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Application of high-content microscopy to investigate membrane trafficking

Lisauskas, Tautvydas

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Membrane trafficking is an abstraction describing material transport within different organelles of the cell. It is one of the areas of interest in molecular and cell biology. The practical interest for the investigation of membrane trafficking is lying in its relation to an increasing list of diseases, such as cancer. Identification of genes involved in membrane trafficking and their regulation within the cell are in particular focus of interest. Microscopy based observation of membrane trafficking events in the native or close to native context is the method of choice. It allows relating different stages of the whole process to specific cellular organelles with spatial and temporal resolution. It also has the advantage of better statistics over other population mean based methods, as results are concluded from the observation of a high number of individual cells. Despite the versatility of fluorescence microscopy, the application of any particular technique for cell biology requires more application layers such as fluorophores and labelling strategies, data acquisition and storage, digital image analysis, data interpretation in a biological context, and last but not least – the biological process of interest itself. Improvements of those application layers or even synergy of different microscopy techniques increases information content what can be extracted from a single experiment. Currently we call this approach as high-content microscopy. During the time of my studies I was working on improving some of application layers as well as creating stronger link between them. This thesis describes my experimental and data analysis work which was done to address some of the specific membrane trafficking related cases in context of high-content microscopy: a) The organization kinetics of the Golgi complex, which is the major membrane trafficking organelle, and the influence of over-expressed membrane proteins on it. Live cell fluorescence screening microscopy was employed to address this question. b) Statistical data analysis of a cargo protein secretion screen. Influence of specific RAB GTPases over-expression on collagen-I secretion was evaluated. c) The development of a comprehensive fluorescence microscopy based platform to investigate messenger RNA (mRNA) and micro RNA (miRNA) interactions in live cells.

Item Type: Dissertation
Supervisor: Wolfrum, Prof. Dr. Jürgen
Date of thesis defense: 31 January 2014
Date Deposited: 12 Feb 2014 07:36
Date: 2013
Faculties / Institutes: Service facilities > Bioquant
Subjects: 540 Chemistry and allied sciences
570 Life sciences
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