Project works

(1) Interaction of pulmonary epithelial cells with a pathogenic fungus (I)
a.    After preliminary work with human pulmonary epithelial cells in projects carried out last season, a murine pulmonary epithelial cell will be used in this project to investigate the interaction with a human/animal pathogenic fungus. Since the number of available antibodies is significantly higher than in human cells, the interaction can be characterised much more precisely. The system is completed by studies of the influence of murine macrophages on epithelial cells. The following investigation methods are available for this project and offer a comprehensive insight into laboratory techniques
i.    Cell Culture
ii.    TEER (measurement of transepithelial cell resistance)
iii.    FITC-Dextran measurement (measurement of the diffusion rate through the epithelium by fluorescence photometry)
iv.    Flow cytometric analyses (use of a modern multi-colour flow cytometer to determine molecular alterations of selected marker proteins of epithelial cells)
b.    The data obtained are recorded in a laboratory book and then evaluated with software support. The necessary programs are available and after a detailed instruction the independent evaluation of the data takes place.
c.    The work is carried out in a block of 96 hours throughout the day. The work concludes with a detailed report on the project and includes an introduction to the topic, a description of the applied methods, the results achieved and a discussion of the results. Furthermore, it is planned to produce a poster which will be presented and defended at the next dies academicus.

(2) Interaction of pulmonary epithelial cells with a pathogenic fungus (IIa)
a.    In this project, the cellular interactions of cells in the lung will be characterized. Different techniques will be used for this purpose. First, lungs of non-infected and infected mice will be removed, filled with agarose, embedded and cut by cryotome. The sections will be examined unfixed to improve antibody binding. The antibodies will be used to identify cells and their position in the lung. These investigations provide information about possible cellular interactions of immune cells with epithelial cells in normal and infection states. The following techniques will be used for the investigations
i.    Section of mice under sterile conditions and treatment of the organ to be examined
ii.    Embedding the specimens and cutting with a cryotome
iii.    Immunofluorescence staining of samples
iv.    Analysis of samples by laser scanning microscopy
b.    The data obtained are recorded in a laboratory book and then evaluated with software support. The necessary programs are available and after a detailed instruction the independent evaluation of the data takes place.
c.    The work is carried out in a block of 96 hours throughout the day. The work concludes with a detailed report on the project and includes an introduction to the topic, a description of the applied methods, the results achieved and a discussion of the results. Furthermore, it is planned to produce a poster which will be presented and defended at the next dies academicus.

(3) Interaction of pulmonary epithelial cells with a pathogenic fungus (IIb)
a.    Using a reporter system, the induction of allergic inflammation in murine lung tissue will be investigated. Lungs of so-called reporter mice, whose lung cells begin to glow green at the beginning of Th2 induction, will be removed and divided into 100-200 µm thick sections by means of a vibratome. These living lung sections are subsequently treated with various stimuli, including red luminescent pathogenic fungi. It will be investigated whether there are differences in the strength of the Th2 response to the fungus depending on the number of fungal cells used in the lung. The evaluation is done by confocal microscopy techniques. The following techniques are presented in this project:
i.    Cell culture
ii.    Sectioning of mice under sterile conditions and preparation of the lungs for making cuts
iii.    Preparation of vital pulmonary slices, so-called precision cut lung slices (PCLS)
iv.    In vitro infection and stimulation of lung tissue
v.    Analysis of samples by laser scanning microscopy
b.    The data obtained are recorded in a laboratory book and then evaluated with software support. The necessary programs are available and after a detailed instruction the independent evaluation of the data takes place.
c.    The work is carried out in a block of 96 hours throughout the day. The work concludes with a detailed report on the project and includes an introduction to the topic, a description of the applied methods, the results achieved and a discussion of the results. Furthermore, it is planned to produce a poster which will be presented and defended at the next dies academicus.

At the moment, please address your inquiries regarding project work to Dr. Nicole Schütze, as Dr. Eschke is on parental leave.

Dr. Protschka offers project work with a focus on "Molecular Genetic Methods in Current Research Projects".

(1) Establishment/optimization of RT-qPCR for the detection of gene expression at RNA level in murine / canine / porcine tissue   

Methods:

    Primer Generation with Primer Blast (NCBI)
    RNA Isolation
    DNAse digestion
    reverse transcription
    RT-qPCR
    Evaluation of the data with Excel/Graph Pad

(2) Establishment/optimization of immunohistological detection of protein expression in murine / canine / porcine tissue   

Methods:

    Preparation of tissue sections (cryo/paraffin)
    Comparison of different fixation methods
    antigen demasking
    Optimization of antibody concentration
    Signal amplification with Biotin/Streptavidin