This thesis describes the development of microRNA-based gene therapies for ALS andSCA3. Other aspects of a successful gene therapy including the administration routes toreach the target organs and... Show moreThis thesis describes the development of microRNA-based gene therapies for ALS andSCA3. Other aspects of a successful gene therapy including the administration routes toreach the target organs and regulation of the transgene expression were also investigated.Due to the permanent nature of gene therapy, a method to modulate the transgeneexpression is desirable. Show less
The interplay between nidoviruses and the infected host cell was investigated. Arterivirus RNA-synthesising activity was shown to depend on intact membranes and on a cytosolic host protein which... Show moreThe interplay between nidoviruses and the infected host cell was investigated. Arterivirus RNA-synthesising activity was shown to depend on intact membranes and on a cytosolic host protein which does not cosediment with the RTC. Furthermore, the immunosuppressant drug cyclosporin A (CsA) blocks replication of EAV and the swine arterivirus PRRSV in cell culture. Cyclophilin A appears to be an important host factor for EAV replication. CsA may be a nidovirus-wide inhibitor of replication since this compound also blocked replication of the coronaviruses SARS-CoV, HCoV-229E, and MHV. We further described a kinase siRNA library screen that identified ninety antiviral and forty proviral hits and signalling pathways involved in the SARS-CoV replicative cycle. PKR (antiviral) and COPB2 (proviral) were validated in follow-up experiments. We also investigated MERS-CoV replication characteristics and we described an assay t o screen for compounds that block MERS-CoV infection. CsA and pegylated IFN-_ (PEG-IFN) significantly inhibited infection, and MERS-CoV was shown to be much more sensitive to PEG-IFN treatment than SARS-CoV, an observation that may have implications for the treatment of MERS-CoV infection. The data presented in this thesis might contribute to better understand virus replication and hopefully provide additional starting points for the development of antiviral strategies. Show less
Puy, L. du; Beqqali, A.; Helena, T.V.T.; Monshouwer-Kloots, J.; Passier, R.; Haagsman, H.P.; Roelen, B.A.J. 2012
Major Histocompatibility Complex class II (MHC-II) antigen presentation is involved in the regulation of immune responses against infections and tumours and strongly linked to autoimmune diseases.... Show moreMajor Histocompatibility Complex class II (MHC-II) antigen presentation is involved in the regulation of immune responses against infections and tumours and strongly linked to autoimmune diseases. To better understand this process we performed a genome-wide RNAi screen. The effect of downregulation of each human gene on MHC class II expression and antigen loading was analyzed. Genes affecting MHC-II antigen presentation were tested for their involvement in MHC-II transcription and transport. Some genes might function in repositioning MHC-II towards the plasmamembrane upon Dendritic Cell maturation. This was confirmed by silencing these genes in immature Dendritic Cells. The novel GTPase ARF7, ARF7EP and MYO1E play a central role in this transport process. MHC-II antigen loading on intraluminal vesicles of the MHC-II compartment is facilitated by chaperone DM. In a confocal FRET study we investigated whether microdomain-forming tetraspanins CD63 and CD82, interacting with MHC-II and DM, could facilitate the MHC-II/DM interaction. MHC-II and DM preferably interacted with CD63 on the intraluminal vesicles, likely facilitating the MHC-II/DM interaction and antigen loading. MHC-II favoured interactions with CD82 on the limiting- and plasmamembrane suggesting a sorting role for CD82. This knowledge will contribute to development of new methodologies to influence MHC class II presentation in patients. Show less
