Document Server@UHasselt >
Education >
School for Life Sciences >
Master theses >

Please use this identifier to cite or link to this item: http://hdl.handle.net/1942/1927

Title: The role of TAK1 in NFkB signaling in macrophages
Authors: HEIJNEN, Bart
Advisors: CURFS, D.M.J.
Issue Date: 2007
Publisher: tUL
Abstract: Macrophages are white blood cells that play an important role in the immune system. They are characterized by their ability to phagocytize pathogens and other foreign material. Macrophages are recruited to infected tissues, where they produce immunological substances like cytokines and transcription factors to introduce an inflammatory response. One of the pathways by which macrophages activate the transcription of cytokines is the NF B signaling pathway. The NF B signaling pathway can be activated by several external stimuli, like LPS. Transforming growth factor activated kinase 1, TAK1, is one of the proteins involved in this pathway and regulates the activation of NF B by activating the IKK complex. Activation of this complex leads to degradation of the I B, which inhibits NF B from going into the nucleus. The freed NF B can now translocate to the nucleus for transcription of several genes like IL10, TNF- , IL6 and IL12. These have all an effect on the inflammatory response. A second pathway that is influenced by TAK1, also known as MAP3K7, is the MAP kinase pathway and in particular the JNK and p38 kinase pathway. Activation of these pathways show pro-inflammatory effects. In this project the emphasis lies on the role of TAK1 in NF B signaling in bone marrow derived macrophages (BMMs). There are several methods for investigating gene expression: overexpression, knockout and silencing by RNA interference of the gene. Because TAK1 knockout is embryonic lethal, RNAi will be used in this project for knocking down the TAK1 gene. During this technique double stranded RNA is inserted into cells and is cleaved by the enzyme Dicer into small interfering RNA, siRNA. This siRNA can degrade the mRNA from the gene of interest. The first goal of this project was to set up and optimize a technique for introducing TAK1 knockdown in BMMs by siRNA. In search of the optimal protocol for performing this technique, the different stadia of the RNAi protocol were observed and optimized. Transfection efficiency, the amount of siRNA added, the volume of transfection reagens used, the incubation period, are all factors influencing the RNAi protocol. This part lead to a protocol which is able to introduce a 35% knockdown of TAK1, but further optimization has still to occur. The second goal was to determine if the created knockdown had actually a biological effect. The applied knockdown seemed to introduce a more than 55% reduction in IL10 and around 30% reduction for TNF- . IL6 and IL12 remained unchanged, but a biological effect has already been shown. With the developed technique the role of TAK1 in the NF B signaling in BMMs can be further investigated.
Notes: Master in de Biomedische wetenschappen - Klinische en moleculaire wetenschappen
URI: http://hdl.handle.net/1942/1927
Category: T2
Type: Theses and Dissertations
Appears in Collections: Master theses

Files in This Item:

Description SizeFormat
N/A3.19 MBAdobe PDF

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.