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Biosystems and Integrative Sciences Institute
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Characterization of microRNAs implicated in maritime pine resistance to pinewood nematode
Publication . Mendes, André Vieira; Miguel, Célia Maria Romba Rodrigues; Castro, Dora Cristina Vicente Batista Lyon de
Pine wilt disease (PWD), caused by the pinewood nematode (PWN) Bursaphelenchus
xylophilus, is a serious threat to coniferous forests worldwide. Post-transcriptional regulation
of gene expression by microRNAs (miRNAs) plays important roles during plant-pathogen
interactions. Recently, a few miRNAs have been identified as differentially expressed
between PWD resistant and susceptible maritime pine (Pinus pinaster Ait.) plants. The aim of
this work was to further characterize the expression patterns of four of these miRNAs during
early stages of PWN infection and evaluate how they relate with maritime pine responses.
P. pinaster 4-year-old plants from half-sib families previously characterized regarding
their predicted survival mean to PWN, were inoculated and symptom development was
recorded. Stem samples were collected before (0 hours) and at 24, 48 and 72 hours post
inoculation. After RNA extraction, reverse transcription quantitative PCR (RT-qPCR) data was
obtained for quantification of miRNA expression in susceptible, resistant and control plant
stem samples. First, reference miRNAs for normalization of expression data were identified
using well-established algorithms. Then, a step toward functional characterization of the
potentially defense-related P. pinaster miR166h, miR951f, miR3627m and Novel_110 was
given by comparing their expression patterns along time between resistant and susceptible
plants. The results show that miR11532_4 and miR396_48 are good reference miRNAs,
being more appropriate than currently used reference genes. Expression analysis suggests
the potential contribution of miR166h and miR951f upregulation to inefficient activation of
susceptible plants’ immunity, as well as the possible involvement of Novel_110 in cell wall
reinforcement of resistant genotypes. Furthermore, the potential regulation of oxidoreductase
activity by miR3627m is discussed.
Overall, valuable information on post-transcriptional regulation of maritime pine
response to PWD was obtained, although further functional characterization of these
miRNAs is needed. Ultimately, this knowledge might be helpful for breeding programs that
aim at developing resistant P. pinaster plants.
Antimicrobial resistance phylodynamics and source attribution under a One Health framework
Publication . Ramos, Beatriz; Cunha, Mónica Vieira; Rosalino, Luís Miguel do Carmo
Antimicrobial resistance (AMR) is a significant public health threat and has recently been recognized as a One Health issue, reflecting the interconnected nature of human, animal, and environmental health. This thesis aimed to increase knowledge on the burden, drivers and transmission dynamics of AMR at the human-animal-environment interfaces, for which Staphylococcus aureus, wild ungulates and genomics were central.
Wastewater-based surveillance (WBS) of urban and hospital sewersheds by shotgun metagenomics depicted microbiota signatures of public health importance, while showing that the urban resistome is not restricted to clinically relevant pathogens, being strongly related with the most consumed antimicrobials in Europe.
Through molecular, phenotypic and ecological modelling analyses, we show that AMR in commensal Staphylococcus aureus strains from wild ungulates in Portugal is driven by agricultural land cover and livestock farming. We then generate and explore a large dataset of S. aureus draft genomes from wild ungulates to address with high resolution the hypotheses that wildlife colonization and AMR occurrence are related with human activities and that host adaptation is accompanied by genome diversification with phenotypic impacts. For source attribution purposes, we dissect host-informative mobile genetic elements (MGE) and, in parallel, perform ancestral host state reconstruction via phylodynamics. Based on cgMLST, we demonstrate high genomic similarity of S. aureus at the animal-human interface, with MGE biomarkers for host adaptation further supporting epidemiological connections. Phylodynamic inferences on relevant molecular types across Iberia indicate that several clonal lineages were widespread among humans before jumping to new hosts, highlighting recent spillover events from livestock to wildlife. Pangenome analyses retrieved S. aureus antimicrobial and heavy metals resistance determinants in the accessory genome.
This thesis provides new insights on AMR transmission across interfaces, encouraging environmental and animal surveillance to help curb AMR, and confirms that genomic data-driven approaches are powerful to track AMR trends and drivers.
Antimicrobial Ceramic Filters for Water Bio-Decontamination
Publication . Ferreira, Olga; Rijo, Patricia; Gomes, João; Santos, Ricardo; Monteiro, Sílvia; Guedes, Rita; Serralheiro, Maria Luísa; Gomes, Marisa; Gomes, Luciana C.; Mergulhão, Filipe J.; Silva, Elisabete R.
Bio-contamination of water through biofouling, which involves the natural colonization of submerged surfaces by waterborne organisms, is a global socio-economic concern, allied to premature materials bio-corrosion and high human health risks. Most effective strategies release toxic and persistent disinfectant compounds into the aquatic medium, causing environmental problems and leading to more stringent legislation regarding their use. To minimize these side effects, a newly non-biocide-release coating strategy suitable for several polymeric matrices, namely polydimethylsiloxane and polyurethane (PU)-based coatings, was used to generate antimicrobial ceramic filters for water bio-decontamination. The best results, in terms of antimicrobial activity and biocide release, showed an expressed delay and a decrease of up to 66% in the population of methicillin-resistant Staphylococcus aureus bacteria on ceramic filters coated with polyurethane (PU)-based coatings containing grafted Econea biocide, and no evidence of biocide release after being submerged for 45 days in water. Biocidal PU-based surfaces were also less prone to Enterococcus faecalis biofilm formation under flow conditions with an average reduction of 60% after 48 h compared to a pristine PU-based surface. Biocidal coated filters show to be a potential eco-friendly alternative for minimizing the environmental risks associated with biofouling formation in water-based industrial systems.
Endogenous VIP VPAC1 receptor activation modulates hippocampal theta burst induced LTP: transduction pathways and GABAergic mechanisms
Publication . Caulino-Rocha, Ana; Rodrigues, Nádia Carolina; Ribeiro, Joaquim A.; Cunha-Reis, Diana
Vasoactive intestinal peptide (VIP), acting on both VPAC1 and VPAC2 receptors, is a key modulator of hippocampal synaptic transmission, pyramidal cell excitability and long-term depression (LTD), exerting its effects partly through modulation GABAergic disinhibitory circuits. Yet, the role of endogenous VIP and its receptors in modulation of hippocampal LTP and the involvement of disinhibition in this modulation have scarcely been investigated. We studied the modulation of CA1 LTP induced by TBS via endogenous VIP release in hippocampal slices from young-adult Wistar rats using selective VPAC1 and VPAC2 receptor antagonists, evaluating its consequence for the phosphorylation of CamKII, GluA1 AMPA receptor subunits and Kv4.2 potassium channels in total hippocampal membranes obtained from TBS stimulated slices. Endogenous VIP, acting on VPAC1 (but not VPAC2) receptors, inhibited CA1 hippocampal LTP induced by TBS in young adult Wistar rats and this effect was dependent on GABAergic transmission and relied on the integrity of NMDA and CaMKII-dependent LTP expression mechanisms but not on PKA and PKC activity. Furthermore, it regulated the autophosphorylation of CaMKII and the expression and Ser438 phosphorylation of Kv4.2 potassium channels responsible for the A-current while inhibiting phosphorylation of Kv4.2 on Thr607. Altogether, this suggests that endogenous VIP controls the expression of hippocampal CA1 LTP by regulating disinhibition through activation of VPAC1 receptors in interneurons. This may impact the autophosphorylation of CaMKII during LTP, as well as the expression and phosphorylation of Kv4.2 K+ channels at hippocampal pyramidal cell dendrites.
Molecular Defense Response of Pine Trees (Pinus spp.) to the Parasitic Nematode Bursaphelenchus xylophilus
Publication . Modesto, Inês; Mendes, André; Carrasquinho, Isabel; Miguel, Célia M.
Pine wilt disease (PWD) is a severe environmental problem in Eastern Asia andWestern
Europe, devastating large forest areas and causing significant economic losses. This disease is caused
by the pine wood nematode (PWN), Bursaphelenchus xylophilus, a parasitic migratory nematode
that infects the stem of conifer trees. Here we review what is currently known about the molecular
defense response in pine trees after infection with PWN, focusing on common responses in different
species. By giving particular emphasis to resistance mechanisms reported for selected varieties
and families, we identified shared genes and pathways associated with resistance, including the
activation of oxidative stress response, cell wall lignification, and biosynthesis of terpenoids and
phenylpropanoids. The role of post-transcriptional regulation by small RNAs in pine response
to PWN infection is also discussed, as well as the possible implementation of innovative RNAinterference
technologies, with a focus on trans-kingdom small RNAs. Finally, the defense response
induced by elicitors applied to pine plants before PWN infection to prompt resistance is reviewed.
Perspectives about the impact of these findings and future research approaches are discussed
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
6817 - DCRRNI ID
Número da atribuição
UIDB/04046/2020