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Selected Publication:

Type of publication: Master Thesis
Type of document:

Year: 2014

Authors: Hochreiter, Bernhard

Title: Studies on the composition and interaction of inflammatory signaling molecules with a focus on high-end microscopy.

Other title: Studien zur Zusammensetzung und Interaktion von Signalmolekülen entzündlicher Prozesse mit einem Fokus auf High-end Mikroskopie

Source: Master Thesis, Vet. Med. Univ. Wien, pp. 82.


Advisor(s):

Pohl Elena

Reviewer(s):
Egerbacher Monika

Vetmed Research Units:
Institute of Physiology, Pathohysiology and Biophysics, Unit of Physiology and Biophysics


Graduation date: 30.04.15


Abstract:
The nuclear factor ‘kappa-light-chain-enhancer' of activated B-cells (NF-κB) -pathway is one of the most important pathways during innate immune reactions and has also been linked to many other pathways within the body. One of its main parts, the Inhibitor of κ-B kinase (IKK) -complex, is composed of three subunits named IKKα, IKKβ and NEMO. The single IKK subunits exhibit many functions beyond the NF-κB pathway and have been linked to numerous biological functions. Although the interactions within the pathway are well studied, not much is known about IKK itself, including most of its conformation and dynamics. One of the main aims of this thesis was to investigate the dynamic changes that happen within the IKK complex during signalling, initiated by Tumor necrosis factor alpha (TNFα). To make IKK subunits visible on endogenous levels, we tested a new approach by combining RNA interference (RNAi) and an overexpression model with each other on one single vector. The single vector was not accomplished, but it was shown that RNAi and overexpression can work perfectly at the same time without interfering with each other. Determination of the dynamics of the IKK complex was done by measuring the behaviour of the fluorescence resonance energy transfer (FRET) between proteins of the IKK complex that were tagged with fluorescent proteins. It was clearly shown that dissociation between all subunits of the IKK complex happened after activation with TNFα. This was also confirmed by a co-immunoprecipitation experiment of IKKβ with IKKα, showing similar results. Further, part of this thesis was focussed on the improvement of FRET measurements with the 3-Filter method. By using different transfection ratios and relating them to a construct with a stable 1:1 ratio, a titration curve could be plotted that was used to determine a stable maximal FRET efficiency at donor saturation. This curve also contained information about the ratio of saturation and protein affinity. Finally, scouting experiments are presented, in which FRET and FRAP microscopy was combined to obtain measures of the dynamics of protein interaction and half-lives of protein complexes. This newly developed method assesses dissociation and association of proteins by following the recovery curve of FRET after bleaching.


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