Zellen als lebende Materialien: Kraftspektroskopische Untersuchung der Mechanotransduktion

Mechanotransduction describes a cellular mechanism of sensing and converting mechanical cues into biochemical signals to regulate cell processes, such as adhesion, migration, proliferation and/or apoptosis. Thus, becoming an ever-growing field of research with high potential for medical applications. I present a new strategy towards reliable microindentation measurements, which is essential for investigating mechanotransduction using soft substrates. I show a precise, reproducible determination of Young’s moduli through an automatic analysis of indentation data. The algorithm presented detects Young’s moduli in a region without dependence on indentation depth while minimizing the fitting error. This strategy is a step towards a comprehensive study of soft materials on a spatial scale similar to cell interactions. It has broad applicability ranging from fundamental research to developing innovative implants that match the in vivo situation. Also, I present novel approaches for multifaceted cellular manipulation. I show that layer thickness of a soft material fixed to a stiff underlying substrate can be crucial for cell adhesion. These findings are pioneer for new implant designs and advanced application fields. I present two atomic force microscopy-based manipulation systems that allow applying specific mechanical stimuli to single cells and a subsequent correlation to whole cell detachment and single bond strengths. The unique AFM-based shear system presented combines application of shear stimuli and cell detachment measurements, whereas the AFM-based modulation system combines oscillatory pushing and pulling with cell detachment measurements. Both shear and oscillatory forces are essential in our body. Thus, the strategies presented in this thesis are of significant medical interest allowing an overarching study of mechanotransduction and may pave the way towards smart stimulation devices that allow cell adhesion on demand

Transfer and accumulation of paralytic and amnesic shellfish toxins in secondary consumers of the marine trophic chain

Tese de mestrado, Ecologia Marinha, 2010, Universidade de Lisboa, Faculdade de CiênciasResumo alargado em português disponível no documentoParalytic and amnesic shellfish toxins can enter in the marine trophic chain by filterfeeding and benthic organisms. Then due to trophic interrelationships the toxins are transferred to theirs predators. In the last instance, top predators (marine mammals and sea birds) can be intoxicated after fed on primary and secondary consumers. The wild horse mackerel (Trachurus trachurus) was collected in NW and S Portuguese coast between September of 2009 and March of 2010. The results show for the first time that horse mackerel can accumulate high concentrations of PSTs in theirs gastrointestinal tract. Which increased the requirement of evaluated the dynamics of accumulation and elimination of PSTs in secondary consumer fish. Thus aquaculture fishes, white sea breams, were fed with toxin-contaminated cockles. After 5 days of toxin exposure, the fishes were fed with non toxic cockles during 10 days. In this feeding experiment, an increasing of toxin concentration over the exposure period was noticed in the fish gastrointestinal tract. B1 and dcSTX were the only toxins continuously detected during the exposure/elimination period, which reveal a slower elimination than the others PSTs. These two PSTs were also the main toxins in the mackerel toxin profile, which may suggest a specific PSTs elimination by fish species. Although AST were not detected in any mackerel specimen, in octopus (Octopus vulgaris) specimens captured in the same fishing area, domoic acid (DA) was detected in the digestive glands. This accumulation of DA in the octopus was previously identified. However this is the first time that DA was detected during periods of absence of ASP events. Plus DA was predominantly found in the cytosol which increased the evidence of a retention mechanism. Since DA as a hydrophilic compound should be easily release. This thesis supplies relevant data to the growing knowledge on the dynamics of accumulation and elimination of PSTs and AST in secondary consumers and on the toxin transfer in the marine food web

Impact of organic solvents on lipid-extracted microalgae residues and wastewater sludge co-digestion

This study investigates the combined production of lipids and biogas via anaerobic mono- and co-digestion of microalgae biomass grown in municipal untreated wastewater. In the co-digestion assays, extracted microalgae and wastewater sludge were mixed at different ratios: 25–75, 50–50, and 75–25% total volatile solids (VS) of each substrate, respectively. The neutral and polar fatty acid methyl esters constituted approximately 13 and 1.5% of the total lipids, respectively. Mono-digestion of lipid-extracted microalgae had a lower biochemical methane potential (BMP) than of non-extracted microalgae. Organic solvents were shown to negatively impact both mono- and co-digestion of extracted microalgae. Co-digestion experiments showed synergy between sludge and microalgae residues, increasing the BMP from 91.4 Normalized mL (NmL) CH4/g VS in the mono-digestion of evaporated extracted microalgae up to 228.6 NmL CH4/g VS in the co-digestion of the mixture with 25% VS of microalgae biomass with 75% VS of sludge

Determinación de toxinas paralizantes en el jurel (Trachurus trachurus) durante una proliferación de Gymnodinium catenatum: la prevalencia de decarbamoilsaxitoxina en la red trófica marina

This study reports the accumulation of paralytic shellfish toxins (PSTs) in Atlantic horse mackerel (Trachurus trachurus) over a bloom of the toxigenic dinoflagellate Gymnodinium catenatum. High levels of toxins, up to 4800 μg STXeq kg–1, were registered at the peak of the bloom (5.0 103 cells l–1). The suite of individual PSTs was examined. Decarbamoylsaxitoxin (dcSTX) and B1 constituted nearly 90% of toxins (on a molar basis) determined in mackerel. This profile of toxins markedly differs from the known profile of toxins produced by G. catenatum strains isolated from the Portuguese coast, which is dominated by N-sulfocarbamoyl toxins, in particular the C1+2 toxins. The prevalence of the potent dcSTX in the pelagic environment and its transfer through the marine food web is highlighted in this study. Atlantic horse mackerel is identified as a high potential vector of PSTs along the Portuguese coast. This fish species has a central position in the marine food web, being an important predator of zooplankton and at the same time an important diet item of top predators. This study reveals bioaccumulation values that are important for evaluating potential impacts of blooms of PST-producing dinoflagellates on marine ecosystems or their components, such as fish.Este estudio reporta la acumulación de toxinas paralizantes de molusco (PSP) en las muestras de jurel (Trachurus trachurus) durante una proliferación do dinoflagelado Gymnodinium catenatum. Los altos niveles de toxinas PSP, alcanzando un valor máximo de 4800 μg STXeq kg–1, se determinaron en el pico de la proliferación (5.0 103 células l–1). El perfil de toxinas se examinó y se reveló que los compuestos dcSTX y B1 constintuían casi 90% de las toxinas (en base molar) encontrados en las muestras de jurel. Este perfil difiere claramente del perfil de toxinas conocido en cepas de G. catenatum aisladas de la costa portuguesa, que está dominado por toxinas N-sulfocarbamoyl, en particular las toxinas C1+2. Este trabajo muestra la prevalencia de dcSTX el ambiente pelágico y la transferencia de este compuesto a través de la cadena alimentaria. El jurel se identifica como un potencial vector de las toxinas PSP a lo largo de la costa portuguesa. Esta especie de pez tiene una posición central en la cadena alimentaria marina, siendo un importante depredador de zooplancton y, al mismo tiempo un elemento importante de la dieta de los depredadores superiores. Este estudio contribuye con valores ecológicos relevantes para evaluar los potenciales impactos de las proliferaciones de dinoflagelados productores de PST en los ecosistemas marinos o sus componentes, como los peces

Chemical composition and species identification of microalgal biomass grown at pilot-scale with municipal wastewater and CO2 from flue gases

The production potential of a locally isolated Chlorella vulgaris strain and a local green-algae consortium, used in municipal wastewater treatment combined with CO2 sequestration from flue gases, was evaluated for the first time by comparing the elemental and biochemical composition and heating value of the biomass produced. The microalgae were grown in outdoor pilot-scale ponds under subarctic summer conditions. The impact of culti-vation in a greenhouse climate was also tested for the green-algae consortium; additionally, the variation in species composition over time in the three ponds was investigated. Our results showed that the biomass produced in the consortium/outdoor pond had the greatest potential for bioenergy production because both its carbohy-drates and lipids contents were significantly higher than the biomasses from the consortium/greenhouse and C. vulgaris/outdoor ponds. Although greenhouse conditions significantly increased the consortium biomass's monounsaturated fatty acid content, which is ideal for biodiesel production, an undesirable increase in ash and chemical elements, as well as a reduction in heating value, were also observed. Thus, the placement of the pond inside a greenhouse did not improve the production potential of the green-algae consortium biomass in the current study infrastructure and climate conditions.info:eu-repo/semantics/publishedVersio

Functional characterization of two enhancers located downstream FOXP2

Background: Mutations in the coding region of FOXP2 are known to cause speech and language impairment. However, it is not clear how dysregulation of the gene contributes to language deficit. Interestingly, microdeletions of the region downstream the gene have been associated with cognitive deficits. Methods: Here, we investigate changes in FOXP2 expression in the SK-N-MC neuroblastoma human cell line after deletion by CRISPR-Cas9 of two enhancers located downstream of the gene. Results: Deletion of any of these two functional enhancers downregulates FOXP2, but also upregulates the closest 3′ gene MDFIC. Because this effect is not statistically significant in a HEK 293 cell line, derived from the human kidney, both enhancers might confer a tissue specific regulation to both genes. We have also found that the deletion of any of these enhancers downregulates six well-known FOXP2 target genes in the SK-N-MC cell line. Conclusions: We expect these findings contribute to a deeper understanding of how FOXP2 and MDFIC are regulated to pace neuronal development supporting cognition, speech and language.Spanish National Research and Development Plan PI14/01884Instituto de Salud Carlos III PI14/01884FEDER PI14/0188

Characterization of Cynara cardunculus L. flower from Alentejo as a coagulant agent for cheesemaking

The cardoon (Cynara cardunculus L.) is a mandatory vegetable coagulant for certain Protected Designation of Origin Portuguese cheeses. It grows wild in Portugal and is used without any type of control regarding flower picking or extract preparation, representing some uncertainty in cheese manufacture. The variability in technological properties, in the context of traditional cheese manufacture, of cardoon flower ecotypes from the Alentejo region was evaluated, including milk clotting and proteolytic activities, coagulation properties and potential cheesemaking yield of flower extracts. Multivariate statistics highlighted the variability of flower properties for cheesemaking, but allowed the aggregation of the ecotypes into five groups under the major influence of milk clotting activity and effect on gel firmness and micellar aggregation rate, followed by proteolytic activity. These differences may have an impact on cheese properties and therefore can allow the selection of cardoon flower for the manufacture of different types of cheese

LC-HRMS profiling of paralytic shellfish toxins in Mytilus galloprovincialis after a Gymnodinium catenatum bloom

Saxitoxin and its more than 50 analogues are a group of naturally occurring neurotoxins collectively designated as paralytic shellfish toxins (PSTs). PSTs are toxic to humans and maximum legal limits in seafood have been implemented by regulatory authorities worldwide. In the European Union, monitoring of PSTs is performed using the AOAC Official Method 2005.06, based on liquid chromatography coupled with fluorescence detection (LC- FLD). However, this method has been suggested to not effectively detect the emerging C-11 hydroxyl (M-toxins) and benzoate (GC-toxins) analogues, with these analogues currently not being surveyed in monitoring programs. In this study, a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was used to search for these emerging PSTs in mussels, Mytilus galloprovincialis, contaminated following an intense Gymnodinium catenatum bloom in the Tagus estuary (Lisbon, Portugal). Five M-toxins (M1, M2, M6, dcM6, and dcM10), but no GC-toxins, were detected in the mussels’ whole-soft body tissue. Moreover, the classical PSTs (C1 to C4, GTX 4 to GTX6, dcGTX1 to dcGTX4, dcSTX, dcNEO, and STX) were also found and comprised the largest fraction of the PSTs’ profile. The presence of unregulated PSTs in edible mussel samples suggests potential seafood safety risks and urges further research to determine the frequency of these analogues in seafood and their contribution to toxicityinfo:eu-repo/semantics/publishedVersio

No β-N-Methylamino-L-alanine (BMAA) was detected in stranded cetaceans from Galicia (North-West Spain)

The neurotoxin β-N-methylamino-L-alanine (BMAA), a non-proteinogenic amino acid produced by several species of both prokaryotic (cyanobacteria) and eukaryotic (diatoms) microorganisms, has been proposed to be associated with the development of neurodegenerative diseases. At first, BMAA appeared to be ubiquitously present worldwide in various organisms, from aquatic and terrestrial food webs. However, recent studies, using detection methods based on mass spectrometry, instead of fluorescence detection, suggest that the trophic transfer of BMAA is debatable. This study evaluated BMAA in 22 cetaceans of three different species (Phocoena phocoena, n = 8, Delphinus delphis, n = 8, and Tursiops truncatus, n = 6), found stranded in North-West Spain. BMAA analysis of the liver, kidney, or muscle tissues via sensitive liquid chromatography with tandem mass spectrometry did not reveal the presence of this compound or its isomers. The absence recorded in this study highlights the need to better understand the trophic transfer of BMAA and its anatomical distribution in marine mammals.info:eu-repo/semantics/publishedVersio

Quantitation overcoming Matrix effects of Lipophilic toxins in Mytilus galloprovincialis by liquid chromatography-full scan high resolution mass spectrometry analysis (LC-HR-MS)

The analysis of marine lipophilic toxins in shellfish products still represents a challenging task due to the complexity and diversity of the sample matrix. Liquid chromatography coupled with mass spectrometry (LC-MS) is the technique of choice for accurate quantitative measurements in complex samples. By combining unambiguous identification with the high selectivity of tandem MS, it provides the required high sensitivity and specificity. However, LC-MS is prone to matrix effects (ME) that need to be evaluated during the development and validation of methods. Furthermore, the large sample-to-sample variability, even between samples of the same species and geographic origin, needs a procedure to evaluate and control ME continuously. Here, we analyzed the toxins okadaic acid (OA), dinophysistoxins (DTX-1 and DTX-2), pectenotoxin (PTX-2), yessotoxin (YTX) and azaspiracid-1 (AZA-1). Samples were mussels (Mytilus galloprovincialis), both fresh and processed, and a toxin-free mussel reference material. We developed an accurate mass-extracted ion chromatogram (AM-XIC) based quantitation method using an Orbitrap instrument, evaluated the ME for different types and extracts of mussel samples, characterized the main compounds co-eluting with the targeted molecules and quantified toxins in samples by following a standard addition method (SAM). An AM-XIC based quantitation of lipophilic toxins in mussel samples using high resolution and accuracy full scan profiles (LC-HR-MS) is a good alternative to multi reaction monitoring (MRM) for instruments with HR capabilities. ME depend on the starting sample matrix and the sample preparation. ME are particularly strong for OA and related toxins, showing values below 50% for fresh mussel samples. Results for other toxins (AZA-1, YTX and PTX-2) are between 75% and 110%. ME in unknown matrices can be evaluated by comparing their full scan LC-HR-MS profiles with those of known samples with known ME. ME can be corrected by following SAM with AM-XIC quantitation if necessary.info:eu-repo/semantics/publishedVersio