cellular mechanisms

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Imaging cellular mechanisms of presynaptic structural plasticity

Imaging cellular mechanisms of presynaptic structural plasticity

To investigate cellular mechanisms of axonal varicosity turnover, I tested the effect of acute protein synthesis and degradation blockade on the loss and gain of varicosities. My experiments reveal that turnover of axonal varicosities was independent from acute protein synthesis and degradation. As the rates of gain and loss remain unaltered during acute protein synthesis and degradation blockade, the data indicates that new varicosities can emerge without the generation of new proteins, and that axonal varicosities can disassembly without degrading proteins. Also, the relative fractions of the distinct turnover types remained unchanged, indicating that none of the required protein synthesis or degradation. The data infers that the constituents for axonal varicosities preexisted before their formation and that they can persist after their disassembly, implying a shared pool of presynaptic varicosity proteins. This hypothesis is substantiated by reports that single molecules and synaptic vesicles can be shared amongst synapses (Chi, Greengard et al. 2001; Darcy, Staras et al. 2006; Tsuriel, Geva et al. 2006). Alternatively, it would also be possible that presynaptic constituents directly recycle without contributing to a ‘buffering’ pool. However, the loss of an axonal varicosity was not always accompanied by the gain of another varicositiy in the same axon and vice versa, and varicosity turnover was not balanced within single axons, arguing against this hypothesis. Moreover, the presynaptic turnover rates were maintained during acute blockade of protein synthesis or degradation for one or four hours, suggesting that the available protein pool is large and stable enough to persist even when it is not replenished for longer periods. The hypothesis of a presynaptic protein pool leads to new hypotheses. For example, a shared pool of proteins may lead to competition amongst synapses for the same proteins - a similar mechanism has been reported on the postsynaptic side (Fonseca, Nägerl et al. 2004).
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Elucidation of the molecular and cellular mechanisms of FKBP51-selective inhibitors

Elucidation of the molecular and cellular mechanisms of FKBP51-selective inhibitors

It has to be noted, that high throughput screening assays for PROTACs, especially with respect to concentration dependence and time course of the degradation, are hardly available so far, but first steps have been made by genetically labeling the protein of interest with a luciferase fragment tag [58]. Taken together, PROTACs are a promising concept of drug development. Besides conventional small molecules and monoclonal antibodies, PROTACs exhibit an often unpredictable difference regarding selectivity, efficacy and applicability compared to its small molecule warhead. Although they are proven to be powerful tools in molecular and cellular biology, clinical administration is in its earliest stage of development. Just recently, the first phase I study started by Arvinas to test in vivo degradation of the androgen receptor by their substance ARV-110 in stage 4 prostate cancer patients [59]. Due to the fact, that FKBP51 related pathologies are connected to elevated expression levels [3], the model of FKBP51 as scaffolding protein and a number of FKBP51 interactions, that have been found to be ligand insensitive [25], the research of FKBP51 PROTACs is promising to elucidate the biological role of this protein.
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Cellular Mechanisms Regulating the Enzymatic Activities of cGMP-Producing Natriuretic Peptide Receptors : Studies in Mus musculus Cell Lines

Cellular Mechanisms Regulating the Enzymatic Activities of cGMP-Producing Natriuretic Peptide Receptors : Studies in Mus musculus Cell Lines

That prolonged exposure to ANP can induce GC-A desensitization has been well established in a variety of studies performed in different GC-A-transfected cell lines (Potter and Garbers, 1992; Koller et al., 1993; Potter and Garbers, 1994; Joubert et al., 2001; Bryan and Potter, 2002). In addition, homologous desensitization of GC-A has been identified in cultured vascular smooth muscle cells (Cahill et al., 1990; Yasunari et al., 1992). The present findings, elucidating GC-A desensitization in MA-10 Leydig cells, support that this kind of regulation represents a widespread phenomenon, presumably active in many cell types. Consistent with other investigations (Kato et al., 1991; Vieira et al., 2001; Fan et al., 2005), GC-A desensitization in MA-10 cells is not due to receptor endocytosis and/or degradation but seems to be entirely explained by a decreased receptor hormone- responsiveness. Receptor-mediated endocytosis is the cellular mechanism by which clearance (C) type receptors of natriuretic peptides exert their clearance function (Cohen et al., 1996).
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Hypoxic pulmonary vasoconstriction in intact mouse lungs: new insights into cellular mechanisms and therapeutic targets

Hypoxic pulmonary vasoconstriction in intact mouse lungs: new insights into cellular mechanisms and therapeutic targets

TRPV4 as a mechanosensitive ion channel with high expression in the pulmonary endothelium, our group studied the role of TRPV4 in hydrostatic lung edema formation. In a seri[r]

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Identification of cellular mechanisms interfering with the Helicobacter pylori cag type IV secretion system

Identification of cellular mechanisms interfering with the Helicobacter pylori cag type IV secretion system

The presence of the cagA gene as a representative of the cag PAI divides H. pylori species into two groups: Type I and Type II Helicobacter pylori. Type I strains encode a cagA gene, whereas Type II strains do not (Xiang Z et al., 1995) (Tummuru MKR et al., 1993). This classification gained its importance when the pathogenic power of CagA was discovered. Epidemiological data shows, that it is far more likely to isolate a Type I strain or Type I and II strains from a patient with gastric disorders due to H. pylori infections than Type II strains alone (Azuma T et al., 2004). Correspondingly, asymptomatic individuals with H. pylori infection are more likely to host Type II strains than Type I H. pylori (Xiang Z et al., 1995) (Covacci A et al., 1997). Additionally, CagA's interference with essential intracellular mechanisms of its target cells (see chapter IV.2. ) supports the idea of CagA as a main cause of gastric diseases.
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Proteotoxicity of polyglutamine expansion proteins: Cellular mechanisms and their modulation by molecular chaperones

Proteotoxicity of polyglutamine expansion proteins: Cellular mechanisms and their modulation by molecular chaperones

Discussion 101 Although a number of different cellular events are known to be associated with HD, the actual pathogenic mechanisms remain unclear. Transcriptional dysregulation is hypothesized to be a common theme in polyQ pathogenesis (Cha, 2000; Sakahira et al., 2002; Sugars and Rubinsztein, 2003). Such dysregulation might be mediated by several mechanisms. PolyQ proteins can directly interact with numerous transcription regulators, which have been found in polyQ inclusions. The expanded polyQ stretch in mutant Htt may result in sequestration of these proteins into perinuclear aggregates, leading to their functional depletion (Kazantsev et al., 1999). However, given the evidence for a protective role of inclusions in disease, toxicity might be exerted by soluble aggregate precursors via aberrant interaction with transcription factors, thereby rendering them non-functional and disrupting transcriptional pathways. Molecular chaperones might counteract these aberrant protein interactions, presumably by shielding hydrophobic surfaces. Finally, the association of aggregating polyQ proteins with the quality control machinery may itself contribute to cellular toxicity (Sherman and Goldberg, 2001; Sakahira et al., 2002). Misfolded proteins may sequester and/or non-productively engage components of the chaperone (and degradation) systems and consequently reduce their activity in the cell. The increased expression of chaperones frequently relieves toxicity associated with aggregation diseases.
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Gutenberg Open Science: Cellular mechanisms of antigen-specific immunotherapy of autoimmune disease

Gutenberg Open Science: Cellular mechanisms of antigen-specific immunotherapy of autoimmune disease

Several molecules have previously been suggested to mediate cell contact-dependent suppression by PMN-MDSCs. To investigate the molecules that may be involved in the suppressive effect of PMN-MDSCs, blocking antibodies targeting molecules expressed on the surface of PMN-MDSCs, namely Galectin-9, PD-L1, CD40, CD80 and CD86, or anti-IL-10R were added to the co-culture. In most of these conditions was the suppressive activity of PMN-MDSCs abrogated (Figure 4.35. A). The addition of anti-Galectin-9 or PD-L1 alone did not abrogate the suppressive activity of PMN-MDSCs while the co-blockade of both ligands restored CD4 + T cell proliferation indicating a synergistic effect of Galectin-9 and PD-L1 in CD4 + T cell suppression. IL-10 receptor signalling is not essential for the suppressive activity of PMN-MDSCs because in our in vitro suppression assay blockade of IL-10R signalling by an antibody (Figure 4.35 A) did not alleviate CD4 + T cell suppression. The addition of the blocking antibodies targeting the surface molecules CD40, CD80 and CD86 restored CD4 + T cell proliferation as well. Soluble factors may still be involved in mediating suppression of cells which are in close proximity. Therefore, the possible role of iNOS and arginase-1 in mediating the suppression of CD4 + T cell proliferation by the PMN-MDSCs was investigated further by the addition of an inhibitor of iNOS, (NG-monomethyl-L-arginine (L-NMMA) or the inhibitor of arginase-1, (S-(2-boronoethyl)-L-cysteine (BEC) to the suppression assay at 500 μM (Figure 4.35. B). The addition of the arginase-1 inhibitor BEC abrogated the suppressive effect of PMN-MDSCs indicating that PMN-MDSCs use arginase-1 rather than iNOS for their suppressive activity. More recently, other mechanisms of suppression deployed by MDSCs were discovered, including the production of the enzyme indoleamine 2,3-dioxygenase (IDO). However, the addition of 1-methyl analogue of tryptophan (1-MT) to block IDO in our
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Characterization of the molecular and cellular mechanisms in vomeronasal signal transduction in mice

Characterization of the molecular and cellular mechanisms in vomeronasal signal transduction in mice

100  territorial marking (Luo and Katz, 2004; Brennan, 2004). Moreover, the ability to recognize individuals based on their unique body odor plays an important role in mammalian social behavior (Brennan et al., 1990; Brennan and Keverne, 1997). The vomeronasal pathway forms a relatively direct route by which chemosensory stimuli mediate effects on endocrine status and / or stereotyped behavior. VSNs send their axons to glomeruli in the AOB where they innervate mitral and tufted (M/T) cells. M/T cells project on to the cortical-medial regions of the amygdala. The flow of information proceeds via the medial hypothalamus to the tuberoinfundibular dopaminergic neurons in the arcuate nucleus of the hypothalamus (Li et al., 1989, 1990, 1992). As sustained activity already occurs at the first level of signal transduction in the periphery, the prolonged action potential output to the AOB may result in changes in hormonal secretion. Single molecules or complex mixtures that shall induce an effect in endocrine status might trigger a prolonged discharge pattern and, thereby, induce second-order activity patterns that lack temporal precision (Luo and Katz, 2003). This unusual temporal pattern is then forwarded via the amygdala to the hypothalamus resulting in specific hormonal secretion patterns. One of the best characterized examples of such pheromonal effects is the selective pregnancy block (Bruce effect; Bruce, 1965) that is associated with the formation and maintenance of an olfactory recognition memory by the vomeronasal system (Halpern and Martinez-Marcos, 2003; Kelliher et al., 2006). A newly mated female mouse learns the individual identity of the male's urinary chemosignals during mating. Exposure to the odor of an unfamiliar male activates neuroendocrine mechanisms leading to pregnancy failure by means of the AOB, amygdala, and medial hypothalamus.A possible 'location' for this 'memory' could be the AOB. It has been reported that electrical stimulation of the AOB is effective in blocking pregnancy (Li et al., 1994). Synaptic plasticity in the AOB could be part of the anatomical substrate for the chemosensory memory underlying the Bruce effect. Unusual output pattern of VSNs induced by long-lasting stimulation shown in this study might lead to synaptic changes in the AOB.
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Investigation of cellular mechanisms of hippocampal LTP and LTD

Investigation of cellular mechanisms of hippocampal LTP and LTD

and favourably at a temperature of 4 o C to minimize cellular metabolism and to avoid irreversible intracellular phase changes. It is well known from studies investigating ischemic and/or hypoxic influences on brain function that ischemic episodes with a duration of longer than 3 min as well as glutamate receptor-dependent calcium release during preparation can result in an irreversible prevention of protein synthesis in nervous tissue (Erdogdu et al., 1993;Djuricic et al., 1994;Djuricic et al., 1995). Furthermore, to obtain these morphological and functional characteristics we use always a cleaned new razor blade for each preparation of not more than 3-4 slices from a single hippocampus from one animal to obtain hippocampal CA1-slices (dorsal part of the right hippocampus of males). When slices are taken out with proper care the responses, observed on stimulation are similar to those seen in intact animals. Slices were incubated within an interface chamber (Fig. 4) at 32°C (carbogenated incubation medium contained 124 mM NaCl, 4.9 mM KCl, 1.2 mM KH 2 PO 4 , 2.0 mM MgSO 4 , 2.0 mM CaCl 2 , 24.6mM NaHCO 3 ,
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Molecular and cellular mechanisms of vomeronasal signaling in mammals

Molecular and cellular mechanisms of vomeronasal signaling in mammals

4.2.1. Vomeronasal sensory neurons are activated by acidic solutions In every organism, one basic principle of cellular homeostasis is the regulation and maintenance of acid-base balance. However, several physiological and pathophysiological events induce deviations from physiological pH (Chesler, 1990; Chesler and Kaila, 1992; Cobbe and Poole-Wilson, 1980; Issberner et al., 1996; Kumar et al., 2007; Steen et al., 1995; Tombaugh and Sapolsky, 1993). Thus, proton-sensitivity has evolved as a critical property of different neuronal cell types to detect intra- and extracellular pH alterations. In the initial project phase, we demonstrated that mouse urine can create an acidic environment for urinary compounds important for vomeronasal signaling. To investigate if such acidic conditions play a role in vomeronasal chemosensory signaling, we first analyzed proton-sensitivity of VSNs using the whole-call patch-clamp technique. We found that acidic stimuli elicit two different response types (Fig. 3.9). On the one hand, we detected sustained and abundantly occurring responses (Fig. 3.9 A, B), which are discussed in the following sections. On the other hand, we recorded transient events elicited by acidic solutions (Fig. 3.9 C to E). These responses were only found in a subset of cells (26 %). Furthermore, they show fast activation kinetics and rapid inactivation during stimulus application. Thus, we conclude that these responses are mediated by another mechanism, which represents an additional possibility to detect extracellular acidification in the VNO. Given the observed kinetics, we speculate about a possible involvement of ASICs. However, this second mechanism of vomeronasal proton-sensitivity has to be examined in further studies.
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PEG-Shielded and EGF Receptor-Targeted DNA Polyplexes: Cellular Mechanisms

PEG-Shielded and EGF Receptor-Targeted DNA Polyplexes: Cellular Mechanisms

The nuclear membrane presents a major barrier to the entry of free and polyplex- associated DNA from the cytoplasm (68). In several studies, less than 0.1 % of plasmid DNA which had been injected into the cytoplasm was found to be transcribed (69). In general, there are two paths to nuclear entry: one through the “nuclear pore complex” (NPC) and one via nuclear disassembly that occurs during cell division. Entry through the NPC may be passive or active. Passive diffusion through the NPC is determined mainly by the size of the molecule: DNA of up to 300 bp and DNA nanoparticles up to a diameter of 25 nm were found to traverse the NPC (70), since their small size permitted nuclear uptake across the 25 nm nuclear membrane pore. In polyplex-mediated gene delivery, the major path of nuclear entry seems to be via nuclear disassembly during mitosis since dividing cells yield much higher transfection efficiencies than postmitotic cells. For lipoplex- and polyplex-based transfections, highest reporter gene expression levels have been recorded for cells in the G2/M phase with a 30- to 500-fold increase as compared to G1 cells (71). Interestingly, polymer-mediated transfection using linear PEI was far less dependent on cell cycle compared to branched PEI (72). In order to enhance nuclear uptake, signal-mediated active transport mechanisms have been explored for trafficking vectors to the nucleus. Covalent coupling of a single nuclear localization signal (NLS) peptide to a CMV-Luciferase gene led to an up to 1000-fold enhancement in transfection efficiency presumably attributed to improved nuclear translocation by the nuclear import machinery (73).
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Cellular Dynamics of Network Memory 

Cellular Dynamics of Network Memory 

Although the reasons for this behavior of the model at a fine temporal resolution were unclear, the phenomenon obliged us to take a closer look at the discharge of real - inferotemporal - neu­ rons in the visual memory task. By analyzing spike trains at a fine temporal resolution, as we had not done previously in our experiments, we discovered similar rapid changes between alternating fre­ quencies as those that the model exhibited. We began to think that those changes were inherent in recurrent networks and resulted precisely from their re-entry property. We found literature sup­ port for that way of thinking. Cowan (1971) was one of the first to show that recurrent networks of nonlinear units, like ours, will tend to drift toward certain frequencies called fixed-point “attractors.“ An unresolved question is whether there are any physiological or anatomical constraints, in­ stead of or in addition to recurrence, that could account for what has been called “attractor dy­ namics“ in cortical networks actively retaining in­ formation. One possibility is that attractor dy­ namics is at least in part determined by intrinsic cellular mechanisms of cortical cells that cannot
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Changes in Solvolysis Mechanisms

Changes in Solvolysis Mechanisms

Chapter 2: Direct Observation of the Ionization Step in Solvolysis Reactions: Electrophilicity versus Electrofugality of Carbocations.. Table 2.2..[r]

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Mechanisms of change

Mechanisms of change

Morison’s case study of ‘Gunfire at Sea’ shows (at least) three mechanisms of change at work: while Scott’s discovery favoured the prepared mind and happened to take place in a conducive environment (serendipity), it was Sims’ goal orientation (teleology) combined with his determination being in contradiction to the established structure (dialectics) leading to a major change in the field of naval gunnery. The case shows that structures reinforce conservative forces and are slow to change. Vested in- terests oppose even promising innovations especially if these are associated with far- reaching consequences for the overall structure. In parts the case study suggests that the implementation of continuous aim-firing was a cumbersome if in the end success- ful campaign of one individual against the establishment. But as a matter of fact, the campaign only was successful because the structures contained conservative forces as well as progressive forces aiming at overcoming outdated solutions. Such forces were the technological impetus of industrialization arriving in the US navy, the strong posi- tion of officers allowing them to pursue their projects and finally the possibility to gain support beyond the established chain of command.
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Strategically Simple Mechanisms

Strategically Simple Mechanisms

simple mechanisms may be more attractive to a mechanism designer concerned with fairness, efficiency, or revenue, than dominant strategy mechanisms. To illustrate our idea, it is best to consider an example. Suppose that a mechanism designer wants to determine the terms of trade between two agents, a seller and a buyer. It is known from Hagerty and Rogerson [ 19 ] that the only dominant strategy mechanisms that are ex post budget balanced and individually rational are posted price mechanisms. In a posted price mechanism, the designer chooses a (possibly random) price, without taking into account any of the agents’ private information, and then agents decide whether to agree or not to agree to trade at this price. Trade comes about only when both agents agree. Obviously, this is a rather unappealing mechanism for a welfare maximizing mechanism designer.
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Cellular automata with sparse communication

Cellular automata with sparse communication

Two of the common techniques to show undecidability results are reduc- tions of Post’s Correspondence Problem or reductions of the emptiness and finiteness problem on Turing machines using the set of valid computations. Both techniques have been used successfully to obtain results for variants of cellular automata [10–12, 14]. Taking a closer look at these known techniques, it is not clear yet whether they can be applied to MC(1)-OCAs. Here, we first show that emptiness is undecidable for real-time MC(1)-OCAs by reduction of Hilbert’s tenth problem which is known to be undecidable. The problem is to decide whether a given polynomial p(x 1 , . . . , x n ) with integer coefficients
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Cellular reactions to patterned biointerfaces

Cellular reactions to patterned biointerfaces

  High  water  content  hydrogels  are  an  attractive  material  for  developing  artificial  biomimetic  ECM as they can meet a range of architectural characteristics and mechanics relevant for many  soft  tissues  [5].  Hydrogels  are  water  swellable,  yet  water  insoluble,  crosslinked  networks  that  provide  the  embedded  cells  with  a  highly  hydrated  environment  permeable  for  oxygen,  nutrients and cellular wastes [6‐8]. Hydrogel matrices for in vitro applications include a variety  of  naturally  derived  proteins  and  polysaccharides  (e.g.  collagen,  fibrin),  as  well  as  synthetic  polymers  (e.g.  poly(ethylene  glycol)  (PEG),  polylactide,  polyacrylamide)  (for  review  see  references [9‐12] and Chapter 3). While natural materials have an inherent bioactivity and are  commonly  degradable  by  cellular  enzymes,  synthetic  materials  also  have  potential  for  in  vivo  applications  as  their  properties  can  be  readily  tailored  with  specific,  desirable  biochemical  or  mechanical  properties.  Many  synthetic  hydrogels  are  formed  under  mild,  cytocompatible  conditions  and  can  be  modified  to  display  specific  cell  adhesive  ligands  or  degradation  sites.  Moreover,  variations  in  the  synthetic  gels’  compliance  can  be  achieved  by  adjusting  the  crosslinking  density  [9,  10].  Hydrogels  based  on  PEG  in  particular  have  attracted  interest  as  synthetic  ECM  analogs  in  different  tissue  engineering  applications  and  have  been  widely  explored as substrates for cell encapsulation. Various types of cells, such as osteoblasts, smooth  muscle  cells  and  chondrocytes  were  shown  to  survive,  grow  and  proliferate  in  differently  modified PEG matrices [11‐14].  
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On Pressure-Actuated Cellular Structures

On Pressure-Actuated Cellular Structures

For the sliced construction of a PACS cell body, a series of panel cuttings is produced in a laser cutting process with a CO2 cutting system from Eurolaser GmbH. The cross section of the cellular structure is therefore cut out of PA12 sheets with a thickness of 10 𝑚𝑚. Due to the divergence of the laser beam for increasing distance from its focal point, an hourglass- or cone-shaped cut contour results. This effect is strengthened by the refraction of the beam in the polymeric material. A minimum wall thickness of 𝑡 𝐻,𝑚𝑖𝑛 = 1.0 𝑚𝑚 for a sheet thickness of 10 𝑚𝑚 is identified. For a decrease of sheet thickness to 5 𝑚𝑚, the accuracy of this process is sufficient to manufacture wall thicknesses of about 0.5 𝑚𝑚. With this, the laser cutting is outnumbered by the water cutting process and thus rejected. For the demand for quantity manufacturing, injection moulding is investigated in theoretical manner. The company RICONE GmbH is commissioned to perform feasibility studies about the manufacturability of a double row PACS in an injection moulding process. Simulations showed that the mould cavity can be filled completely with the recommended material DuPont Hytrel 7246. Draft angles normally lie above the minimum of 1°. For a depth of 𝑑 = 500 𝑚𝑚, this limit value would result in a thickness reduction of ∆𝑡 = 8.7 𝑚𝑚 for hinges and cell sides. It can be seen that such a boundary condition is not compatible with the assumption of an approximately constant hinge thickness. High demoulding forces allow avoiding draft angles for limited structural depths but increase the risk for production defects. Further, unavoidable casting seams reduce the strength of flexure hinges substantially. For this degradation of performance, a process- and material-dependent range of 10 % to 90 % is given by RICONE GmbH. Shrinkage of 1.6 % can be expected for DuPont Hytrel 7246 [183]. Extensive efforts regarding the tool design and the determination of process parameters may lead to a successful implementation, but are not economically viable at the present state of concept.
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Cellular Automata on Group Sets

Cellular Automata on Group Sets

The present dissertation studies cellular automata whose cell spaces are not as usual the integers or groups but merely sets that are acted trans- itively upon by groups. These cell spaces can be continuous like the real numbers acted upon by translations or the sphere acted upon by rotations, or discrete like vertex-transitive graphs acted upon by graph automorphisms or uniform tilings of the hyperbolic plane acted upon by tiling-respecting bijections. As usual all cells have a state which they change synchronously in discrete time steps depending on the states of neighbouring cells as described by a local transition function. This global behaviour is represented by the global transition function, which maps global states of the automaton, so-called global configurations, to such.
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Bystander Effects in the Cellular Radiation Response

Bystander Effects in the Cellular Radiation Response

To analyze influences of secreted soluble factors, cells were irradiated with X-rays (200 kV) and incubated with fresh culture medium for 24 h. The conditioned medium was then transferred onto non-irradiated cells and incubated for 24 h (bystander treatment). Bystander responses concerning cellular survival were determined with the colony forming ability test. NF- κB activation and DNA double strand break (DSB) response were visualized by immunofluorescence staining (p65, γH2AX).

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