Aktuelle Master Projekte
Characterization of phase I and II biotransformation enzymes in fish cell lines
The aquatic environment is significantly impacted by a constant introduction of chemical contaminants. Upon exposure, aquatic animals, like fish, display different mechanisms of defense, collectively known as the chemical defensome. Among these mechanisms, biotransformation pathways are key components of an organism’s ability to cope with pollution. For example, biotransformation directly influences the bioaccumulation of chemicals. To date, the evaluation of biotransformation pathways and the assessment of bioaccumulation relies extensively on the use of live animals (e.g. OECD TG305 and TG319), despite the availability of novel animal alternatives, specifically fish cell lines. It is important, therefore, to explore the biotransformation ability of established fish cell lines to advance their application in the assessment of bioaccumulation and overall responses to chemical exposure, while continuing to support the reduction, refinement, and potential replacement of whole-animal models.
Characterize the kinetics and magnitude of activity of different phase I and II biotransformation enzymes in established fish cell lines, and increase their applicability in the evaluation of bioaccumulation potential.
Fish cell lines represent a robust in vitro model for the evaluation of chemical exposure. Therefore, well-established cell lines such as the rainbow trout (Oncorhynchus mykiss) RTL-W1 (liver), RTgill-W1 (gill), RTgutGC (gut) and RTbrain (brain) will be assessed for their ability to display different phase I and II biotransformation enzymes. Additional cell lines from other organs and fish species will be considered.
The characterization of the kinetics and overall activity of biotransformation enzymes will follow standardized colorimetric and fluorescence-based bioassays to explore real-time biotransformation activity in a short time.
The methodologies in the proposed work will involve (i) routine cell culture techniques, (i) protein quantification, (iii) spectrophotometry, and (iv) instrumental analysis (e.g. HPLC-FLD, LC-MS).
Suitable candidates for this project are expected to hold a BSc degree in biology, biochemistry, environmental science, or a related discipline. Moreover, candidates should have a strong interest for advancing animal alternatives in toxicology.
For further information, please contact Dr. Marco E. Franco
This work will be performed at the Department of Environmental Toxicology at Eawag in Dübendorf, Switzerland.
Identification of molecular pathways mediating the toxicity of insecticides in fish
In agricultural areas, insecticides comprise a considerable part of the chemical mixture present in the aquatic environment: they reach water bodies via run-off or leaching after spraying events. While designed to be neurotoxic to insects, insecticides can have adverse effects on a multitude of organisms due to the high conservation of the nervous system among phyla. To assess the risk of insecticides for the environment and estimate their long-term ecological effects, it is important to investigate their impact on non-target organisms such as fish. Toxic effects elicited by insecticides can occur at different organizational levels and may range from easily observable lethal to very subtle behavioral effects. The molecular pathways behind these effects, however, are still largely unknown, but are important to understand in order to develop predictive models, biomarkers and in vitro tests.
We would like to gain a better understanding of neurotoxic effects in fish by linking observed effects on organism-level to underlying molecular events.
Due to its exceptional suitability for molecular and developmental studies, the zebrafish, Danio rerio, will be used as a test organism. Based on previous experiments, in which the effects of insecticides on the behavior and neuromuscular structure of zebrafish larvae were investigated, gene expression analysis will be conducted focusing on genes involved in development and functioning of the nervous system and muscles.
The techniques applied include (i) breeding and handling of zebrafish, (ii) design of primers, (iii) RNA isolation, (iv) qPCR, and eventually (v) CRISPR-Cas9 gene editing (if time allows).
Suitable candidates for this project are expected to hold a BSc degree in biology, environmental sciences or a related discipline and should have a genuine interest in neuroscience and molecular biology.
For further information please contact Colette vom Berg and Sarah Könemann.
This work will be performed at Eawag at the Department of Environmental Toxicology in Dübendorf.
Molecular mechanisms behind reduction of internal calcium concentrations in zebrafish embryos exposed to copper
Metals are widespread aquatic contaminants and affect aquatic wildlife in different ways. Copper, for instance, although an element essential to life, can be detrimental in excess. Preliminary studies in our laboratory have shown that zebrafish embryos exposed to sub-lethal concentrations of copper have reduced levels of internal calcium and other elements. Although the interplay of copper and calcium has been documented in many species, the exact mechanisms of copper uptake and calcium reduction are not well understood.
The aim of this project is to
- Decipher the interplay between uptake of copper and calcium
- Characterize the internal distribution sites and accumulation of these two metals in zebrafish embryos upon copper exposure
- Investigate molecular mechanisms behind lower calcium uptake and consequences of reduction in internal calcium levels
Methods and Techniques applied
Zebrafish maintenance and breeding, zebrafish embryo toxicity assay, histochemical staining to visualize bones and cartilage, microscopy, pharmacological and/or molecular inhibition of transport proteins and channels, qPCR to measure gene expression, ICP-MS to measure metal concentrations
If you are interested in this project, please contact Ksenia Groh, Kristin Schirmer or Colette vom Berg.
This work will be performed at Eawag at the Department of Environmental Toxicology in Dübendorf.
Mechanistic insights into the toxicity of tire particles applying fish cell lines
Tire and Road Wear Particles (TRWP), which are generated by the erosion of tires while driving, have recently been estimated as representing up to 28.3% of all primary microplastics released in the aquatic environment, for an estimated mass of 424 Ktons/year. This finding raises questions regarding the toxicological impact of these TRWP and associated chemicals on aquatic organisms.
This study aims at determining the toxicity mechanisms induced by TRWP and associated chemicals to fish using different rainbow trout cell lines (Oncorhynchus mykiss).
We will use several rainbow trout cells lines (e.g., from gill, gut and brain), which will be subject to different exposure scenarios.
You will gain experience with routine cell culture techniques (how to work in sterile conditions and culture cells). You will moreover learn how to set up and perform different assays: cell viability assays to determine the cytotoxicity of chemicals (seeding cells using different substrates, exposure to chemicals and multi-endpoint viability assay), bioavailability assays to quantify how much of a chemical has entered the cells (mass spectrometry) and gene expression analysis (RT-PCR) to identify molecular mechanisms induced. Finally, you will learn how to produce, interpret and present scientific data.
If you are interested in this particular research topic and are excited to know more about environmental toxicology research in general, please contact William Dudefoi or Kristin Schirmer.
This research will be performed at the department of Environmental Toxicology, Eawag, in Dübendorf.
Bioaccumulation of charged chemicals in Rainbow Trout Cell Lines
Preferred starting date: flexible
It has been demonstrated that permanent cell lines from rainbow trout have the potential to predict the bioaccumulation in fish of uncharged chemicals, such as polyaromatic hydrocarbons. In such an exposure test, the biotransformation rate of the chemical in exposed cell cultures is measured over time. This information serves as an input for toxicokinetic models, which simulate the bioaccumulation in fish. However, whether the cell lines could as well be used to predict biotransformation rates of charged chemicals, such as pharmaceuticals, pesticides or agents in personal care products, is not yet known.
This project focusses on the bioaccumulation assessment of selected charged chemicals, such as, for example, the primary amine, dodecylamine, in rainbow trout cell lines. The aim is to compare the in vitro-derived biotransformation rates and the modelled bioaccumulation to data available from in vivo studies with rainbow trout. The tasks comprise 1.) the conduction of exposure experiments in rainbow trout cell lines from different tissues (gill, gut and liver) 2.) the determination of the chemical concentrations in the cells and 3.) the modelling and interpretation of data.
You will acquire knowledge and skills in a diverse range of disciplines:
- In vitro cell culture techniques
- Design of kinetic experiments with cells
- Sample preparation and measurement using liquid-chromatography-high resolution tandem mass spectrometry
- Toxicokinetic modelling and data interpretation
Supervision by: Fabian Balk (PhD student), Prof. Dr. Kristin Schirmer, Prof. Dr. Juliane Hollender
Contact: Fabian.firstname.lastname@example.org, Kristin.Schirmer@cluttereawag.ch, Juliane.Hollender@cluttereawag.ch
Influence of mTOR pathway modulation on growth in fish cells
In mammals, mechanistic target of rapamycin (mTOR) pathway is known to be a central cellular hub that mediates regulation of cell growth and proliferation in response to a host of endogenous and exogenous stimuli. The mTOR is a serine-threonine kinase that, upon activation, initiates a cascade of phosphorylation events for its downstream targets, which are further involved in regulating transcription, translation, autophagy, metabolism, and cytoskeletal organization, among many other essential cellular processes. Interestingly, exposure to chemicals can influence many of the upstream signals sensed by the mTOR, such as energy levels, oxidative stress, proteotoxic stress, genotoxicity, or inflammation. The role of the mTOR in regulating cellular responses to stress has indeed been recognized, but so far rarely considered with regard to chemical toxicity, in fish even less so than in mammals. Therefore, the research in my group seeks to understand the role of mTOR in regulating fish growth in response to chemical exposures and other environmental factors such as temperature. The project described here will characterize the baseline relationships between the mTOR activity levels and growth responses in a fish cell line.
This master thesis will investigate the correlation between the mTOR pathway activity and growth in cultured fish cells. For this, fish cells will be exposed to specific pharmacological inhibitors or activators of the mTOR pathway and its activity will be monitored by measuring changes in phosphorylation status of mTOR substrate proteins. In parallel, growth and several other endpoints will be assessed in the exposed cells to understand their correlation with the observed phosphorylation-based signaling dynamics.
Experimental work on the proposed master thesis will provide training in basic cell culture techniques, chemical exposure, fish cell growth assay, and microscopy/imaging. The central analytical task will include performing targeted phosphoproteomic analysis of selected proteins using mass spectrometry. Our phosphoproteomic assays constitute a novel tool to study phosphorylation-based signaling in fish, where this mechanistic aspect has been so far largely neglected due to challenges with obtaining species-specific antibodies for (de)phosphorylated proteins of interest. The workflow consists of protein extraction from cultured cells, protein digestion, phosphopeptide enrichment, and measurement on our state of the art LC-MS/MS instrumentation.
If you are interested in this project please contact: Ksenia Groh.
Influence of temperature on the toxicological response in rainbow trout cell lines
Rainbow trout (Oncorhynchus mykiss) derived cell lines, similar to rainbow trout, can survive in temperature ranges between 4◦C to 24◦C, but are routinely cultured and maintained at 19◦C. However, several lines of evidence suggest that temperature could exert an effect on cellular responses to chemicals in vitro. Firstly, phospho-lipids in membranes adapt to lower temperatures by decreasing permeability, and to higher temperatures by increasing permeability. This can result in temperature-dependent changes in uptake and bioaccumulation of substances. Furthermore, biotransformation of chemicals in cells is effected by enzymes, such as CYP450, whose kinetics are also influenced by temperature.
In order to establish cell lines as a robust in vitro system for environmental risk assessments, it is important to explore whether outcomes can be predicted for complex exposure scenarios, such as with temperature as an additional stressor. Furthermore, understanding the influence of temperature on sensitivity is becoming increasingly important in the context of climate change.
The aim of this Master thesis is to investigate the effect of temperature on the sensitivity and biotransformation capacity of rainbow trout cell lines, and to assess whether trends observed are comparable to those known from experiments with fish in in vivo, and in the environment.
Research comprises training in:
• Basic cell culture techniques
• In vitro cell line based toxicological assays (cell viability assays, biotransformation assays)
• Chemical analysis by Mass Spectrometry (MS)
• Physiologically Based Toxico Kinetic (PBTK) modelling
If you are enthusiastic about exploring the relevance of temperature stress on cellular responses to chemicals, and learning useful skills and methods in environmental toxicology in the process, please contact Kristin Schirmer or Gayathri Jaikumar. This research will be performed at the department of Environmental Toxicology, Eawag, in Duebendorf.
Identification of attachment factors for the development of serum-free medium of RTgill-W1 cell line
Permanent fish cell lines of rainbow trout (Oncorhynchus mykiss) have great potential as alternatives to conventional fish tests in chemical safety testing. While several strategies and assay procedures are being developed that use fully defined media for chemical exposure, the routine culture of these cells, and certain chemical exposure assays, still require fetal calf serum (FBS) for cell maintenance and proliferation.
On this background, we aim to develop a fully transparent serum-free cell culture medium for cell lines from rainbow trout. Cultures of a rainbow trout gill cell line (RTgill-W1) are known to be anchorage dependent cells, meaning attachment to solid surface is prerequisite for successful cell proliferation. Optimal cell attachment of RTgill-W1 is dominantly achieved by culturing cells in FBS. Whereas omission of serum leads to poor cell attachment and stopped cell proliferation. While desirable combination of externally supplemented growth factors may contribute to overcoming cell proliferation limitation under serum-free conditions, appropriate attachment of cells on substrate still needs to happen prior to cell grow. Thus, identification of supplements or coating materials that promote appropriate cell attachment are still major events that needs to be unravelled on the road to chemically defined serum-free media formulation.
For this Master Project, we propose to identify cell attachment factors and develop a cell attachment assay for the RTgill-W1 cell line using the 96-well plate format. A high-throughput screening approach that quantifies impact of selected attachment supplements, alone and in combination, on fish cell attachment over seven days will be used to identify the optimal composition of the serum-free media. Big emphases will be put on time-dependent studying of cell attachment and cell lose during 7 days period of cell cultivation of either serum-free or FBS-containing media.
Throughout this master thesis, the candidate will first learn how to routinely culture RTgill-W1 cell lines, gain experience in cell viability assay and/or cell-growth assay. Subsequently the candidate will have the opportunity to apply various protocols for cell staining and have some training in basic techniques, such as fluorescent microscopy and Cell Imagining Multi-Mode Reader imaging and high-content/through-put screening. Experimental design, data presentation and analyses are an integral part of this project as well.
Interested? If you are excited about this line of research please contact Kristin Schirmer (Kristin.Schirmer@eawag.ch) or Barbara Jozef (Barbara.Jozef@eawag.ch). The work will be performed at Eawag in the department of Environmental Toxicology in Dübendorf.
Development and testing of molecular biomarkers in stream biofilms
In nature the majority of microorganisms are found in complex communities, so called biofilms. Stream biofilms consist of different algal, bacterial and fungal species. Herbicides that reach the streams can lead to structural and functional alterations of stream biofilms, which in turn might affect the whole ecosystem functioning. Development and application of biomarkers is a powerful molecular tool for early detection of hazardous exposure in the environment.
The aim of this Master thesis is to develop and test molecular biomarkers for the early detection of herbicide exposure in stream biofilms.
Research comprises training in: (1) various bioinformatics techniques (databases search, multiple sequence alighment, in silico primer design), (2) broad spectrum of molecular techniques (RNA extraction, reverse transcription and RT-qPCR) and (3) how to work sterile, culture and chemically expose stream biofilms.
If you are eager to learn and implement new molecular technologies and passionate about environmental toxicology, please contact Olga Lamprecht. This research will be performed at the department of Environmental Toxicology, Eawag, in Dübendorf.
Data base of microplastic particle optical properties based on flow cytometry
Detection and identification of microplastic particles in complex environmental samples or tissues is challenging due to the similarity in size and chemistry of natural and plastic materials in these samples. Measurement by flow cytometry and data analysis by a viSNE-based protocol developed at Eawag1,2 has proven useful to detect microplastic particles in stream biofilms, stream water, tissue and feces. viSNE. Originally developed for leukemia research, it is a tool to map high-dimensional cytometry data onto 2D while conserving high-dimensional structure and is based on the t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm3. Data is interpreted and particle types are quantified by comparison to reference data sets. Metrics for comparison include light scattering and fluorescence, which, when combined together, may provide a “fingerprint” of microplastics in complex samples. The detection limits of this approach in terms of both particle size and resolution of polymer identity are still unknown. By developing a database using a suite of known microplastic particles with various polymers, both in simple and complex media, this would provide a platform on which to analyse natural samples where unknown microplastics are present.
Within the framework of a Master’s thesis, the proposed project aims to establish a database of optical properties of microplastic particles based on flow cytometry data. Data analysis will be done by the viSNE-based protocol to characterize the detection limits in environmental samples with a focus on stream biofilms. To test the viSNE system, a relevant environmental exposure scenario will be constructed to assess microplastic particles in complex media. Stream biofilms are a potential sink for microplastic particles that sediment and interact with the extracellular polymeric substances excreted by biofilm-associated microorganisms, and thus make for an ideal proof of concept framework. Field sampling and controlled growth (setup available at Eawag) of biofilms will be realized in collaboration with Dr Ahmed Tlili/Eawag Environmental Toxicology Department. Guidance in analysis of samples by flow cytometry and data evaluation will be provided by Alexandra Kroll (Ecotox Centre). Microplastic particles from different origin (primary and secondary microplastic particles) will be selected to cover relevant sizes, aspects (e.g. spheres, fibres), polymer types, and additives in collaboration with Dr Denise Mitrano/ Eawag Process Engineering Department.
Suitable candidates for this project are expected to hold a BSc degree in biology, environmental sciences or a related discipline. For further information, please contact Alexandra Kroll, Ahmed Tlili or Denise Mitrano.
1 Sgier L, Freiman R, Zupanic A, Kroll A. (2016) Flow cytometry combined with viSNE for the analysis of microbial biofilms and detection of microplastics. Nat Comm 7(11587).
2 Sgier, L., Merbt, S. N., Tlili, A., Kroll, A., Zupanic, A. (2018) Characterization of Aquatic Biofilms with Flow Cytometry. J Vis Exp (136), e57655, doi:10.3791/57655.
3 Amir el-AD, Davis KL, Tadmor MD, Simonds EF, Levine JH, Bendall SC, Shenfeld DK, Krishnaswamy S, Nolan GP, Pe'er D. (2013) viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia. Nat Biotechnol. 31(6):545-52.
Establishment of a CRISPR gene-editing strategy in rainbow trout cell lines
BACKGROUND: One of the main goals in the field of toxicology is the development of Adverse Outcome Pathways (AOPs): an emerging tool aimed at understanding the biochemical and signal transduction pathways that are affected when an organism is exposed to a chemical. An early step in the development of AOPs is the assessment of the toxic action of a chemical at the molecular level. The gene-editing CRISPR/Cas9 technology could help in this field by allowing scientists to specifically modify genes involved in different outcome pathways and observe how the response to the chemical exposure changes.
AIM: The aim of this Master thesis is to establish a CRISPR/Cas9 gene editing system in rainbow trout (Oncorhynchus mykiss) cell lines and use this technology in order to shed light on the molecular processes underlying the chemical action.
METHODS: Research comprises training in: (1) basic molecular biology techniques (e.g. DNA, RNA and plasmid DNA extraction, PCR) and molecular cloning strategies (e.g. Gibson assembly and E. coli transformation), (2) gene editing using the CRISPR/Cas9 system (3) how to culture, transfect and chemically expose fish cell lines (4) fluorescence microscopy and FACS analysis.
If you are eager to learn and implement new technologies and passionate about molecular biology and environmental toxicology, please contact Kristin Schirmer (Kristin.Schirmer@eawag.ch) or Marina Zoppo (email@example.com). This research will be performed at the department of Environmental Toxicology, Eawag, in Dübendorf.
Impact of nano- and microplastic fibers on a stream biofilm-grazer system
Particulate plastics (microplastics, fibers, and nanoplastics) are produced either intentionally for their commercial use in consumer products or unintentionally as breakdown fragments of larger synthetic materials (e.g. plastic bottles, polyester textiles, rubber tire wear). Currently, there is little information on the fate, transport and bioavailability of nanoplastics and microplastic fibers in water bodies. Importantly, knowledge on their potential to interact with organisms and to impact food-webs is largely lacking.
AIMS: Here we aim to quantify interactions of nanoplastics and microplastic fibers with stream microbial biofilms, as well as their potential transfer from biofilms to grazers. Focus will be on examining for transgenerational effects on the snail offspring fitness. Unique metal-doped particulate plastics developed in-house will be used through all experiments, which will both expedite analysis times in determining biota-plastic burden and provide more precise and accurate quantification of plastics throughout the exposure system.
METHODS: The candidate can expect to learn (1) how to set up and maintain experimental mesocosms in a laboratory setting, (e.g. growth and sampling of biofilms and snails), (2) how to register changes in biofilm and snail biology (e.g. growth, respiration, reproduction), and (3) how to quantify particulate plastics in various media, including techniques for particle characterization (e.g. Dynamic light scattering, Nanoparticle tracking analysis, Transmission electron microscopy) and analytical chemistry (e.g. Inductively coupled plasma mass spectrometry)).
Suitable candidates for this project are expected to hold a BSc degree in biology, environmental sciences or a related discipline. For further information please contact Ahmed Tlili (firstname.lastname@example.org). This work will be performed at Eawag (Dübendorf) in the Department of Environmental Toxicology, and in collaboration with Denise Mitrano (email@example.com) in the Department of Process Engineering.
Molecular responses to herbicide exposures in stream biofilms
Stream biofilms are complex communities of algae, bacteria and fungi, which play a fundamental ecological role in aquatic ecosystems. Microorganisms composing biofilms are primary targets for herbicides, which can lead to structural and functional alterations of the community, with potential negative consequences for ecosystem functioning. Yet, molecular processes underlying functional and structural responses of biofilms to herbicides are still largely unknown.
AIM: By establishing a set of genes that are specifically regulated in biofilms upon exposure to herbicides, we aim in this project to (i) examine toxicity and adaptive cellular processes, on the transcriptome level, in aquatic biofilms and (ii) link these processes to community functional and structural alterations.
METHODS: Candidates will learn (1) how to culture and sample biofilms both in the field and in laboratory mesocosms , (2) various techniques for gene expression (mRNA extraction and quantification, qPCR), molecular and functional diversity analyses (genomic DNA extraction and amplification, DGGE, flowcytometry), and (3) quantification of the herbicides in water and biofilms.
Suitable candidates for this project are expected to hold a BSc degree in biology, environmental sciences or a related discipline.
For further information please contact Ahmed Tlili (firstname.lastname@example.org). This work will be performed at Eawag in the department of Environmental Toxicology in Dübendorf.
Wir sind aktiv in der Lehre an der ETHZ und der EPFL tätig.
|Kurstitel ||Kursnummer ||Personen |
|Advanced Ecotoxicology (ETHZ) ||701-1312-00L ||Rik Eggen, Elisabeth Janssen, |
Kristin Schirmer, Ahmed Tlili
|Praktikum Advanced Ecotoxicology Laboratory (ETHZ)||701-1330-00P || |
Kristin Schirmer, Colette vom Berg, Ksenia Groh
|Introduction to Toxicology (ETHZ)||752-1300-00L||Rik Eggen|
|Ecotoxicology (EPFL)||ENV-306||Kristin Schirmer|
|Sustainability and Water Resources (ETHZ)||118-0111-00L ||Darcy Molnar, Paolo Burlando, |
Kristin Schirmer, (Ahmed Tlili)