138883/2018.info:eu-repo/semantics/publishedVersio

Uncovering the metabolic capacities of H. pylori 26695 using 13C labeling experiments

The determination of nutritional requirements of pathogenic organisms is of great significance for understanding host-pathogen interactions. Despite the knowledge obtained so far concerning amino acid requirements in H. pylori, it is still unclear which are the metabolic pathways used for biosynthesis and catabolism. Thus, information on the carbon flow in this organism is required. Glutamate is a very important metabolite in bacterial metabolism that can be used as a carbon and nitrogen source. 13C flux analysis has been largely applied to characterize phenotypes by quantifying in vivo the carbon fluxes. One of the most important applications of this approach is the identification of active pathways in less-studied organisms. Thus, in order to clarify the metabolic pathways used by H. pylori 26695, 13C labeling experiments with 13C-glutamate were conducted and labeled amino acids in biomass hydrolysates were analyzed by GC-MS. The obtained results confirmed L-glutamate as a potential sole and effective carbon source for H. pylori. Overall, all non-essential amino acids, except proline, presented a 13C labeling pattern. We hypothesized that L-proline is produced from L-arginine, while L-alanine is probably produced from pyruvate by alanine dehydrogenase. Additionally, the full usage of complete TCA cycle, under the conditions used, was also demonstrated

Habitações em CC – Uma Mudança de Paradigma em Direção a Uma Maior Eficiência Energética

Todas as pessoas, a um certo nível, estão dependentes da energia, sendo que uma das formas de energia à qual mais estamos habituados é a energia elétrica em Corrente Alternada (CA), de tal forma é esta habituação que nem pensamos que tipo de energia elétrica estamos a usar em nossas casas, assumimos que seja CA e sempre deva ser CA. O grande objetivo desta dissertação é dizer que não tem obrigatoriamente de ser assim, existem outras formas de gerar, transportar e consumir energia elétrica, isto é, utilizar a energia elétrica em Corrente Contínua (CC), e esta forma de utilização de energia elétrica pode mesmo trazer consigo diversas vantagens, sendo algumas dessas vantagens um comprovado aumento da Eficiência Energética (EE), menor necessidade de equipamentos conversores, melhor qualidade de energia consumida ou mesmo um menor risco para a saúde, resultante de uma menor exposição a campos eletromagnéticos. Embora seja vantajosa a utilização de CC, como é possível ver na dissertação esta utilização nem sempre é a mais viável, sendo apontados aspetos a mudar e um rumo a seguir de modo a promover a utilização de CC e com isto uma maior EE. Num momento em que se fala, e se discutem soluções, de Energias Renováveis (ER), EE, microgrids, smart grids, edifícios Net Zero Energy Buildings (NZEB) e Internet of Things (IoT), pretende-se estudar as vantagens e desvantagens que a utilização de CC poderia trazer, tal como analisar os normativos existentes, em maior pormenor o normativo Português, de modo a analisar que alterações devem ser consideradas quando se pretende fazer um projeto de infraestrutura elétrica de uma habitação em CC ao invés de CA. É igualmente dado grande ênfase ao estudo do estado político ao qual se pode associar a implementação da energia elétrica em CC, pois mesmo que em termos técnicos a CC se apresente como a melhor solução, sem a devida conjuntura política e social a melhor solução não se consegue desenvolver e difundir com tanta facilidade

FISH and chips: a review of microfluidic platforms for FISH analysis

Fluorescence in situ hybridization (FISH) allows visualization of specific nucleic acid sequences within an intact cell or a tissue section. It is based on molecular recognition between a fluorescently labeled probe that penetrates the cell membrane of a fixed but intact sample and hybridizes to a nucleic acid sequence of interest within the cell, rendering a measurable signal. FISH has been applied to, for example, gene mapping, diagnosis of chromosomal aberrations and identification of pathogens in complex samples as well as detailed studies of cellular structure and function. However, FISH protocols are complex, they comprise of many fixation, incubation and washing steps involving a range of solvents and temperatures and are, thus, generally time consuming and labor intensive. The complexity of the process, the relatively high-priced fluorescent probes and the fairly high-end microscopy needed for readout render the whole process costly and have limited wider uptake of this powerful technique. In recent years, there have been attempts to transfer FISH assay protocols onto microfluidic lab-on-a-chip platforms, which reduces the required amount of sample and reagents, shortens incubation times and, thus, time to complete the protocol, and finally has the potential for automating the process. Here, we review the wide variety of approaches for lab-on-chip-based FISH that have been demonstrated at proof-of-concept stage, ranging from FISH analysis of immobilized cell layers, and cells trapped in arrays, to FISH on tissue slices. Some researchers have aimed to develop simple devices that interface with existing equipment and workflows, whilst others have aimed to integrate the entire FISH protocol into a fully autonomous FISH on-chip system. Whilst the technical possibilities for FISH on-chip are clearly demonstrated, only a small number of approaches have so far been converted into off-the-shelf products for wider use beyond the research laboratory

Inconsistencies in conventional culture vs molecular approaches: unveiling distinct dynamics in cystic fibrosis polymicrobial communities

Book of Abstracts of CEB Annual Meeting 2017info:eu-repo/semantics/publishedVersio