Student projects - Joseph Lorent

Cold atmospheric plasma (CAP)

Due to increasing numbers of antibiotic resistances, new treatments for bacterial infections are indispensable. Cold atmospheric plasma (CAP) has shown to be effective against resistant topical bacterial infections in first clinical trials and its main activities are related to the cell membrane. The two main question which regroup all the student projects are how does CAP and reactive oxygen and nitrogen species (RONS) affect cellular membranes and how a selective effect on certain membrane types is achieved. In these projects, students will learn how biophysical membrane properties are implicated in cellular homeostasis, and how these are modulated in an oxidative environment. These effects are not only important during CAP treatment but also involved in physiological oxidative processes such as the respiratory burst in immune cells, neurotoxicity and aging.

Decrease in E.coli membrane packing (GP) upon CAP treatment
Figure 1 Decrease in E.coli membrane packing (GP) upon CAP treatment by spectral confocal microscopy

Project 1: Effects of CAP on bacterial membranes and homeostasis (in collaboration with Eefjan Breukink)

Students are going to study the effects of CAP on membrane components such as lipids and proteins, the effects on biophysical membrane properties including permeability and membrane fluidity (Figure 1), and the consequences for bacterial cell homeostasis.

Potassium release from LUV upon CAP treatment
Figure 2 Potassium release from LUV upon CAP treatment

Project 2: What are the detailed mechanisms on how CAP and RONS permeabilize membranes?

As many bactericidal substances, CAP is able to permeabilize membranes. We therefore want to investigate how permeabilization is generated by CAP and RONS and how it could be modulated by changing plasma or membrane properties.

Oxidation of lipids and proteins by RONS
Figure 3 Oxidation of lipids and proteins by RONS

Project 3: Interplay of membrane proteins and lipids during oxidation by CAP

Membrane proteins constitute around half of the mass of cell membranes and occupy 30-50% of the membrane surface. It has been shown that their interaction with membrane lipids is of major importance in oxidative processes upon aging, neurotoxicity and cell death. The conclusions of this study could lay a foundation to the understanding of how proteins and lipids interact in an oxidative membrane environment.

Spectral imaging with generalized polarization (GP-scale) or membrane packing of membrane domains in Giant unilamellar vesicles
Figure 4 Spectral imaging with generalized polarization (GP-scale) or membrane packing of membrane domains in Giant unilamellar vesicles

Project 4: Effect of lipid and protein oxidation on lateral membrane domains?

Lateral lipid domains such as membrane rafts are formed because of collective lipid interactions of lipids. They host proteins and constitute signaling platforms important in immune cell activation, cell death and more. Lipid oxidation has shown to enhance raft formation in Giant unilamellar vesicles (GUVs) and could be a potential driver of raft formation in cells.

Project 5: Establishment of a protein oxidation map in different bacterial and mammalian membranes (bioinformatics approach in combination with another project)

In this study, students will use a bioinformatic approach to establish an oxidative map of membrane proteins in different species.

Techniques

The main techniques which are used in these projects can also be found on the (view profile page, Figure 3). Briefly, students will gain a profound understanding of membrane models and how to examine biophysical membrane properties in simple artificial and more biological complex membranes such as bacteria and eukaryotic cells. These methods are mainly based on fluorescence spectroscopy and enhanced microscopy. Depending on the project, students will also use qualitative and quantitative analytical methods such as LC-MS, GC, HP-TLC or FTIR. The project involving proteins will also require the use of circular dichroism, SDS gels and western blots. Projects which involve microscopy will include enhanced image treatment and statistical analysis of images via Matlab®. The bioinformatics project will require the use of programming languages such as python and R.