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|Title: ||The role of TAK1 in NFkB signaling in macrophages|
|Authors: ||HEIJNEN, Bart|
|Advisors: ||CURFS, D.M.J.|
|Issue Date: ||2007|
|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|
|Type: ||Theses and Dissertations|
|Appears in Collections: ||Master theses|
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