Utilizing the polymeric platform of polypept(o)ides, this thesis describes synthesis and investigation of novel triblock copolymers to obtain carrier systems with multiple compartments for... Show moreUtilizing the polymeric platform of polypept(o)ides, this thesis describes synthesis and investigation of novel triblock copolymers to obtain carrier systems with multiple compartments for efficient siRNA delivery. Although the individual microstructure of nanoparticles differs depending on the polymeric building blocks, desired application and cargo, the final nanoparticles always combine a polysarcosine (pSar) shell with a polypeptide core, providing the ability of siRNA complexation by a polycationic segment. In addition, a third block enabled either covalent cross-linking, hydrophobic / π- π-stacking mediated stabilization or co-encapsulation of small hydrophobic drugs. Broadening the structural variety of such polypept(o)ides, a novel synthetic procedure was introduced to access AA'B- and ABC-type miktoarm star polymers.Investigations have been dedicated to the design of novel polymeric structures based on polypept(o)ides, to improve the delivery of siRNA by Polyion Complex Micelles (PICMs), provide access to different polymeric architectures, and to establish novel synthetic methods for the synthesis of these materials. Covering aspects from the synthesis of novel polymeric species up to advanced drug delivery strategies for siRNA in vivo, developments throughout this thesis extent the accessibility of the polypept(o)ide platform for nucleic acid delivery, highlight their potential in nanomedicine and further elaborate delivery strategies for next-generation nanomedical applications. Show less
Essen, M.F. van; Schlagwein, N.; Gijlswijk-Janssen, D.J. van; Anholts, J.D.H.; Eikmans, M.; Ruben, J.M.; ... ; COMBAT Consortium 2020
Gene silencing using small interfering ribonucleic acids (siRNA) is a powerful method to interfere with gene expression, allowing for the functional exploration of specific genes. siRNA... Show moreGene silencing using small interfering ribonucleic acids (siRNA) is a powerful method to interfere with gene expression, allowing for the functional exploration of specific genes. siRNA interference can be applied in both cell lines, as well as in primary, non-dividing cell types like dendritic cells. However, the efficacy in different cell types is variable and requires optimization. Here, we showed that the type of culture medium used during lipid-based siRNA-mediated transfection acts as a critical factor, affecting dendritic cell activation. Transfection of immature monocyte-derived dendritic cells in RPMI medium, but not in IMDM, showed increased transcript levels of pro-inflammatory cytokines. Moreover, the expression of co-stimulatory molecules was enhanced, thereby increasing the T cell stimulatory capacity. Our data demonstrates that the choice of medium should be critically examined as one of the variables while optimizing cell transfection. Show less
Heestermans, M.; Jong, A. de; Tilburg, S. van; Reitsma, P.H.; Versteeg, H.H.; Spronk, H.M.; Vlijmen, B.J.M. van 2019
Thrombosis is considered to be a multifactorial disease in which both genetic and acquired risk factors are involved to cause disease. Over the past years epidemiological studies have revealed a... Show moreThrombosis is considered to be a multifactorial disease in which both genetic and acquired risk factors are involved to cause disease. Over the past years epidemiological studies have revealed a number of acquired risk factors that increase the risk of venous thrombosis. Some acquired risk factors of venous thrombosis are associated with a hypercoagulable state, which may be to a certain extent dependent on the dysregulation of gene expression in the liver, as the liver is the major organ that produces coagulation factors. Coagulation gene transcription can be modulated at different levels through hepatic transcription factors, co-regulatory or intermediate proteins, however, the exact contribution of these modulators to coagulation gene transcription is largely unknown. We aimed to study the mechanisms by which hepatic coagulation gene transcription is regulated, in order to increase our understanding of how thrombotic risks conditions coincide with hypercoagulable state. RNA interference (via synthetic small interfering RNA; siRNA) was used as a tool to study genes involved in coagulation and coagulation control in mice. Studies described in this dissertation may contribute to a better understanding of which genes are involved in coagulation (control) and how thrombotic risk factors result in a hypercoagulable state Show less
The results in this thesis showed for the first time doublecortin-like (DCL)-specific expression in the adult mouse brain. Besides the expected regions with the capacity to generate new neurons ... Show moreThe results in this thesis showed for the first time doublecortin-like (DCL)-specific expression in the adult mouse brain. Besides the expected regions with the capacity to generate new neurons (hippocampus and olfactory forebrain), DCL expression was found in three novel brain areas namely hypothalamic tanycytes, suprachiasmatic nucleus and Islands of Calleja. A state of the art conditional shRNA expressing mouse model was used to target DCL mRNA. The analysis of these DCL knockdown animals using qPCR and Western blot revealed strong reduction of DCL protein expression. Subsequent stereological analysis using BrdU and several stem cell and neuronal markers revealed increased progenitor proliferation, but impaired neurogenesis in the hippocampus. This impaired neurogenesis was associated, however, with an apparent normal spatial and contextual fear memory formation in circular hole board and in a contextual fear conditioning paradigm. Therefore, DCL-regulated adult neurogenesis seems not crucial for hippocampus-dependent learning. However, more subtle functions like pattern separation and context distinction might be regulated by DCL. DCL knockdown also increased D2 activity within the hypothalamus. Altogether, the DCL-KD mouse seems a good working model to study adult neurogenesis and the role of DCL in this process. Show less
Formation of the epidermal barrier is a complex process involving the tight regulation of the expression of structural proteins, enzymes and cytokines, implementing different signaling pathways... Show moreFormation of the epidermal barrier is a complex process involving the tight regulation of the expression of structural proteins, enzymes and cytokines, implementing different signaling pathways depending on the differentiation stage of KC. Alterations of skin barrier formation occur as consequence of many skin diseases but can also be the primary event leading to skin diseases. Organotypic skin models have greatly contributed to delineate molecular mechanisms underlying the epidermal differentiation process. In this thesis we have adapted a human organotypic skin model to knock-down individual genes and study their impact on the differentiation of epidermal KC in vitro. Using this model we have provided new insights in the formation of the skin barrier of human skin. Show less
