14,858 research outputs found

    Impact of polyester and cotton microfibers on growth and sublethal biomarkers in juvenile mussels

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    Anthropogenic microfibres are a prevalent, persistent and globally distributed form of marine debris. Evidence of microfibre ingestion has been demonstrated in a range of organisms, including Mytilus spp. (mussels), but the extent of any impacts on these organisms are poorly understood. This study investigates, for the first time, the effect of exposing juvenile mussels to polyester and cotton microfibres at environmentally relevant concentrations (both current and predicted future scenarios) over a chronic timescale (94 days). Sublethal biomarkers included growth rate, respiration rate and clearance rate. Mussels were exposed to polyester (median length 149 µm) and cotton (median length 132 µm) microfibres in three treatments: polyester (~ 8 fibres L−1), polyester (~ 80 fibres L−1) and cotton (~ 80 fibres L−1). Mussels exposed to 80 polyester or cotton microfibres L−1 exhibited a decrease in growth rate of 35.6% (polyester) and 18.7% (cotton), with mussels exposed to ~ 80 polyester microfibres L−1 having a significantly lower growth rate than the control population (P < 0.05). This study demonstrates that polyester microfibres have the potential to adversely impact upon mussel growth rates in realistic future scenarios, which may have compounding effects throughout the marine ecosystem and implications for commercial viability

    Pollution-induced community tolerance in freshwater biofilms – from molecular mechanisms to loss of community functions

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    Exposure to herbicides poses a threat to aquatic biofilms by affecting their community structure, physiology and function. These changes render biofilms to become more tolerant, but on the downside community tolerance has ecologic costs. A concept that addresses induced community tolerance to a pollutant (PICT) was introduced by Blanck and Wängberg (1988). The basic principle of the concept is that microbial communities undergo pollution-induced succession when exposed to a pollutant over a long period of time, which changes communities structurally and functionally and enhancing tolerance to the pollutant exposure. However, the mechanisms of tolerance and the ecologic consequences were hardly studied up to date. This thesis addresses the structural and functional changes in biofilm communities and applies modern molecular methods to unravel molecular tolerance mechanisms. Two different freshwater biofilm communities were cultivated for a period of five weeks, with one of the communities being contaminated with 4 μg L-1 diuron. Subsequently, the communities were characterized for structural and functional differences, especially focusing on their crucial role of photosynthesis. The community structure of the autotrophs was assessed using HPLC-based pigment analysis and their functional alterations were investigated using Imaging-PAM fluorometry to study photosynthesis and community oxygen profiling to determine net primary production. Then, the molecular fingerprints of the communities were measured with meta-transcriptomics (RNA-Seq) and GC-based community metabolomics approaches and analyzed with respect to changes in their molecular functions. The communities were acute exposed to diuron for one hour in a dose-response design, to reveal a potential PICT and uncover related adaptation to diuron exposure. The combination of apical and molecular methods in a dose-response design enabled the linkage of functional effects of diuron exposure and underlying molecular mechanisms based on a sensitivity analysis. Chronic exposure to diuron impaired freshwater biofilms in their biomass accrual. The contaminated communities particularly lost autotrophic biomass, reflected by the decrease in specific chlorophyll a content. This loss was associated with a change in the molecular fingerprint of the communities, which substantiates structural and physiological changes. The decline in autotrophic biomass could be due to a primary loss of sensitive autotrophic organisms caused by the selection of better adapted species in the course of chronic exposure. Related to this hypothesis, an increase in diuron tolerance has been detected in the contaminated communities and molecular mechanisms facilitating tolerance have been found. It was shown that genes of the photosystem, reductive-pentose phosphate cycle and arginine metabolism were differentially expressed among the communities and that an increased amount of potential antioxidant degradation products was found in the contaminated communities. This led to the hypothesis that contaminated communities may have adapted to oxidative stress, making them less sensitive to diuron exposure. Moreover, the photosynthetic light harvesting complex was altered and the photoprotective xanthophyll cycle was increased in the contaminated communities. Despite these adaptation strategies, the loss of autotrophic biomass has been shown to impair primary production. This impairment persisted even under repeated short-term exposure, so that the tolerance mechanisms cannot safeguard primary production as a key function in aquatic systems.:1. The effect of chemicals on organisms and their functions .............................. 1 1.1 Welcome to the anthropocene .......................................................................... 1 1.2 From cellular stress responses to ecosystem resilience ................................... 3 1.2.1 The individual pursuit for homeostasis ....................................................... 3 1.2.2 Stability from diversity ................................................................................. 5 1.3 Community ecotoxicology - a step forward in monitoring the effects of chemical pollution? ................................................................................................................. 6 1.4 Functional ecotoxicological assessment of microbial communities ................... 9 1.5 Molecular tools – the key to a mechanistic understanding of stressor effects from a functional perspective in microbial communities? ...................................... 12 2. Aims and Hypothesis ......................................................................................... 14 2.1 Research question .......................................................................................... 14 2.2 Hypothesis and outline .................................................................................... 15 2.3 Experimental approach & concept .................................................................. 16 2.3.1 Aquatic freshwater biofilms as model community ..................................... 16 2.3.2 Diuron as model herbicide ........................................................................ 17 2.3.3 Experimental design ................................................................................. 18 3. Structural and physiological changes in microbial communities after chronic exposure - PICT and altered functional capacity ................................................. 21 3.1 Introduction ..................................................................................................... 21 3.2 Methods .......................................................................................................... 23 3.2.1 Biofilm cultivation ...................................................................................... 23 3.2.2 Dry weight and autotrophic index ............................................................. 23 3.2.4 Pigment analysis of periphyton ................................................................. 23 3.2.4.1 In-vivo pigment analysis for community characterization ....................... 24 3.2.4.2 In-vivo pigment analysis based on Imaging-PAM fluorometry ............... 24 3.2.4.3 In-vivo pigment fluorescence for tolerance detection ............................. 26 3.2.4.4 Ex-vivo pigment analysis by high-pressure liquid-chromatography ....... 27 3.2.5 Community oxygen metabolism measurements ....................................... 28 3.3 Results and discussion ................................................................................... 29 3.3.1 Comparison of the structural community parameters ............................... 29 3.3.2 Photosynthetic activity and primary production of the communities after selection phase ................................................................................................. 33 3.3.3 Acquisition of photosynthetic tolerance .................................................... 34 3.3.4 Primary production at exposure conditions ............................................... 36 3.3.5 Tolerance detection in primary production ................................................ 37 3.4 Summary and Conclusion ........................................................................... 40 4. Community gene expression analysis by meta-transcriptomics ................... 41 4.1 Introduction to meta-transcriptomics ............................................................... 41 4.2. Methods ......................................................................................................... 43 4.2.1 Sampling and RNA extraction................................................................... 43 4.2.2 RNA sequencing analysis ......................................................................... 44 4.2.3 Data assembly and processing................................................................. 45 4.2.4 Prioritization of contigs and annotation ..................................................... 47 4.2.5 Sensitivity analysis of biological processes .............................................. 48 4.3 Results and discussion ................................................................................... 48 4.3.1 Characterization of the meta-transcriptomic fingerprints .......................... 49 4.3.2 Insights into community stress response mechanisms using trend analysis (DRomic’s) ......................................................................................................... 51 4.3.3 Response pattern in the isoform PS genes .............................................. 63 4.5 Summary and conclusion ................................................................................ 65 5. Community metabolome analysis ..................................................................... 66 5.1 Introduction to community metabolomics ........................................................ 66 5.2 Methods .......................................................................................................... 68 5.2.1 Sampling, metabolite extraction and derivatisation................................... 68 5.2.2 GC-TOF-MS analysis ............................................................................... 69 5.2.3 Data processing and statistical analysis ................................................... 69 5.3 Results and discussion ................................................................................... 70 5.3.1 Characterization of the metabolic fingerprints .......................................... 70 5.3.2 Difference in the metabolic fingerprints .................................................... 71 5.3.3 Differential metabolic responses of the communities to short-term exposure of diuron ............................................................................................................ 73 5.4 Summary and conclusion ................................................................................ 78 6. Synthesis ............................................................................................................. 79 6.1 Approaches and challenges for linking molecular data to functional measurements ...................................................................................................... 79 6.2 Methods .......................................................................................................... 83 6.2.1 Summary on the data ............................................................................... 83 6.2.2 Aggregation of molecular data to index values (TELI and MELI) .............. 83 6.2.3 Functional annotation of contigs and metabolites using KEGG ................ 83 6.3 Results and discussion ................................................................................... 85 6.3.1 Results of aggregation techniques ........................................................... 85 6.3.2 Sensitivity analysis of the different molecular approaches and endpoints 86 6.3.3 Mechanistic view of the molecular stress responses based on KEGG functions ............................................................................................................ 89 6.4 Consolidation of the results – holistic interpretation and discussion ............... 93 6.4.1 Adaptation to chronic diuron exposure - from molecular changes to community effects.............................................................................................. 93 6.4.2 Assessment of the ecological costs of Pollution-induced community tolerance based on primary production ............................................................. 94 6.5 Outlook ............................................................................................................ 9

    Comparing the succession of microbial communities throughout development in field and laboratory nests of the ambrosia beetle Xyleborinus saxesenii

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    Some fungus-farming ambrosia beetles rely on multiple nutritional cultivars (Ascomycota: Ophiostomatales and/or yeasts) that seem to change in relative abundance over time. The succession of these fungi could benefit beetle hosts by optimal consumption of the substrate and extended longevity of the nest. However, abundances of fungal cultivars and other symbionts are poorly known and their culture-independent quantification over development has been studied in only a single species. Here, for the first time, we compared the diversity and succession of both fungal and bacterial communities of fungus gardens in the fruit-tree pinhole borer, Xyleborinus saxesenii, from field and laboratory nests over time. By amplicon sequencing of probed fungus gardens of both nest types at three development phases we showed an extreme reduction of diversity in both bacterial and fungal symbionts in laboratory nests. Furthermore, we observed a general transition from nutritional to non-beneficial fungal symbionts during beetle development. While one known nutritional mutualist, Raffaelea canadensis, was occurring more or less stable over time, the second mutualist R. sulphurea was dominating young nests and decreased in abundance at the expense of other secondary fungi. The quicker the succession proceeded, the slower offspring beetles developed, suggesting a negative role of these secondary symbionts. Finally, we found signs of transgenerational costs of late dispersal for daughters, possibly as early dispersers transmitted and started their own nests with less of the non-beneficial taxa. Future studies should focus on the functional roles of the few bacterial taxa that were present in both field and laboratory nests

    Bacterial community responses to planktonic and terrestrial substrates in coastal northern Baltic Sea

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    Bacteria are major consumers of dissolved organic matter (DOM) in aquatic systems. In coastal zones, bacteria are exposed to a variety of DOM types originating from land and open sea. Climate change is expected to cause increased inflows of freshwater to the northern coastal zones, which may lead either to eutrophication or to increased inputs of refractory terrestrial compounds. The compositional and functional response of bacterial communities to such changes is not well understood. We performed a 2-day microcosm experiment in two bays in the coastal northern Baltic Sea, where we added plankton extract to simulate eutrophication and soil extract to simulate increased inputs of refractory terrestrial compounds. Our results showed that the bacterial communities responded differently to the two types of food substrates but responded in a similar compositional and functional way in both bays. Plankton extract addition induced a change of bacterial community composition, while no significant changes occurred in soil extract treatments. Gammaproteobacteria were promoted by plankton extract, while Alphaproteobacteria dominated in soil extract addition and in the non-amended controls. Carbohydrate metabolism genes, such as aminoglycan and chitin degradation, were enriched by plankton extract, but not soil extract. In conclusion, the coastal bacterial communities rapidly responded to highly bioavailable substrates, while terrestrial matter had minor influence and degraded slowly. Thus, in the northern Baltic Sea, if climate change leads to eutrophication, large changes of the bacterial community composition and function can be expected, while if climate change leads to increased inflow of refractory terrestrial organic matter the bacterial communities will not show fast compositional and functional changes. Degradation of terrestrial organic matter may instead occur over longer periods of time, e.g. years. These findings help to better understand the ability of bacterial communities to utilize different carbon sources and their role in the ecosystem

    Terapia fotodinâmica na inativação de bacteriófagos com porfirina e potenciadoresem águas residuais

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    Pathogenic viruses are frequently introduced into marine and estuarine waters through the discharge of treated and untreated sewage, since current treatments are unable to provide virus-free wastewater (WW) effluents, affecting the receiving waters quality and, consequently, human health. The removal of harmful constituents by the conventional treatments comprises a combination of chemical, physical and biological methods. Usually, WW from urban areas is secondarily, rarely tertiary, treated. Although the secondary effluent contains high concentrations of microorganisms, the effect of water dilution makes it acceptable in terms of quality indicators. In tertiary treatment, chlorination is the most common method used to ensure microbiological safety in tertiarily treated effluents. However, its massive utilization, both in free and combined chlorine forms, may lead to the formation of chemical disinfection by-products though the reaction with organic matter present in the effluents, being those chemicals toxic to aquatic organisms, representing potential health hazards. Unfortunately, these conventional methods are limited and may not be adequate to reach the quality levels specified by the guidelines. Photodynamic therapy (PDT) with porphyrins may be a promising approach for the inactivation of pathogens as they are effective in inactivating microorganisms without the formation of potentially toxic products. Some studies have reported an enhancer effect on antimicrobial photodynamic therapy (aPDT) by the combined used of some photosensitizer (PS) with potassium iodide (KI) and hydrogen peroxide (H2O2). The main objective of this study was to evaluate the aPDT efficacy of a PS based on a low-cost formulation constituted by five cationic porphyrins (Form) and its potentiation effect by KI and H2O2 in the inactivation of a T4-like bacteriophage in WW. The experiments were done in phosphate buffered saline and in filtered and non-filtered contaminated wastewater. The aPDT assays in filtered WW (0.45 μm pore-size) were performed with different concentrations of Form (1.0 to 10 μM). In a second phase was evaluated the effect of KI (100 mM) in the photodynamic action of Form (1.0 to 10 μM). The results of these experiments demonstrated that Form is efficient in filtered WW treatment and that the efficacy of bacteriophage photoinactivation is correlated with the concentration of the used PS. When combined with KI, the Form is clearly less effective to inactivate the bacteriophage. To evaluate if the organic matter present in water influences the efficiency of PS, the WW was filtered using three different pore-sized membranes (0.45, 0.30 and 0.22 μm). The results demonstrated that the increase of organic matter promote a significant decrease in the efficiency of Form. In order to evaluate if the efficiency of aPDT to inactivate bacteriophages is maintained when the treatments are performed in non-filtrated WW, the effect of Form alone (10 μM) and combined with H2O2 (2, 5 and 9%) in non-filtered WW was evaluated. The Form alone proved to be an efficient PS to photoinactivate the bacteriophage in non-filtered WW, but the presence of H2O2 enhanced the photodynamic effect. The FORM can be an effective alternative to control viruses in WW, particularly if combined with H2O2.Os vírus patogénicos são frequentemente introduzidos nas águas marinhas e estuarinas através da descarga de esgoto tratado e não tratado, uma vez que os tratamentos atuais não inativam os vírus presentes nas águas residuais (WW), afetando a qualidade das águas recetoras e, consequentemente, a saúde humana. Nos tratamentos convencionais, a remoção de constituintes nocivos consiste no uso de métodos químicos, físicos e biológicos. Geralmente, a WW de áreas urbanas é tratada secundariamente e não terciariamente. Embora o efluente secundário contenha altas concentrações de microrganismos, o efeito da diluição na água torna-o aceitável em termos de indicadores de qualidade. A cloração é o método mais comum usado para garantir a segurança microbiológica em efluentes tratados terciariamente. No entanto, a sua utilização maciça, tanto na forma de cloro livre como combinada, pode levar à formação de subprodutos químicos como resultado da reação com a matéria orgânica presente nos efluentes, sendo esses produtos químicos tóxicos para os organismos aquáticos, apresentando riscos para a saúde. Os métodos convencionais são limitados e podem não ser adequados para manter os níveis de qualidade especificados nas diretrizes. As porfirinas quando usadas como fotossensibilizadores (PS) na terapia fotodinâmica (PDT) podem ser desinfetantes promissores para a inativação de microrganismos patógenicos, pois são eficazes na inativação de microrganismos sem formação de produtos tóxicos. Alguns estudos mostraram efeito potenciador de alguns PS usados em terapia fotodinâmica antimicrobiana (aPDT) quando estes são usados em combinação com iodeto de potássio (KI) e peróxido de hidrogénio (H2O2). O principal objetivo deste estudo foi avaliar a eficácia da aPDT de um PS baseado numa formulação de baixo custo constituída por cinco porfirinas catiónicas (Form) e o seu efeito potenciador por KI e H2O2 na inativação de um bacteriófago tipo T4. As experiências foram realizadas em solução salina tamponada com fosfato e em água residual contaminada filtrada e não filtrada. Os ensaios de aPDT em WW filtrada (tamanho do poro de 0,45 μm) foram realizados com diferentes concentrações de Form (1,0 a 10 μM). Numa segunda fase foi avaliado o efeito do KI (100 mM) na ação fotodinâmica da FORM (1,0 a 10 μM). Os resultados dessas experiências demonstraram que a Form é eficiente no tratamento de WW filtrada e que a eficácia da fotoinativação de bacteriófagos está correlacionada com a concentração do PS usado. Quando combinada com o KI, a Form é claramente menos eficaz na inativação do bacteriófago. Para avaliar se a matéria orgânica presente na água influencia a eficiência do PS, a WW foi filtrada usando três membranas com tamanho de poros diferentes (0,45, 0,30 e 0,22 μm). Os resultados mostraram que o aumento da matéria orgânica promove uma diminuição significativa na eficiência da Form. Para avaliar se a eficiência da aPDT para inativar bacteriófagos é mantida quando os tratamentos são realizados em WW não filtrada, o efeito da Form sozinha (10 μM) e combinado com H2O2 (2, 5 e 9%) em WW não filtrada foi avaliado. A Form por si só provou ser um PS eficiente para fotoinativar o bacteriófago em WW não filtrada, mas a presença de H2O2 aumentou significativamente o efeito fotodinâmico. A Form pode ser uma alternativa eficaz para controlar vírus na WW, principalmente se combinada com H2O2.This work was supported by funding FEDER through COMPETE – Programa Operacional Factores de Competitividade, and by National funding through Fundação para a Ciência e Tecnologia (FCT) and Marine Studies (CESAM).Mestrado em Biologia Molecular e Celula

    Relations between soil organic carbon, soil structure and physical processes in an agricultural topsoil : The role of soil mineral constituents

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    A better understanding of the interactions between soil organic carbon (SOC) and mineral constituents (e.g. clay and reactive oxide phases) and their consequences for soil structure and physical processes is important for assessing the potential for, and benefits of, carbon sequestration in arable soils. This thesis investigated the factors determining topsoil SOC content at the field scale for an arable field with large var-iations in soil properties. Relationships between SOC, soil pore size distributions, macropore network characteristics, water flow and solute transport were also exam-ined using intact soil samples from the field. The spatial variation in SOC content at the Bjertorp field was mainly explained by the oxalate-extractable aluminum (Alox) content followed by carbon input from crops that was estimated from crop yield. In contrast, clay and oxalate-extractable iron (Feox) seemed not to play a major role in SOC stabilization/accumulation, pos-sibly due to the occurrence of stagnant water in soils with larger clay contents. It was concluded that reactive Al phases may be important for physico-chemical stabiliza-tion of SOC for arable topsoils in humid continental climates. Multiple linear regression analysis revealed that an increase of SOC was associ-ated with relatively large increases of porosities in the 0.2–5 µm and 480–720 µm diameter classes, which can contribute to enhancing both water supply to crops and water flow rates. The degree of preferential solute transport under steady state near-saturated conditions was reduced with larger volumes of small macropores (240–480 µm diameter) and mesopores (30–100 µm diameter), whereas it was not corre-lated with measures of macropore connectivity. The statistical analysis indicated that SOC had only limited effects on the degree of preferential transport, being overshad-owed by the large variation in clay content across the field

    RNA pull-down-confocal nanoscanning (RP-CONA), a novel method for studying RNA/protein interactions in cell extracts that detected potential drugs for Parkinson’s disease targeting RNA/HuR complexes

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    MicroRNAs (miRNAs, miRs) are a class of small non-coding RNAs that regulate gene expression through specific base-pair targeting. The functional mature miRNAs usually undergo a two-step cleavage from primary miRNAs (pri-miRs), then precursor miRNAs (pre-miRs). The biogenesis of miRNAs is tightly controlled by different RNA-binding proteins (RBPs). The dysregulation of miRNAs is closely related to a plethora of diseases. Targeting miRNA biogenesis is becoming a promising therapeutic strategy. HuR and MSI2 are both RBPs. MiR-7 is post-transcriptionally inhibited by the HuR/MSI2 complex, through a direct interaction between HuR and the conserved terminal loop (CTL) of pri-miR-7-1. Small molecules dissociating pri-miR-7/HuR interaction may induce miR-7 production. Importantly, the miR-7 levels are negatively correlated with Parkinson’s disease (PD). PD is a common, incurable neurodegenerative disease causing serious motor deficits. A hallmark of PD is the presence of Lewy bodies in the human brain, which are inclusion bodies mainly composed of an aberrantly aggregated protein named α-synuclein (α-syn). Decreasing α-syn levels or preventing α-syn aggregation are under investigation as PD treatments. Notably, α-syn is negatively regulated by several miRNAs, including miR-7, miR-153, miR-133b and others. One hypothesis is that elevating these miRNA levels can inhibit α-syn expression and ameliorate PD pathologies. In this project, we identified miR-7 as the most effective α-syn inhibitor, among the miRNAs that are downregulated in PD, and with α-syn targeting potentials. We also observed potential post-transcriptional inhibition on miR-153 biogenesis in neuroblastoma, which may help to uncover novel therapeutic targets towards PD. To identify miR-7 inducers that benefit PD treatment by repressing α-syn expression, we developed a novel technique RNA Pull-down Confocal Nanoscaning (RP-CONA) to monitor the binding events between pri-miR-7 and HuR. By attaching FITC-pri-miR-7-1-CTL-biotin to streptavidin-coated agarose beads and incubating them in human cultured cell lysates containing overexpressed mCherry-HuR, the bound RNA and protein can be visualised as quantifiable fluorescent rings in corresponding channels in a confocal high-content image system. A pri-miR-7/HuR inhibitor can decrease the relative mCherry/FITC intensity ratio in RP-CONA. With this technique, we performed several small-scale screenings and identified that a bioflavonoid, quercetin can largely dissociate the pri-miR-7/HuR interaction. Further studies proved that quercetin was an effective miR-7 inducer as well as α-syn inhibitor in HeLa cells. To understand the mechanism of quercetin mediated α-syn inhibition, we tested the effects of quercetin treatment with miR-7-1 and HuR knockout HeLa cells. We found that HuR was essential in this pathway, while miR-7 hardly contributed to the α-syn inhibition. HuR can directly bind an AU-rich element (ARE) at the 3’ untranslated region (3’-UTR) of α-syn mRNA and promote translation. We believe quercetin mainly disrupts the ARE/HuR interaction and disables the HuR-induced α-syn expression. In conclusion, we developed and optimised RP-CONA, an on-bead, lysate-based technique detecting RNA/protein interactions, as well as identifying RNA/protein modulators. With RP-CONA, we found quercetin inducing miR-7 biogenesis, and inhibiting α-syn expression. With these beneficial effects, quercetin has great potential to be applied in the clinic of PD treatment. Finally, RP-CONA can be used in many other RNA/protein interactions studies
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