Rund 300 Millionen Alveolen sorgen in der menschlichen Lunge für den Gasaustausch. Eine essentielle Rolle spielen dabei die Alveolarepithelzellen, die die erste Barriere der Luft gegenüber dem Blut bilden. Man unterteilt diese in die kubisch förmigen Typ-II-Epithelzellen, die den wichtigen stabilisierenden Surfactant bilden und in die flacheren Typ-I-Zellen, die aufgrund von engen Zell-Zell Kontakten miteinander eine funktionelle Barriere zwischen Luft und Blut (Blut-Luft-Schranke) ermöglichen. In der Pathophysiologie von verschiedenen Lungenerkrankungen, wie z.B. dem ARDS, spielt die Störung dieser Barriere eine essentielle Rolle. Ziel der vorliegenden Promotionsarbeit war es, ein in vitro-Modell dieser Barriere, basierend auf der humanen Epithellzelllinie NCI H441, zu etablieren. Die Epithelzellen wurden hierzu erfolgreich auf Transwelleinsätzen gezüchtet. Um die Barriereeigenschaften zu überprüfen, wurden mit TER Messungen und Fluoreszenzpermeabilitätsmessungen zwei verschiedene Verfahren eingesetzt. Für die Bewahrung der Konfluenz wurden die Zellen mit Dexamethason – einem potenten Glukokortikoid – behandelt. Dexamethason wurde idealerweise bei jedem Medienwechsel ab Tag 5 apikal hinzugefügt und zeigte einen barrierefördernden Effekt. Die optimale hinzugefügte Dexamethason–konzentration erwies sich als 100 nM. Hierunter bildete die Zellschicht – sowohl mittels TER-Messung als auch mittels Fluoreszenzpermeabilitätsmessung überprüft – eine starke Barriere aus, die am Tag 14-15 nach Zellaussaat ihr Maximum erreichte. Auf mRNA Ebene konnten zu diesem Zeitpunkt unter 100 nM Dexamethason jeweils relevante Mengen zellspezifischer Proteine von Alveolarepithelzellen Typ-I und II nachgewiesen werden. Mittels Immunfluoreszenzmikroskopie konnte zusätzlich visualisiert werden, dass die zwei Zelltypen koexistieren. Mittels Immunfluoreszenzmikroskopie zeigte sich weiterhin ein ausgeprägtes Netz an Occludensjunktionsproteinen (Claudin -1,-3,-4, Occludin, ZO-1). In weiteren Versuchen wurde der Einfluß von Endothelzellfaktoren auf das epitheliale Lungenkulturmodell untersucht. Es zeigte sich ein stark ausgeprägter gewebsspezifischer Effekt: Die Faktoren der Lungenendothelzellen stärkten die Barriere des Modells, hingegen die Hirnendothelzellfaktoren sie stark schwächten. Dieser Effekt zeigte sich sowohl durch die gemessenen TER-Werte als auch durch die gemessenen Fluoreszeinpermeabelitätswerte. Erste Belastungsversuche des Modell durch Hypoxie und reaktive Sauerstoffspezies zeigten eine gewisse Widerstandsfähigkeit der Barriere gegenüber Noxen. Das vorliegende Zellkulturmodell des Lungenalveolarepithels, basierend auf der NCI H441 Zelllinie, kann folglich genutzt werden, um die Regulation der Barriere genauer zu erforschen und mögliche neue Therapiestrategien zu entwickeln. / The blood–air barrier in the lung consists of the alveolar epithelium, the underlying capillary endothelium, their basement membranes and the interstitial space between the cell layers. Within the framework of this thesis an in vitro-Transwell model of the alveolar epithelium based on human cell line H441 was established and the influence of conditioned medium obtained from human lung endothelial cell line HPMEC-ST1.6R on the barrier properties of the H441 layers were investigated. As control for tissue specificity H441 layers were exposed to conditioned medium from human brain endothelial cell line hCMEC/D3. Addition of dexamethasone was necessary to obtain stable H441 cell layers. Moreover, dexamethasone increased expression of cell type I marker caveolin-1, and cell type II marker SP-B, whereas decreased the transepithelial electrical resistance (TEER) in a concentration dependent manner. Soluble factors obtained from the lung endothelial cell line increased the barrier significantly proven by TEER values and fluorescein permeability on the functional level and by the differential expression of tight junctional proteins on the molecular level. In contrast to this, soluble factors derived from brain endothelial cells weakened the barrier significantly. In conclusion, soluble factors from lung endothelial cells can strengthen the alveolar epithelium barrier in vitro, which suggests communication between endothelial and epithelial cells regulating the integrity of the blood–air barrier. With regard to the clinical context, influencing the alveolar permeability may be a future target in the treatment of different lung diseases such as ARDS and this in-vitro model may be a helpful tool for it.

Neuromuscular synapse formation is driven by two nerve-derived molecules, agrin and neuregulin. Agrin is believed to signal through a Muscle Specific Kinase (MuSK)/Lrp4 receptor complex to cluster existing acetylcholine receptors (AChRs) in the postsynaptic membrane via a rapsyn-mediated mechanism, while neuregulin signals via ErbB kinase receptors to induce synaptic gene transcription. Recent studies suggest that neuregulin-ErbB signalling may also cross-signal to the agrin-MuSK pathway to modulate agrin’s ability to cluster AChRs. This thesis aimed to further elucidate this idea. Results of this thesis present two novel findings. First, there is a direct interaction between two tyrosine kinase signalling pathways at the neuromuscular synapse and second, neuregulin plays an important role in modulating, modifying and refining AChRs at developing synapses. Here I show that neuregulin can modulate two distinct processes. In the presence of agrin, neuregulin was able to potentiate both agrin-induced AChR clustering and agrin-induced AChR cluster dispersal, and this modulation by neuregulin occurred independently of any transcriptional mechanism. In vitro, I observed a marked effect by neuregulin on the number and size of AChR clusters that were induced by agrin. Treatment of myotubes for 4hrs with agrin and neuregulin led to a significant potentiation in agrin-induced AChR clustering compared to agrin treatment alone. Neuregulin on its own had no measurable effect on AChR clustering. When incubation times were much longer (12hrs), neuregulin promoted a further significant decrease in AChR cluster number compared to agrin treatment alone. Thus at 12hrs, rather than inhibit AChR clustering, as has been previously suggested, neuregulin promoted the dispersal of AChRs from pre-existing agrin-induced clusters. Follow-up in vivo studies into the potentiating ability of neuregulin in agrin-induced AChR clustering showed that the injection of exogenous neuregulin into developing mouse sternomastoid musculature led to an increase in the size of AChRs. Collectively, these data suggest interactions between the signalling pathways initiated by agrin and neuregulin. Subsequent investigation into the second messengers downstream of agrin-MuSK and neuregulin-ErbB signalling revealed that cyclin-dependent kinase 5 (Cdk5) and Shp2 played a role in neuregulin’s modulation of AChR cluster formation and dispersal. It appears that neuregulin enhances the phosphorylation status of MuSK by inhibiting the Shp2-dependent negative feedback loop on MuSK phosphorylation, thereby leading to an increase in AChR cluster numbers. By contrast, the way in which neuregulin disperses agrin-induced AChR clusters seems to occur partially, via a Cdk5 signalling-dependent mechanism. While it is accepted that neuregulin acts in a transcriptional manner during neuromuscular synapse formation, real-time PCR and immunoblot results suggest that transcriptional regulation was not involved in neuregulin’s modulation of agrin-induced AChR clustering.

Thesis (M.S.)--University of Delaware, 2006. / Principal faculty advisors: Robert E. Akins, Jr. and Deni S. Galileo, Dept. of Biological Sciences. Includes bibliographical references.

Malachite green(MG), an N-methylated diaminotriphenylmethane dye, has been widely used as an antifungal agent in aquaculture. Malachite green is reduced to and persists as leucomalachite green(LMG) in the tisssues of fish. Concern over MG and LMG are due to the potential for consumer exposure, suggestive evidence of tumor promotion in rodent liver, and suspicion of carcinogenicity based on structure-activity relationships. Several hepatotxicants and liver carcinogens have been shown to alter cell-cell signaling by interference with gap junction intercellular communication (GJIC).This study wanted to determine if disruption of cell-cell interactions occurs in rat liver epithelial cells in response to MG and LMG treatment. Rat liver epithelial cells(WB) were treated with LMG(0~6£gg/ml) or MG.(0~5£gg/ml) for one hour and gap junction was analyzed using the scrape- loading/dye transfer assay. The viability and proliferation of rat liver epithelial cells treated with LMG or MG were determined by MTT and colony forming efficiency. In addition, expression and intracellular localization of connexin43, E-cadherin,£]-catenin,£\-tubulin, ZO-1 and occludin were determined by immunoblot and immunostain analysis. A clear decrease in the distance of dye transfer was evident following treatment with MG(0~5£gg/ml) or LMG(0~6£gg/ml). Treatment with LMG and MG at different concentrations resulted in a decrease in cell viability and cell proliferation. Preincubation of cells with protein kinase C(PKC) inhibitor decreased the inhibition of GJIC by 5ug/ml MG and the specific MEK 1 inhibitor decreased substantially the inhibition of GJIC by 5£gg/ml MG and 5£gg/ml LMG. On the other hand, the specific PI3 kinase inhibitor decreased the inhibition of GJIC only by 3£g/mlLMG and we treated WB cells with EDTA to chelate extracellular calcium ion. The decrease of free calcium ion caused the expression of GJIC. At the transcriptional level, 10£gg/ml LMG and 10£gg/ml MG after treatment for one hour caused no change in the level of connexin43 mRNA. At the translational level, the different concentrations of MG or LMG after treatment for one hour or 24 hours caused a decrease in the level of the concentrations of connexin43 protein, E-cadherin protein,£]-catenin protein,£\-tubulin protein, ZO-1 protein and changed the distribution of occludin and ZO-1. Therefore, these data speculated the hypothesis that disruption of cell-cell signaling by interference with GJIC may contribute to LMG and MG toxicity, carcinogenicity and apoptosis.

Homologous recombination is the primary DNA repair pathway in bacteria and it is immensely important in repairing DNA double strand breaks. Components of the homologous recombination pathway have been well conserved throughout evolution as an essential part of cell survival. Homologous recombination plays an important role in cellular processes like DNA repair as well as exchange of genetic information through chromosomal crossover. During homologous recombination, DNA strand exchange leads to formation of a heteroduplex joint between the invading and displaced DNA strands. This hetereoduplex joint is called a Holliday Junction. Resolution of the Holliday Junction proceeds via one of two pathways. In the presence of RuvC and/or RecG, Holliday Junction resolution proceeds via a cut and paste pathway where the invading DNA strand replaces a region of homologous DNA on the target DNA. In the absence of RuvC and RecG, Holliday Junction resolution takes place via a copy and paste pathway during which DNA synthesis needs to be primed at Holliday Junction intermediates formed during strand invasion. In an effort to separate this myriad of different requirements, I have attempted to develop a novel ends-in recombination assay system using E. coli as a model organism. This ends-in system would allow recombinant molecule formation by DNA synthesis of approximately 200 to 2000 bp size interval between the two converging ends of an invading linear dsDNA substrate oriented just like the greek letter Ù, but with the arms pointing inwards. In this study, a number of linear dsDNA assay templates were constructed and analyzed. All the constructs had two arms of homology to the chromosome pointing inwards i.e. in the ends-in orientation. Using this ends-in system, it was demonstrated that the presence of chi (Crossover Hotspot Initiator) sites was an important requirement for ends-in recombination in wild type E. coli cells. Our studies also showed that ends-in homologous recombination did not occur if chi sites were placed at or very near to the ends of the incoming linear dsDNA molecule, suggesting that the chi site recognition is efficient only if the incoming dsDNA has chi sites internal to the ends. Moreover, it was shown that neither RuvC nor RecG were required for successful recombinant product formation using the ends-in assay. This finding reinforces previous observations that suggest the idea that Holliday Junctions can be resolved independent of both RuvC and RecG.

<p>Topoisomerase III&alpha; (Top3&alpha;) is an essential component of the double Holliday junction (dHJ) dissolvasome complex in metazoans. Previous work has shown that Top3&alpha; and Bloom's helicase (Blm) are able to convergently migrate the dHJ to create solely non-crossover products, thus preserving genomic integrity. However, many questions remain about the details of this process. Using a combination of biochemical and genetic tools, including dHJ substrate assays, gel electrophoresis, EMSA, pulldowns, fly crosses, and electron microscopy, this work expands our knowledge of the dissolution reaction. Tail mutants of Top3&alpha; were created and tested in a series of <italic>in vitro</italic> assays. Through these experiments, I discovered that the C-terminus of Top3&alpha; is important for binding Blm, interacting with DNA, conveying RPA stimulation, and <italic>in vivo</italic> functionality. I also observed that dissolution is an extremely processive reaction, with no accumulation of intermediates prior to product formation. When a non-specific topoisomerase was used (Top1, a type IB), accumulation of an intermediate was evident; however, contrary to predicted models, direct observation revealed that this intermediate is not a hemicatenane structure and still requires branch migration. Modifications were also made to the dHJ substrate creation method so that multiple types of HJ substrates could be produced efficiently.</p> / Dissertation

Homologous recombination is the primary DNA repair pathway in bacteria and it is immensely important in repairing DNA double strand breaks. Components of the homologous recombination pathway have been well conserved throughout evolution as an essential part of cell survival. Homologous recombination plays an important role in cellular processes like DNA repair as well as exchange of genetic information through chromosomal crossover. During homologous recombination, DNA strand exchange leads to formation of a heteroduplex joint between the invading and displaced DNA strands. This hetereoduplex joint is called a Holliday Junction. Resolution of the Holliday Junction proceeds via one of two pathways. In the presence of RuvC and/or RecG, Holliday Junction resolution proceeds via a cut and paste pathway where the invading DNA strand replaces a region of homologous DNA on the target DNA. In the absence of RuvC and RecG, Holliday Junction resolution takes place via a copy and paste pathway during which DNA synthesis needs to be primed at Holliday Junction intermediates formed during strand invasion. In an effort to separate this myriad of different requirements, I have attempted to develop a novel ends-in recombination assay system using E. coli as a model organism. This ends-in system would allow recombinant molecule formation by DNA synthesis of approximately 200 to 2000 bp size interval between the two converging ends of an invading linear dsDNA substrate oriented just like the greek letter Ù, but with the arms pointing inwards. In this study, a number of linear dsDNA assay templates were constructed and analyzed. All the constructs had two arms of homology to the chromosome pointing inwards i.e. in the ends-in orientation. Using this ends-in system, it was demonstrated that the presence of chi (Crossover Hotspot Initiator) sites was an important requirement for ends-in recombination in wild type E. coli cells. Our studies also showed that ends-in homologous recombination did not occur if chi sites were placed at or very near to the ends of the incoming linear dsDNA molecule, suggesting that the chi site recognition is efficient only if the incoming dsDNA has chi sites internal to the ends. Moreover, it was shown that neither RuvC nor RecG were required for successful recombinant product formation using the ends-in assay. This finding reinforces previous observations that suggest the idea that Holliday Junctions can be resolved independent of both RuvC and RecG.

This research successfully created an ultra-small orifice utilizing undercut fabrication process in a droplet-based microfluidics chip. The proposed novel T-junction structure with ultra-small orifice has a lot of advantages, including long-term stability for uniform droplets formation, reproducible ultra-small size droplet and tunable droplet size. The hydraulic diameter of the orifice is under 2 £gm, and the size of micro droplet produced from the orifice can be tuned to less than 10 £gm in diameter. Chitosan droplet can be produced by the proposed chip, which is usually adopted for medical applications. Surface modification technique was applied to modify the surface of microchannel to be hydrophobic for eaily producing hydro-droplets. Experimental results show that the ultra-small orifice microfluidics chip can steadily produce water-in-oil droplets only by controlling the flow ratio between dispersed phase and continuous phase flow rates. The size of the water-in-oil droplets can be tunable from 22 £gm to 6.5 £gm in diameter by adjusting the flow rate ratio of the continuous and disperse phase flows from 1 to 3.5 and the hydraulic diameter of the orifice is 1.1 £gm. And the size of the chitosan-in-oil droplets also can be tunable from 59 £gm to 27 £gm by adjusting the flow rate ratio of the continuous and disperse phase flows from 4 to 8. The proposed microchip has advantages including ease of control, low cost, and high throughput. The proposed technique can be widely applied on emulsion and micro droplet generation.

Polycrystalline p-Cu2O were fabricated by reactive rf magnetron sputtering . we found that The electrical, optical, and crystallographical properties of films were strongly dependent on the deposition condition . Grant size increasing in the range from 10 to 45nm , A hole concentration increasing in the range from 1016 to 1017 cm-3 and a mobility increasing on the order of 10-1 cm2/V s were obtained in the cuprous oxide thin film prepared by controlling work pressure (Argon partial partial pressure ) . Fabricated thin-film heterojunction diodes consisting of a p-type cuprous oxide combined with and n-type Al-doped ZnO and ITO exhibited a rectifying current-voltage characteristic .

We used self-assembling tunnel junctions (SATJs) to study the electron transport through films of the molecular magnets, Mn12-Acetate. Pulse laser deposition (PLD) was used to deposit two monolayers of Mn12-Acetate on thin Pt wires (diameter 0.001 in). The electron tunneling current was measured with typical bias voltages from -1 to 1 V at liquid helium temperature, 4.2 K. I, dI/dV , and d2I/dV 2 signals were directly acquired with the aid of a current amplifier and two lock-in-amplifiers. Results show that the differential conductance is approximately 10â 6 S for bias voltages 0.04 V < or =| V |< or = V and exhibits a strong voltage dependence. In the region | V |< or = 0.04 V, we find a zero-bias feature (ZBF) in which the differential conductance is suppressed. In some samples, we observe I -V staircases which we attribute to electrons "hopping" between the electrodes and the molecules. The observed hysteresis was attributed to the slow relaxation of molecules re-orienting within the junction. Abrupt conductance jumps at a bias voltage of -0.12 V were also observed and may indicate state transitions in the Mn12-Acetate molecules. Furthermore, we observed that the zero bias feature (ZBF) can switch from an enhancement to a suppression of the differential conductance. A dip and dry (DAD) method was also used to form films of Mn12-Acetate on Al and Pt wires. Although the conductances were similar to those obtained using the PLD method, there were some subtle differences. In particular, we did not observe the I -V staircases and the state jumps were more ambiguous. The differential conductance for the Mn12-Acetate films on Al wires were typically 10- 7 S, which we attributed to the oxide layer on Al surfaces. We have also found substantial changes in the I - V characteristics when the Pt wires coated with the Mn12-Acetate films were stored in 10-2 Torr for 6 months. In particular, we observed many new features such as peaks in the conductance as a function as the bias voltage. We believe that these effects may be caused by the slow oxidation of the Mn12-Acetate molecules.