Der neuronale Zelluntergang bei einer Vielzahl von Krankheiten des ZNS, wie z.B. Morbus Alzheimer (AD) und Temporallappenepilepsie (TLE), wird mit oxidativem Stress sowie Fehlfunktionen von Kaliumkanälen in Verbindung gebracht. In dieser Studie soll die selektive neuronale Sensitivität auf oxidativen Stress durch die Messung der oxidativen Modulation von Kaliumströmen untersucht werden. Dabei werden sternförmige Neuronen der zweiten Schicht des entorhinalen Kortex (EC) (bei AD bereits früh geschädigt) mit pyramidalen Neuronen der dritten Schicht des EC (früh geschädigt bei TLE) sowie hippocampalen pyramidalen Neuronen der CA1 Region (bei AD und TLE erst spät geschädigt) miteinander verglichen. Mittels patch-clamp Ganzzellmessung zeigt diese Studie die differentielle Hemmung spannungsabhängiger transienter (IA) und „delayed-rectifier“ K+-Ströme (IK(V)) durch Arachidonsäure (AA) und Wasserstoffperoxid (H2O2). Die intrazelluläre Applikation von AA (1 pM) reduzierte IA in Neuronen des entorhinalen Kortex signifikant stärker verglichen mit Neuronen des CA1. ETYA imitiert diesen Effekt, dies schliesst die Metabolite der AA als Mediatoren des Effekts auf Kaliumkanäle aus. Weder AA noch ETYA reduzierten IK(V). Im Gegensatz dazu reduzierte H2O2 IA in Neuronen des CA1 effektiver als in Neuronen der Schichten II und III des entorhinalen Kortex. Die Reduktion des IA, vermittelt durch AA, wurde durch Radikalfänger (Glutathion, Ascorbinsäure, Vitamin E Analogon Trolox) blockiert. Dabei verstärkten manche dieser Antioxidantien den Effekt der AA, dies legt eine komplexere Modulation dieser Ströme in Schnitten verglichen mit Kulturen nahe. Dies sollte bei der Entwicklung antioxidativer Therapien von AD und TLE berücksichtigt werden. Bei der heterologer Expression von Kv1.4 und Kv4.2 in HEK-293 Zellen wurden funktionelle Kanäle gebildet und A-Typ Ströme ausgelöst. Diese Ströme wurden nach der Applikation von 1 pM AA stark reduziert. ROS scheinen neben ihrer zellschädigenden Wirkung physiologische Prozesse zu regulieren, indem sie eine Reihe von Signalwegen beeinflussen. Da spannungsabhängige Kaliumkanäle vielen wichtigen zellulären Funktionen zugrundeliegen, könnte die Modulation dieser Kanäle durch ROS einen Mechanismus für die Feinabstimmung zellulärer Prozesse darstellen. / Oxidative stress and dysfunction of potassium channels are believed to play a role in neuronal death in a number of CNS diseases (e.g. Alzheimer’s disease, epilepsy). The present study addresses selective neuronal vulnerability to oxidative stress by studying oxidative modulation of potassium channels in entorhinal cortex (EC) layer II stellate neurons (cell loss early in AD) and layer III pyramidal neurons (early damage in TLE), in comparison to hippocampal CA1 pyramidal neurons (late damage in TLE and AD). Using whole-cell patch-clamp, differential inhibition of transient IA and delayed rectifier K+-currents IK(V) by arachidonic acid (AA) and H2O2 was demonstrated. Intracellular AA (1 pM) reduced IA in EC neurons significantly stronger than in CA1 neurons. AA affected the voltage dependence of steady-state inactivation as well. ETYA mimicked the effect of AA, excluding its metabolites as mediators of IA modulation. Neither AA nor ETYA reduced IK(V). In contrast, a non-lipid oxidizing agent, H2O2 reduced IA more effectively and robustly attenuated IK(V) in CA1, compared to EC neurons. AA-mediated reduction of IA was blocked by free radical scavengers (glutathione, ascorbic acid, Trolox). Antioxidants did not simply inhibit AA and H2O2 effects. In particular, they even enhanced AA effects, suggesting more complex modulation of these currents in slices, compared to culture. Moreover, intracellular antioxidants, themselves, influenced maximal conductance and voltage-conductance characteristics of IA and IK(V). This should be considered in design of anti-oxidative therapies in AD and TLE. Heterologous expression of Kv1.4 and of Kv4.2 cDNA in HEK-293 cells formed functional channels and elicited A-type currents, which shared similar biophysical characteristics with native IA from the hippocampus. These currents were strongly decreased upon administration of 1pM AA, demonstrating that at least one of multiple sites for AA action is situated on the pore-forming alfa-subunit of the A-channel. In conclusion, beside contribution to cell damage, ROS may regulate physiological processes by acting on different signalling pathways. Since voltage-gated K+-channels underlie many important cellular functions modulation of these channels by ROS would represent a mechanism for fine tuning of cellular processes.

Ca(2+)-Ionen sind ein universeller sekundärer Botenstoff in eukaryotischen Zellen und übertragen Information durch wiederholte, kurzzeitige Erhöhungen der cytosolischen Ca(2+)-Konzentration (Ca(2+) Spikes). Ein bekannter Mechanismus, der solche Ca(2+)-Signale erzeugt, beinhaltet die Freisetzung von Ca(2+)-Ionen aus dem endoplasmatischen Retikulum durch IP3-sensitive Kanäle. Puffs sind elementare Ereignisse der Ca(2+)-Freisetzung durch einzelne Cluster von Ca(2+)-Kanälen. Intrazelluläre Ca(2+)-Dynamik ist ein stochastisches System, allerdings konnte bisher keine vollständige stochastische Theorie entwickelt werden. Die vorliegende Dissertation formuliert die Theorie mit Hilfe von Interpuffintervallen und Pufflängen, da diese Größen im Gegensatz zu den Eigenschaften der Einzelkanäle direkt messbar sind. Die Theorie reproduziert das typische Spektrum bekannter Ca(2+)-Signale. Die Signalform und das durchschnittliche Interspikeinterval (ISI) hängen sensitiv von den genauen Eigenschaften und der räumlichen Anordnung der Cluster ab. Im Gegensatz dazu hängt die Beziehung zwischen Mittelwert und Standardabweichung der ISI weder von den Clustereigenschaften noch von der räumlichen Anordnung ab, sondern wird lediglich von globalen Feedbackprozessen im Ca(2+)-Signalweg reguliert. Diese Beziehung ist essentiell für die Funktion des Signalwegs, da sie trotz der Zufälligkeit der ISI eine Frequenzkodierung ermöglicht und den maximalen Informationsgehalt der Spikesequenzen bestimmt. Neben der theoretischen Analyse enthält die vorliegende Arbeit auch experimentelle Puff- und Spikemessungen an lebenden HEK-Zellen, die wichtige Ergebnisse verifizieren. Insgesamt wird durch die integrierte theoretische und experimentelle Untersuchung auf verschiedenen Stufen molekularer Organisation gezeigt, dass stochastische Ca(2+)-Signale verlässliche Informationsträger sind, und dass der Mechanismus durch globalen Feedback an die spezifischen Anforderungen eines Signalpfads angepasst werden kann. / Ca(2+) is a universal second messenger in eukaryotic cells transmitting information through sequences of concentration spikes. A prominent mechanism to generate these spikes involves Ca(2+) release from the endoplasmic reticulum Ca(2+) store via IP3-sensitive channels. Puffs are elemental events of IP3-induced Ca(2+) release through single clusters of channels. Intracellular Ca(2+) dynamics are a stochastic system, but a complete stochastic theory has not been developed yet. As a new concept, this thesis formulates the theory in terms of interpuff interval and puff duration distributions, since unlike the properties of individual channels, they can be measured in vivo. This leads to a non-Markovian description of system dynamics, for which analytical solutions and efficient stochastic simulation techniques are derived. The theory reproduces the typical spectrum of Ca(2+) signals. Signal form and average interspike interval (ISI) depend sensitively on detailed properties and spatial arrangement of clusters. In difference to that, the relation between the average and the standard deviation of ISIs does not depend on cluster properties and cluster arrangement, and it is robust with respect to cell variability. It can only be regulated by global feedback processes in the Ca(2+) signalling pathway. That relation is essential for pathway function, since it ensures frequency encoding despite the randomness of ISIs and determines the maximal spike train information content. Apart from the theoretical investigation, this thesis verifies key results by live cell imaging of Ca(2+) spikes and puffs in HEK cells. Hence, this work comprises a systems level investigation of Ca(2+) signals, integrating data and theory from different levels of molecular organisation. It demonstrates that stochastic Ca(2+) signals can transmit information reliably, and that the mechanism can be adapted to the specific needs of a pathway by global feedback.

Recombinant proteins belong to considerable biofarmaceutics products used in biomedical research and in the treatment of human disease. Recombinant protines can be produced by stable transfection in big amount or by faster transient transfection with smaller amounts. To provide regular biological activity, it is necessary for the protein to be properly folded and post-translationally modified. As these modifications can be accurately performed only in mammalian cells, they have become the major host for complex r-protein expression. In this thesis is described transient transfection HEK 293 EBNA1 cells with linear polyethylenimines. These cells has been adapted to suspension cultivation in serum free medium. The cells were transfected with pcDNA3.1, pCI, pEBSV1, pCEP4, pEAK8 a pcDNA5/FRT/TO plasmids, everyone contained repoter gene SEAP. Concentration of SEAP in cell culture supernatants were determined in order to compare efficiencies of individual transfections. DNA:PEI ratio was another factor which was optimised and two different PEIs were compared. Highest achieved expresion was 50 mg per litre with transfection in 24 well plate when DNA:PEI ratio was 1:5. Comparison of six different plasmids give the bigest expresion pCEP4/SEAP, in well plate as well as in scaled up system.

Protein solubility plays an important role whether for biophysical and structural studies, or for production and delivery of therapeutic proteins. Poor solubility could lead to protein aggregation, which is an undesired physicochemical mechanism at any stage of recombinant proteins production. To date, more than half of all recombinant therapeutic proteins are produced in mammalian cells, mainly due to the high similarity of the final product to human protein structures. However, poor secretion can occur, due to misfolded proteins or aggregates leading to cellular stress and proteolysis. Another widely-used expression system is E. coli, which can offer a cost-efficient alternative. This system has an important limitation, since proteins tends to form insoluble protein aggregates in the cytoplasm upon heterologous overexpression. Several strategies are being implemented to improved soluble expression, ranging from culture conditions to solubility enhancing tags. However, there is no universal approach or technology that solves protein aggregation. In this thesis two recently published hypotheses from our group have been applied. One stated that soluble expression of proteins was inversely correlated with the size of the largest positively-charged patch on the protein surface. The second hypothesis (of protein solubility), arose from the finding that the relative content of lysine and arginine residues separated E. coli proteins by solubility. Both hypotheses arose from a study of an extensive dataset of experimental solubilities determined for cell-free expression of E. coli proteins. In combination with other widely used strategies, such as lowering expression temperature and inducer concentration, decreasing non-charged (hydrophobic) patches and addition of helical capping for increasing stability, a rational understanding for directed alteration of solubility in a variety of recombinant proteins has been explored. This includes three protein models to test: (i) recombinant human erythropoietin (rHuEPO) (one of the top selling therapeutics) (ii) recombinant 6-Phosphofructo-2-Kinase/fructose-2,6-bisphosphatase (rPFKFB3) (a product for which over-expression has been sought for characterisation and insight into possible cancer therapy) and (iii) a set of three selected E. coli proteins containing high ratios of lysines to arginines: thioredoxin-1 (TRX), cold shock-like protein cspB (cspB), and the histidine-containing phosphocarrier protein (HPr). It was found that single or multiple point mutations (changing amino acids from positive to negative charge or vice versa; or lysines to arginines) verified the predicted effect on rHuEPO, rPFKFB3, TRX, cspB, and HPr solubility (experimentally defined as the distribution between soluble and total fractions) for expression in E. coli. In addition, the redesigned set of rHuEPO transiently expressed in HEK 293-EBNA cells, suggesting that positively-charged patch size may also influence protein secretion. Further application of these computational and experimental approaches could provide a valuable tool in the design and engineering of proteins, with enhanced solubility, stability and secretion.

Gene therapy involves the use of nucleic acids, either DNA or RNA for the treatment, cure, or prevention of human diseases. Synthetic cationic polymers are promising as a tool for gene delivery because of their high level of design flexibility for biomaterial construction and are capable of binding and condensing DNA through electrostatic interactions. Our lab has developed a novel polymer (poly (polyethylene glycol-dodecanoate) (PEGD), a polyester of polyethylene glycol (PEG) and dodecanedioic acid (DDA). PEGD is a linear viscous polymer that self-assembles into a vesicle upon immersion in an aqueous solution. A copolymer of dodecanedioc acid and polyethylene glycol (PEG) was synthesized at a 1:1 ratio. Furmaric (FA) or itaconic acid (IA) was used to suppress DDA in the PEGD copolymer at an 80:20 ratio (DDA: furmaric/itaconic acid) to form the PEGDF/I variant. PEGDF/I are then modified through the Michael addition of Protamine Sulfate (PEGDF/I-PS) and Cys-Arg8 (PEGDF/I-CA) peptide to the carbon-carbon double bond on the polymer backbone to introduce a positive charge. The modified PEGDF/I polymers were capable of binding and condensing DNA. Transfection of HEK 293 cells with pTurboGFP plasmid using modified PEGDF/I polymers was successful but showed varied efficiency. The PEGDF/I-CA polymer had around 30% transfection efficiency and was shown to be non-cytotoxic.

Cell assay that evaluates a biologics’ potency and efficacy is an important part in the discovery and development of drug candidates. However, it requires regular maintenance of cell cultures, and the cell assays can only be performed when the cells have reached 70-80% confluency. By instead using assay-ready cells, the drugs can be screened at any time by simply thawing the cells. This creates a more flexible assay, while saving time, labor and materials in addition to removing day-to-day variability. In this report, the freezing conditions for HEK-Blue™ CD40L cells are evaluated using the assay-ready cell method compared to a continuous culture. Via colorimetric detection, the CD40 receptors’ activation can be determined and a dose-response curve of a CD40 agonist can be produced. The optimal freezing condition for the assay-ready cells were determined to be 10% DMSO and a cell concentration between 1-30 million cells/mL. After reproducibility, robustness and screening tests, it could be concluded that the method generally produced results that had no significant difference to a continuous culture. Some of the assay-ready cells display a higher background which can affect the value of the efficacy. The source of the background will have to be evaluated in future studies. The potency, on the other hand, is stable regardless of cell method or high background.

Nicotinic acetylcholine receptors (nAChR) are found widely throughout the body. Like all members of the cys-loop family of receptors, nAChRs are composed of five protein subunits, each with a large extra-cellular domain and four transmembrane domains. Together these subunits form a binding domain, transmembrane pore, and selectivity filter. Neuronal nicotinic acetylcholine receptors, formed exclusively from α2-10 and β2-4 subunits, can form in many arrangements and stoichiometries. Each arrangement can have varying binding affinities and channel kinetics, resulting in great modulatory control. α3 and β2 subunit mRNA is found in CA1 interneurons in the stratum radiatum and stratum oriens of the rat hippocampus, and in surprising expression frequency and ratios. Further study of α3 and β2 subunit mRNA injected into Xenopus laevis oocytes yields interesting results about the potential for two α3β2 subtypes. These results were in intriguing, and prompted further study to better characterize and screen the α3β2 nAChR. In order to do so, a model was needed where the α3β2 nAChR could be studied in a more physiologically relevant mammalian environment, with consistent control over α3 and β2 subunit expression ratios, and sufficient protein expression and functionality. To this end, we created a doxycycline inducible HEK-293 cell line, stably transfected with the genetic sequences for the α3 and β2 subunits and NACHO, a transmembrane protein of the neuronal endoplasmic reticulum, which has been shown to mediate the assembly of α3β2 and other nAChRs. This new model is able to induce expression various ratios between α3 and β2 subunits in a consistent, manner, proving to be valuable tool in the characterization and screening of the α3β2 nAChR.

No description available.

Single-cell real-time quantitative RT-PCR was used to characterize the mRNA expression of rat neuronal nicotinic acetylcholine receptor (nAChR) subunits α3 and β2 in CA1 hippocampus stratum radiatum and stratum oriens interneurons. α3β2 co-expression was detected in 43% of interneurons analyzed. The nAChR subtype α3β2 was transiently expressed in cells derived from the human embryonic kidney cell line 293 at mRNA levels found in the CA1. The functional properties of α3β2 in HEK-293 cells were characterized by whole-cell patch clamping using acetylcholine (ACh) as an agonist. The kinetics of α3β2 channels were further analyzed by altering the level of α3 DNA transfected into HEK-293 cells. Varying the α3 concentration by more than 100,000 fold did not significantly alter the majority of the kinetics; the 10%-90% rise-time was the main characteristic found to be significantly different. A decrease in α3 concentration illustrated a significant increase in rise time. This and future studies will further our understanding of the extensive role neuronal nAChRs play in modulating hippocampal activity and consequently influencing cognition and memory.

No description available.