Department Environmental Toxicology

Current master projects

Influence of mTOR pathway modulation on growth in fish cells

Background

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.

Aims

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.
 

Methods

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

1 Background
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.

2 Aim
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.

3 Methods
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

PROJECT SUMMARY

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.

AIMS

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.

APPROACH/EXPERIMENTAL OUTLINE

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

BACKGROUND
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.

AIM
The aim of this Master thesis is to develop and test molecular biomarkers for the early detection of herbicide exposure in stream biofilms.

METHODS
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

Background

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.

Proposed workflow

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.   


References

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 (marina.zoppo@eawag.ch). This research will be performed at the department of Environmental Toxicology, Eawag, in Dübendorf.

The impact of metal exposure on zebrafish:
Investigating the mechanism of hair cell toxicity in zebrafish larvae by behavioral, structural and molecular analysis

Metals are widespread aquatic contaminants and affect aquatic wildlife in different ways. For instance, copper ions have been shown to specifically impair hair cells of the lateral line organ in fish. These cells are sensors of hydrodynamic flows helping the fish to orient, detect predators and prey and communicate with conspecifics. As a consequence, some behavioral responses, such as e.g. rheotaxis, a natural behavioral reaction of fish to orient counter-flow in order to hold a fixed position in a stream, are severely affected in copper-exposed fish.

AIM: The aim of this Master thesis is to investigate the effects of different metals on hair-cell mediated behavior of fish and to explore the mechanism by which these cells are specifically affected.

METHODs: To tackle this question, we are using zebrafish (Danio rerio) larvae, because of several reasons: genomic resources and genetic tools are available, their transparent larval stages enable different optical techniques and their small body size allows the continuous measurement of behavior with full control of the environment.

The impact of metals on hair cells will be elucidated from different angles: 1) Behavioral tests of metal-exposed zebrafish larvae will be performed, 2) hair cell structure and metal distribution will be investigated by different staining techniques followed by bright-field or confocal imaging and 3) the molecular basis of metal transport mechanisms will be studied by whole mount in situ hybridization in zebrafish larvae, which allows to localize gene expression of relevant transporters to specific tissues such as e.g. hair cells. Gene expression will form the basis for further loss-of-function experiments. Depending on the progression, the Master thesis could be extended with such functional tests.

Up to two Master thesis can be offered about this topic, and the project can be tailored to the candidate’s interests.

Suitable candidates for this project are expected to hold a BSc degree in biology, environmental sciences or a related discipline and to have experience in laboratory work.

For further information please contact Colette vom Berg (Colette.vomberg@eawag.ch) or Michael Burkard (Michael.Burkard@eawag.ch) . This work will be performed at Eawag in the department of Environmental Toxicology in Dübendorf.

Impact of insecticides on the fish developing nervous system

BACKGROUND: Insecticides are extensively used in Switzerland and all over the world to control pests and pathogens in medicine, households, and agriculture. Via spray drift, leaching or run-off they find their way into the aquatic environment where they pose a risk to non-target organisms, such as fish. Toxic effects from insecticides can occur at different organizational levels and may range from easily observable lethal to very subtle behavioral effects. As most insecticides are designed to interfere with neuronal signaling, they are able to adversely affect sensory processing and motor outputs in the fish with extensive ecological consequences.

AIM: We would like to understand the causes underlying the potential locomotion defects in fishes elicited by developmental insecticide exposure. Moreover, we will investigate whether there are critical periods during the development and to what extent adverse effects can be reversed.

METHODS: We will use zebrafish larvae in this study, because behavioral responses can easily be measured and their transparent brain and body allows the use of imaging techniques for the assessment of neuronal defects. We will measure locomotor behavior of larval zebrafish, which have been exposed to insecticides during different stages of their development using a fully automated video tracking system. Neuromuscular defects will be investigated by fluorescent immunohistochemistry and confocal imaging analysis.

The candidate will learn how to 1) breed zebrafish and handle eggs and larvae; 2) measure and analyze zebrafish behavior; 3) perform fluorescent immunostainings, confocal microscopy and imaging analysis.

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.

For further information please contact Colette vom Berg (Colette.vomBerg@eawag.ch) and Sarah Könemann (Sarah.Koenemann@eawag.ch). This work will be performed at Eawag in the department of Environmental Toxicology 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 (ahmed.tlili@eawag.ch). This work will be performed at Eawag (Dübendorf) in the Department of Environmental Toxicology, and in collaboration with Denise Mitrano (denise.mitrano@eawag.ch) 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 (ahmed.tlili@eawag.ch). This work will be performed at Eawag in the department of Environmental Toxicology in Dübendorf.

 

Contact

Prof. Dr. Kristin SchirmerHead of departmentTel. +41 58 765 5266Send Mail
Dr. Ahmed TliliGroup LeaderTel. +41 58 765 5330Send Mail

Teaching

We are actively involved in formal course instructions and thesis supervision.

 

Course Title

Course Number     Persons Involved

Advanced Ecotoxicology (ETHZ)

701-1312-00L

Rik Eggen, Elisabeth Janssen,Kristin Schirmer, Marc Suter, Ahmed Tlili

Practical Course in Molecular Ecotoxicology (ETHZ)

701-1330-00L

Kristin Schirmer, Colette vom Berg, Stephan Fischer (Aquatox Solutions)

Introduction to Toxicology (ETHZ)

752-1300-01L

Rik Eggen, Melanie Erzinger, Shana Sturla

Ecotoxicology (EPFL)

ENV-306

Kristin Schirmer, Michael Burkhard, Julita Stadnicka

Chemistry of Aquatic Systems (ETHZ)

701-0423-00L

Lenny Winkel, Ahmed Tlili

Computational Biology and Bioinformatics Seminar (ETHZ)

636-0704-00L

Jörg Stelling, Manfred Claassen, Dagmar Iber, Tanja Stadler, Anze Zupanic

Grundlagen der Umweltchemie und Ökotoxikologie (ETHZ)

529-0037-01L

Kathrin Fenner, Juliane Hollender, Colette vom Berg

Seminar für Bachelor-Studierende: Biogeochemie (ETHZ)

701-0419-01L

Gerhard Furrer, Ruben Kretzschmar, Colette vom Berg

Division Analytical Sciences - Courses

Marc Suter is the current vice president of the Division Analytical Sciences (DAS) of the Swiss Chemical Society (SCS)which offers a wide range of further education courses in the fields of analytical sciences such as separation, spectroscopy, analytical applications, methods of biotechnology and life sciences as well as quality assurance and information management.

From beginners to experts, all levels of users are targeted. The courses are organized by Esther Wolff, and offered in collaboration with industrial partners to ensure the relevance of application.

A great number of courses are held at Eawag.

The complete list of courses can be found HERE.

 

 

 

Contact

Dr. Marc SuterFormer Deputy Department HeadTel. ---Send Mail