Het begrijpen van belangrijke processen en structuren in de cel op moleculair niveau, uitgaande van chemische en biologische principes. Voorbeelden van dergelijke processen zijn celdeling, de vermenigvuldiging van het erfelijke materiaal, eiwittransport, signaaltransductie, energievoorziening en metabolisme. Voorbeelden van belangrijke structuren zijn celorganellen, membranen, cytoskelet en chromosomen. De volgende praktische vaardigheden worden geleerd: Steriel werken, kweken van bacteriën, cel preparaten maken, gebruik van microscopen, beeldanalyse, analytisch pipetteren, eiwitzuivering, enzym kinetiek.

Medical Biochemistry and Molecular Biology (BM005) <part of Master Medical Biochemistry>

Signaling with G-force
Master: Biomolecular, Biomedical, Biological Sciences or Systems Biology,, Supervisor: Joachim Goedhart

The Gq-PLCbeta signaling pathway plays an important role in the physiology of the brain and the heart. Our current knowledge on Gq mediated signaling is mainly derived from molecular biological and biochemical studies. These approaches do not allow to study (local) signaling in highly organized, differentiated cells of which the brain and heart are comprised. To understand the Gq-mediated signaling pathway in living cells we have tagged the components of the signaling pathway with Fluorescent Proteins. These fusion proteins will be used to study activation and interactions of Gq in living (neuronal) cells by fluorescence microscopy.
Technical skills/methods: For this research a variety of techniques will be used including, molecular biology (cloning), eukaryotic cell culture and (advanced) fluorescence microscopy (FRET, FRAP, TIRF)

Cell wall synthesis regulation
Master: Life Sciences, Master: Chemistry, Physics, Biomolecular, Biomedical or Biological Sciences, Supervisor: Tanneke den Blaauwen 

Gram-negative bacteria are very difficult to combat because of their impermeable envelope. For this reason many antibiotics (e.g. vancomycin) that kill gram-positive bacteria are futile against gram-negative bacteria. The peptidoglycan layer that is sandwiched between the two membranes that surround the bacterial cytoplasm determines the shape of the bacterium and because of its strength it also protects the bacterium against osmotic pressure and mechanical damage. The well-known penicillins target the enzymes (Penicillin-binding proteins or PBPs) that synthesize the peptidoglycan layer. Recently two outer membrane proteins LpoA and LpoB have been discovered (Typas, A., et al (2010) Cell 143:1097) that regulate the activity of the PBPs. These proteins are promising targets for new antibiotics. Precise analysis and knowledge of the mode of action of the Lpo proteins is essential to be able to design the new antibiotics. In the master project a technique will be developed to analyze the mode of action of the Lpo proteins.

Effects of lipids and anti-cancer drugs on PKC
Master: Biomolecular, Biomedical, Biological Sciences or Systems Biology,  Supervisor: Joachim Goedhart

Protein kinase C (PKC) plays a key role in signal transduction cascades that involve phospholipid hydrolysis and has been implicated in many processes including proliferation, differentiation, carcinogenesis and apoptosis. The PKC family is divided in three classes; classical, novel and atypical. Both the classical and novel PKC isoforms have C1-domains that can bind an important signaling lipid; diacylglycerol (DAG). The C1-domains recruit PKC to membranes where DAG is formed, thereby activating its kinase activity. At this moment, several drugs that target C1 domains are currently studied as potential anti-cancer drugs in clinical trials. So far binding studies with PKC and isolated C1-domains have mainly been done in vitro. To obtain a better understanding of how the drugs and natural lipids act on PKC and the C1-domains we study this process in the relevant environment, i.e. the living cell. To this end, green fluorescent protein (GFP) is fused to these proteins and the constructs are expressed in cells. The (trans)location of GFP-tagged proteins is studied by fluorescence microscopy in real time with high spatial resolution. Cutting-edge microscopy methods are used to follow multiple proteins in a single cell by using spectrally different fluorescent proteins. This approach allows to study in detail the mechanism by which lipids and drugs bind to PKC in living cells, shedding light on the role of PKC in signaling and cancer.
Technical skills/methods: For this research a variety of techniques will be used including, molecular biology (cloning), eukaryotic cell culture and (advanced) fluorescence microscopy (FRET, FRAP, TIRF).
 

One for all and all for one – Fluorescent protein development for single-color multi-protein imaging
Master: Chemistry, Biomolecular, Biomedical, Biological Mark Hink  

Cloning of the gene encoding green fluorescent protein (GFP) from the jellyfish Aequorea victoria initiated a revolution in cell biology. GFP (and its color variants) can be used as a "lightbulb" to track proteins in cells, tissue and whole organisms. By fluorescence imaging we can distinguish up to four different color variants. Since we want to image molecular complexes, that consist of five to ten different proteins, only color discrimination is not sufficient. Therefore we want to develop fluorescent protein variants with different fluorescence lifetimes. To develop these variants we use site-directed random mutagenesis and screening methods based on fluorescence. The aim of this project is to obtain lifetime variants of the red fluorescent proteins, characterize their properties and test their usefulness in fluorescence lifetime correlation spectroscopy.
Technical skills/methods: Dependent on the length of the project and the interest of the student, one has the possibility to work on several different disciplines, including molecular biological (cloning) and biochemical work (protein isolation and characterization), cell culturing, advanced fluorescence microscopy (FLIM, FCS and FRAP) and data analysis.

Localization studies.
Master: Biomolecular, Biomedical, Biological Supervisor: Tanneke den Blaauwen  

Brighten up your life with fluorescent proteins
Master: Biomolecular, Biomedical, Biological Supervisor: Joachim Goedhart

Cloning of the gene encoding green fluorescent protein (GFP) from the jellyfish Aequorea victoria initiated a revolution in cell biology. This is illustrated by the 2008 Nobel Prize in Chemistry which was awarded for the discovery, cloning and application of GFP. GFP (and its derivatives) can be used as a "lightbulb" to track proteins in cells, tissue and whole organisms. To improve the brightness of fluorescent proteins we use site-directed random mutagenesis and screening methods based on fluorescence. By applying novel screening methods we have recently identified the brightest cyan fluorescent protein (mTurquoise). The aim of this project is to identify brighter fluorescent proteins of other colors and to characterize their properties.
Technical skills/methods: For this research a variety of techniques will be used including, molecular biology (cloning), eukaryotic cell culture, biochemistry (protein isolation and characterization), and (advanced) fluorescence microscopy (FRET, FRAP, TIRF).

Cell wall synthesis regulation
Master: Life Sciences, Master: Chemistry, Physics, Biomolecular, Biomedical or Biological Sciences, Supervisor: Tanneke den Blaauwen  

Gram-negative bacteria are very difficult to combat because of their impermeable envelope. For this reason many antibiotics (e.g. vancomycin) that kill gram-positive bacteria are futile against gram-negative bacteria. The peptidoglycan layer that is sandwiched between the two membranes that surround the bacterial cytoplasm determines the shape of the bacterium and because of its strength it also protects the bacterium against osmotic pressure and mechanical damage. The well-known penicillins target the enzymes (Penicillin-binding proteins or PBPs) that synthesize the peptidoglycan layer. Recently two outer membrane proteins LpoA and LpoB have been discovered (Typas, A., et al (2010) Cell 143:1097) that regulate the activity of the PBPs. These proteins are promising targets for new antibiotics. Precise analysis and knowledge of the mode of action of the Lpo proteins is essential to be able to design the new antibiotics. In the master project a technique will be developed to analyze the mode of action of the Lpo proteins.

Visualize the invisible- Image correlation analysis of G protein signaling
Master: Chemistry, Physics, Biomolecular, Biomedical, Biological , Supervisor: Mark Hink

We want to quantify the concentration, mobility and degree of interaction of G-protein coupled receptors (GPCR) and their downstream signalling components in living cells. Thereto more sensitive and selective techniques are needed. Fluorescence fluctuation spectroscopy (FFS) methods are promising tools since they can detect fluorescently labeled molecules down to the single-molecule level, even in the living cell. The aim of this project is to use the available (high speed) confocal microscopes in combination with sensitive detectors in order to test, optimize and apply recently developed FFS techniques as RICS, STICS and kICS (Kolin et al., Cell Biochem. Biophys. 49: 141 (2007)) in order to study the fluorescently labeled proteins in living HeLa and HEK cells.
Technical skills/methods: Dependent on the length of the project and the interest of the student, one has the possibility to work on several different disciplines, including cell culturing, advanced fluorescence microscopy (FLIM, FCS and ICS) and data analysis (development).