Isolated singularities, smooth orders and Auslander regularity

In this note we prove that a smooth order satisfying the reverse geometric engineering of singularities conditions in stringtheory (in any compactifying dimension) is Auslander regular. Moreover, we classify the etale local structure of smooth orders over an isolated central singularity.Comment: Section added. Comm. Alg. (to appear

Overcoming intracellular barriers in non-viral gene delivery : chromatin targeting, messenger RNA and non-coding DNA

In principle, gene therapy could increase or restore the expression of virtually any protein in a cell. This in consequence could bring a cure for various inherited or acquired genetic diseases. In spite of years of extensive research, gene delivery by non-viral carriers has not met with these expectations. None of numerous polymeric and lipidic nucleic acid carriers has received approval from the Food and Drug Administration (FDA) and the European Medicines Agency (EMA), which indicates that existing carriers do not possess suitable properties to ensure safe and effective in vivo delivery of nucleic acids. One of the reasons is the low frequency at which plasmid DNA (pDNA) reaches its target, the nucleus. This is mostly due to the presence of multiple extracellular and intracellular barriers which have to be overcome before pDNA can be transcribed. For example, when particles carrying pDNA are administered systemically, they should be stable in the blood stream and be able to reach the target organ. Moreover, these particles should evade clearance by the immune system. Once the particles reach the organ of interest, they should be taken up only by desired cell subpopulations. The internalized particles should then timely escape the endosomal compartment. pDNA in the cytosol should be protected against degradation by nucleases and should be transported toward the nucleus. The nuclear envelope (NE) is one of the most difficult intracellular barriers to overcome. In non-dividing cells the only way for pDNA to enter the nucleus is via the nuclear pore complexes (NPCs). This process is very inefficient due to the size limit of the pores. In dividing cells, the NE is timely disassembled during mitosis allowing pDNA enclosure in the newly formed daughter nuclei. To ensure transfection, intranuclearly located pDNA should be able to reach transcription-active sites. The research presented in this thesis focused on overcoming the intracellular barriers that limit non-viral delivery of pDNA. The first chapter provides a detailed overview on intracellular partitioning of cell organelles, foreign macromolecules and nanoparticles during mitosis. Moreover, it highlights how viruses take advantage of cell division to transfer or segregate their genetic information into the daughter nuclei. It also proposes strategies to enhance non-viral gene delivery in dividing cells. Chapter 2 reports on one of these strategies. Specifically, the nuclear inclusion of fluorescent polystyrene nanospheres and pDNA-containing nanoparticles modified with chromatin-binding peptides was studied. Initially, the nuclear inclusion was followed in the cell-free Xenopus nuclear envelope reassembly (XNER) model in which artificial nuclei are formed. The same particles were also injected and followed in living cells. In chapter 3, the effect of non-coding DNA co-delivered with coding pDNA on transfection efficiency mediated by lipoplexes was studied. Non-coding pDNA in supercoiled and linear form as well as non-coding salmon DNA were evaluated for their influence on transfection efficiency. It was further investigated at which level(s) of the transfection pathway the presence of non-coding DNA becomes essential. In chapter 4, polycationic amphiphilic cyclodextrins (paCDs) were assessed as nucleic acids carriers. mRNA-based transfection was presented as an alternative to pDNAbased transfection in slowly dividing cells. The specific uptake of complexes of galactosylated paCDs and mRNA via asialoglycoprotein receptors (ASGPr) on the surface of hepatocytes was also evaluated

Nuclear inclusion of nontargeted and chromatin-targeted polystyrene beads and plasmid DNA containing nanoparticles

The nuclear membrane is one of the major cellular barriers in the delivery of plasmid DNA (pDNA). Cell division has a positive influence on the expression efficiency since, at the end of mitosis, pDNA or pDNA containing complexes near the chromatin are probably included by a random process in the nuclei of the daughter cells. However, very little is known about the nuclear inclusion of nanoparticles during cell division. Using the Xenopus nuclear envelope reassembly (XNER) assay, we found that the nuclear enclosure of nanoparticles was dependent on size (with 100 and 200 nm particles being better included than the 500 nm ones) and charge (with positively charged particles being better included than negatively charged cr polyethyleneglycolated (PEGylated) ones) of the beads. Also, coupling chromatin-targeting peptides to the polystyrene beads or pDNA complexes improved their inclusion by 2- to 3-fold. Upon microinjection in living HeLa cells, however, nanoparticles were never observed in the nuclei of cells postdivision but accumulated in a specific perinuclear region, which was identified as the lysosomal compartment. This indicates that nanoparticles can end up in the lysosomes even when they were not delivered through endocytosis. To elucidate if the chromatin binding peptides also have potential in living cells, this additional barrier first has to be tackled, since it prevents free particles from being present near the chromatin at the moment of cell division

Switched predictive control design for optimal wet-clutch engagement

Modeling of hydraulic clutch transmissions is far from straightforward due to their nonlinear hybrid dynamics, i.e. switching between three dynamic phases. In this paper we identify a local linear model only for the constrained first phase, based on which a predictive controller is used to track a suitable engagement signal. The robustness of this controller in the latter two phases is guaranteed by making the constraints inactive and pre-tuning the control parameters based on its closed loop formulation and applying robust stability theorem. This controller is then implemented in real-time on a wet-clutch test setup and is shown to achieve optimal engagement