Dissertations / Theses on the topic 'FCCUs'

The control of particle emissions from an oil refinery is often difficult, due to changing operational conditions and the limited range of available treatment options. Excessive particle emissions have often been attributed start up problems with Fluidized Catalytic Cracker Units (FCCU) and little information is available regarding the exact composition and nature of these excessive emissions. Due to the complex nature of a FCCU, it has in the past been difficult to identify and control emissions, without the use of expensive end of pipe technologies. An Australian Oil Refinery, concerned with their catalyst emissions, sponsored this study of FCCU particle emissions. Due to the industrial nature of the project, a holistic approach to the management of emissions was taken, instead of a detailed investigation of a single issue. By looking at the broader range of issues, practical and useful outcomes can be achieved for the refinery. Initially, detailed emissions samplings were conducted to investigate the degree of particle emissions under start up conditions. Stack emissions were collected during a standard start up, and analysed to determine the particle size distribution and metal concentration of the emitted material. Three distinct stages of emissions were discovered, initially a high concentration of larger particles, followed by a peak in the very fine particles and finally a reduction of particle emissions to a more steady or normal operational state. The variation in particle emissions was caused by operational conditions, hardware design and catalyst characteristics. Fluctuations in the gas velocity through the system altered the ability of the cyclones to collect catalyst material. Also, the low bed level allowed air bypass to occur more readily, contributing to the increased emissions levels seen during the initial stage of the start up. Reduced fluidity characteristics of the circulating catalyst also affected the diplegs operations, altering the collection efficiency of the cyclone. During the loading of catalyst into the system, abraded material was quickly lost due to its particle size, contributing to fine particle emissions levels. More importantly, thermal fracturing of catalyst particles occurred when the cold catalyst was fed into the hot regenerator. Catalyst particles split causing the generation of large amounts of fine particle material, which is easily lost from the system. This loading of catalyst directly linked to the period of high concentration of fine particles in the emissions stream. It was found that metals, and in particular iron, calcium and silicon form a thick layer on the outside of the catalyst, with large irregular shaped metal ridges, forming along the surface of the particle. These ridges reduce the fluidity of the catalyst, leading to potential disruptions in the regenerator. In addition to this, the metal rich ridges are preferentially removed via attrition, causing metal rich material to be emitted into the atmosphere. To overcome these high particle emissions rates from the FCCU the refinery should only use calcinated catalyst to reduce the influence of thermal process and particle fracture and generation. Although the calcinated catalyst can fracture when added to the system, it is far less than that obtained with uncalicinated catalyst. To further reduce the risk of particle fracture due to thermal stresses the refinery should consider reducing the temperature gradients between the hot and cold catalyst. Due to the economics involved with the regenerator, possible pre-warming of catalyst before addition into system is the preferred option. This pre-heating of catalyst should also incorporate a controlled attrition stage to help remove the build up of metals on the surface of the particles whilst allowing this material to be collected before it can be released into the atmosphere. The remove of the metal crust will also improve the fluidity of the system and reduce the chance of catalyst blockages occurring. Finally, modelling of the system has shown that control of key parameters such as particle size and gas velocity are essential to the management of air emissions. The refinery should look at adjusting start up procedures to remove fluctuations in these key parameters. Also the refinery should be careful in using correlation found in the literature to predicted operational conditions in the system as these correlations are misleading when used under industrial situations.

The control of particle emissions from an oil refinery is often difficult, due to changing operational conditions and the limited range of available treatment options. Excessive particle emissions have often been attributed start up problems with Fluidized Catalytic Cracker Units (FCCU) and little information is available regarding the exact composition and nature of these excessive emissions. Due to the complex nature of a FCCU, it has in the past been difficult to identify and control emissions, without the use of expensive end of pipe technologies. An Australian Oil Refinery, concerned with their catalyst emissions, sponsored this study of FCCU particle emissions. Due to the industrial nature of the project, a holistic approach to the management of emissions was taken, instead of a detailed investigation of a single issue. By looking at the broader range of issues, practical and useful outcomes can be achieved for the refinery. Initially, detailed emissions samplings were conducted to investigate the degree of particle emissions under start up conditions. Stack emissions were collected during a standard start up, and analysed to determine the particle size distribution and metal concentration of the emitted material. Three distinct stages of emissions were discovered, initially a high concentration of larger particles, followed by a peak in the very fine particles and finally a reduction of particle emissions to a more steady or normal operational state. The variation in particle emissions was caused by operational conditions, hardware design and catalyst characteristics. Fluctuations in the gas velocity through the system altered the ability of the cyclones to collect catalyst material. Also, the low bed level allowed air bypass to occur more readily, contributing to the increased emissions levels seen during the initial stage of the start up. Reduced fluidity characteristics of the circulating catalyst also affected the diplegs operations, altering the collection efficiency of the cyclone. During the loading of catalyst into the system, abraded material was quickly lost due to its particle size, contributing to fine particle emissions levels. More importantly, thermal fracturing of catalyst particles occurred when the cold catalyst was fed into the hot regenerator. Catalyst particles split causing the generation of large amounts of fine particle material, which is easily lost from the system. This loading of catalyst directly linked to the period of high concentration of fine particles in the emissions stream. It was found that metals, and in particular iron, calcium and silicon form a thick layer on the outside of the catalyst, with large irregular shaped metal ridges, forming along the surface of the particle. These ridges reduce the fluidity of the catalyst, leading to potential disruptions in the regenerator. In addition to this, the metal rich ridges are preferentially removed via attrition, causing metal rich material to be emitted into the atmosphere. To overcome these high particle emissions rates from the FCCU the refinery should only use calcinated catalyst to reduce the influence of thermal process and particle fracture and generation. Although the calcinated catalyst can fracture when added to the system, it is far less than that obtained with uncalicinated catalyst. To further reduce the risk of particle fracture due to thermal stresses the refinery should consider reducing the temperature gradients between the hot and cold catalyst. Due to the economics involved with the regenerator, possible pre-warming of catalyst before addition into system is the preferred option. This pre-heating of catalyst should also incorporate a controlled attrition stage to help remove the build up of metals on the surface of the particles whilst allowing this material to be collected before it can be released into the atmosphere. The remove of the metal crust will also improve the fluidity of the system and reduce the chance of catalyst blockages occurring. Finally, modelling of the system has shown that control of key parameters such as particle size and gas velocity are essential to the management of air emissions. The refinery should look at adjusting start up procedures to remove fluctuations in these key parameters. Also the refinery should be careful in using correlation found in the literature to predicted operational conditions in the system as these correlations are misleading when used under industrial situations.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environmental Engineering
Faculty of Environmental Sciences
Full Text

Stripper is an important component of a fluid catalytic cracking (FCCU) unit. It strips and recovers the adsorbed/deposited light hydrocarbons on the catalyst particle surface using steam in a counter current flow process. The common flow problems encountered in an FCC stripper unit include channelling, bridging and maldistribution. In this work the hydrodynamics of industrial scale FCC Strippers was studied and investigated using 3D computational fluid dynamics (CFD) simulations and experiments. The CFD and experimental studies showed that internals and inlet configurations play a vital role in determining the hydrodynamics of the Stripper unit.

Beginning in 1994, the Federal Communication Commission (FCC) started to use auctions for the allocation of scarce radio spectrum licenses. The spectrum auctions have drawn widespread attention as policy makers and economists are interested in how effectively these auctions can raise revenues while promoting efficient allocations. The first chapter provides a broad survey of the PCS auctions and of the developments in Broadband PCS and cellular industries that relate to the PCS auctions. The survey discuss how technology, geography, policy and the firm's industry circumstances affect firms' valuations of the spectrum and are important determinants of the efficient design of the spectrum auctions, as well. The presence of different types of communications providers, cellular providers, wireline providers, and new PCS providers, in the PCS auctions lead to asymmetries in valuations and information. In theory, this structure has a qualitative affect on common value auction outcomes. In laboratory experiments, bidders are found to overbid, much like the findings in previous common value auction experiments, however, the less informed bidders suffer from much stronger overbidding than the informed bidders, and this overbidding can persist over many periods. In the presence of overbidding, additional public information reduced the seller's average revenues. Experience and feedback diminishes but does not eliminate overbidding. In Chapter 3, bidding data from the Interactive Video and Data Service auction, one of the FCC's earliest spectrum auctions are analyzed. Bidding behavior confirms the hypotheses that (1) the larger the area, given the population, the lower the valuation; (2) the larger the population, the higher the income, and the faster the population grows, the higher the valuation; and (3) the number of bidders, the availability of discounts to some bidders, the earlier in the sequence that an auction occurs all positively influence the amount of the winning bid. In addition, licenses in areas with greater population or in auctions with more participating bidders are more likely to be defaulted. Finally, designated entities who receive a bidding discount are significantly more likely to default.

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Bachelors
Sciences
Radio and TV; Broadcast Journalism

Fluorescence spectroscopy and imaging have a very broad spectrum of applicationswithin the life sciences, in particular for detection and characterization ofbiomolecular dynamics and interactions in different environments. This thesis comprisesprojects that strive to further expand the information content extracted fromthe detected fluorescence, leading to sensitive readout parameters for studies ofbiomolecular dynamics and interactions. Two major strategies are presented toachieve this aim. The first strategy is based on the expansion of the availablereadout parameters beyond the "traditional" fluorescence parameters: intensity,wavelength, polarization and fluorescence lifetime. The additional parameters arebased on blinking properties of fluorescent labels. In particular on transitions betweensinglet and triplet states, and transitions between the trans- and cis-isomersof fluorophores. Two publications in the thesis are based on this strategy (paperI and IV). The second strategy is based on the utilization of fluorescence intensityfluctuations in order to detect the oligomerization mechanisms of fluorescentlylabeled peptides and proteins. This strategy combines the intensity fluctuationanalysis and the readout of distance dependent energy transfer between fluorescentmolecules together with the correlation analysis of fluorescence from two labeledproteins emitting at different wavelengths. Another two publications presented inthe thesis are based on the second comprehensive strategy (papers II and III).The work presented in this thesis shows that the blinking kinetics of fluorescentlabels contain significant information that can be exploited by a combination of fluctuationsanalysis with distance dependent excitation energy transfer between thefluorescent molecules, or by analysis of fluorescence covariance between moleculesthat emit at different wavelengths. These fluorescence-based methods have a significantpotential for molecular interaction studies in the biomedical field.

QC 20160527

In order to maintain a multicellular organism cells need to interact and communicate with each other. Signalling cascades such as the Bone Morphogenic Protein (BMP) and Hedgehog (Hh) signalling pathways therefore play essential roles in development and disease. Intercellular signalling also underlies the function of stem cell niches, signalling microenvironments that regulate behaviour of associated stem cells. Range and intensity of the niche signal controls stem cell proliferation and differentation and must therefore be strictly regulated. The testis and ovary of the fruit fly Drosophila melanogaster are established models of stem cell niche biology. In the apical tip of the testis, germ line stem cell (GSCs) and somatic cyst stem cells (CySCs) are arranged around a group of postmitotic somatic cells termed hub. While it is clear which signals regulate GSC maintenance it is unclear how these signals are spatially regulated. Here I show that BMP signalling is specifically activated at the interface of niche and stem cells. This local activation is possible because the transport of signalling and adhesion molecules is coupled and directed towards contact sites between niche and stem cells. I further show that the generation of the BMP signal in the wing disc follows the same mechanism. Hh signalling controls somatic stem cell populations in the Drosophila ovary and the mammalian testis. However, it was unknown what role Hh might play in the fly testis, where the components of this signalling cascade are also expressed. Here I show that overactivation of Hh signalling leads to an increased proliferation and an expansion of the cyst stem cell compartment. Finally, while the major components of the Hh signalling pathway are known, detailed knowledge of how signal transduction is implemented at the cell biological level is still lacking. Here, I show that localisation of the key signal transducer Smo to the plasma membrane is sufficient for phosphorylation of its cytoplasmic tail and downstream pathway activation. Using advanced, microscopy based biophysical methods I further demonstrate that Smo clustering is, in contrast to the textbook model, independent of phosphorylation.

La microscopie de fluorescence est devenue un outil incontournable en biologie. En particulier, il est possible de suivre dans le temps et dans l’espace en cellules vivantes l’activité de protéines en utilisant des biosenseurs FRET génétiquement encodés. Mon travail de thèse a consisté à développer de nouvelles méthodes de microscopie de fluorescence quantitative pour dépasser les limitations rencontrées dans le suivi de biosenseurs FRET. En premier lieu, j’ai eu à développer une méthodologie pour le suivi de deux biosenseurs FRET génétiquement encodés par mesure de durée de vie (FLIM) en simultané avec une seule longueur d’onde d’excitation. En effet, il n’est pas facile de suivre deux activités biochimiques par biosenseurs FRET dans un même échantillon vivant par microscopie de fluorescence à cause de l’existence de fuites spectrales dans les canaux de détection des différentes protéines fluorescentes et l’utilisation de deux longueurs d’onde d’excitation pour chacun des deux donneurs. En combinant les deux couples de protéines fluorescentes mTFP1/ShadowG et LSSmOrange/mKate2, les artefacts de fuite spectrale ont pu être négligés. Il a été possible de suivre les activités kinases des protéines ERK et PKA en simultané par FLIM. À l’aide de cette méthodologie, nous avons pu mettre en évidence une activation de la voie PKA lors d’une stimulation à l’EGF. En second lieu, j’ai développé une méthode pour le suivi de biosenseur FRET par la technique de spectroscopie à corrélation croisée de fluorescence (FCCS). Le suivi d’activité de certaines protéines peut s’avérer difficile du fait de leur faible expression au sein de l’échantillon vivant et de leur localisation. La méthode de FCCS nécessite une faible concentration de fluorophores et peut donc s’adapter à ces échantillons. Le FRET a un effet direct sur l’amplitude de corrélation croisée lorsque celle-ci est mesurée en suivant la co-diffusion de deux protéines fluorescentes attachées à un même biosenseur. Une diminution de ratio d’amplitude des courbes d’autocorrélation (verte ou rouge) sur l’amplitude de la courbe de corrélation croisée correspond à la présence de FRET. Nous avons pu mesurer cette diminution dans des cellules exprimant le biosenseur FRET Aurora A WT (FRET) en comparaison avec un mutant K162M (non FRET). Ces premiers résultats sont très prometteurs pour l’utilisation de cette approche au suivi d’un biosenseur faiblement exprimé en cellules vivantes. L’amélioration du suivi de biosenseur FRET, par les méthodes de microscopie de fluorescence quantitative présentées dans ce travail, doit pouvoir permettre la réponse à des questions d’intérêt biologiques nécessitant le suivi multiplex de FRET ou la mesure de biosenseurs à faible niveau d’expression, là où les techniques conventionnelles se heurtaient à leur limitation
Fluorescence microscopy has become an invaluable tool in biology. In particular, it allows to follow in time and space the activity of proteins, using genetically encoded FRET biosensors, in live cell imaging. In my thesis work, I have developed new quantitative fluorescence microscopy methods to overcome the limitations encountered in monitoring FRET biosensors. First, I developed a methodology to monitor simultaneously two genetically encoded FRET biosensors by lifetime measures (FLIM) with a single excitation wavelength. Previously, it was not easy to follow two biochemical activities by FRET biosensors in the same live sample by fluorescence microscopy. Two reasons for that: the existence of spectral bleed through in the detection channel of each fluorescent proteins and the use of two excitation wavelengths for the two donors. By combining two fluorescent proteins pairs: mTFP1 / ShadowG and LSSmOrange / mKate2, the “spectral bleed through” artifact became negligible. It became then possible to follow the kinase activity of PKA and ERK proteins simultaneously by FLIM. Using this methodology, we were able to show an activation of the PKA pathway upon stimulation with EGF. Second, I developed a method to monitor FRET biosensor by fluorescence cross-correlation spectroscopy technique (FCCS). Monitoring the activity of certain proteins may be difficult due to their low expression in living sample and their sub-cellular localization. The FCCS methods requires a low concentration of fluorophores and can therefore be adapted to these samples. FRET has a direct effect on the cross-correlation amplitude when it is measured by following the co-diffusion of two fluorescent proteins attached to a same biosensor. An amplitude ratio decrease, of the autocorrelation curves (green or red) on the amplitude of the cross-correlation curve, corresponds to the presence of FRET. We were able to measure this ratio decreases in cells expressing the FRET biosensor Aurora A wild type (FRET) compared to the K162M mutant one (non-FRET). These first results are very promising to monitor the activity of a weakly expressed protein in living cells biosensor using this approach. The improvement of FRET biosensor monitoring, by quantitative fluorescence microscopy methods presented in this work, will help to answer biological questions of interest requiring the measurement of multiplex FRET monitoring or biosensors at low level expression, where conventional techniques are facing these limitations

Fluorescence correlation spectroscopy (FCS) analyzes the fluctuations in the fluorescence intensity, which is emitted from a tiny excition volume, to obtain information about the concentration, the mobility, and the molecular interactions of labeled molecules. The more advanced fluorescence cross-correlation spectroscopy (FCCS) increases the precision in the determination of fl ow velocities and binding constants compared to standard FCS. The miniaturization in biomedical and chemical engineering has been developing rapidly, propelled by the vision of a fully functional laboratory on a single chip and its use in human therapeutics, for example, as implanted drug delivery system. A key requirement to fulfill this vision is the ability to handle small fl uid volumes. Handling liquids using the electrohydrodynamical principle circumvents many of the disadvantages of other systems. The complex flow pattern in the active region of such a pump could not be resolved by common tracking techniques. In this thesis, two-focus FCCS (2f-FCCS) was used to map the flow pro file inside a micropump. The high precision of 2f-FCCS in the determination of fl ow measurements even with small fluorescent particles allowed the measurement of the flow velocities induced by electrohydrodynamic forces acting on the solvent, while excluding the effects of dielectrophoretic forces acting on larger particles. Analysis of the fl ow data indicates a fl ow pattern that consists of two vortices of different size and opposite direction of rotation. The flow pattern derived by 2f-FCCS explains the observed complex particle trajectories in the force field and the accumulation of particles in well-de fined regions above the microelectrode array. In the second part of this thesis, the mechanism of RNA interference (RNAi) was studied by dual-color FCCS in vivo. RNAi is an evolutionary conserved gene silencing mechanism, which uses short double-stranded RNA molecules, called short interfering RNAs (siRNAs), as effector molecules. Due to its speci city and simplicity, RNAi yields a great potential for a widespread therapeutic use. To broaden the therapeutic applications, the in vivo stability of siRNAs has to be improved by chemical modi cations, but some of these modi fications inhibit the gene silencing mechanism. The presented FCCS assays are very well suited to investigate the individual assembly steps of RNAi machinery with very high specifi city and sensitivity in real time and to study the cleavage activity of the activated RNAi machinery. A direct correlation between activity of the RNAi machinery and the results from the FCCS measurements could be shown. The in fluence of several chemical modi cations on the assembly and activity of the RNAi machinery was investigated with these assays
Fluoreszenz-Korrelations-Spektroskopie (FCS) analysiert die Fluktuationen im Fluoreszenzsignal eines kleinen angeregten Volumens, um Informationen über die Konzentration, die Bewegung und die Interaktionen der markierten Moleküle zu erhalten. Die Fluoreszenz-Kreuzkorrelations-Spektroskopie (FCCS) erhöht die Genauigkeit bei der Messung von Fließgeschwindigkeiten und Bindungskonstanten im Vergleich zur Standard-FCS. Die Miniaturisierung der Biomedizin und Chemie hat sich rapide entwickelt, angetrieben von der Vision eines kompletten Labors auf einem Chip und dem Einsatz dieses in der medizinischen Therapie, zum Beispiel als implantierter Medikamentenspender. Ein Schlüsselelement zur Erfüllung dieser Vision ist der Transport von kleinsten Flüssigkeitsmengen in diesen miniaturisierten Systemen. Der Transport von Flüssigkeiten mittels des elektrohydrodynamischen Prinzips umgeht viele Nachteile von anderen Systemen, allerdings zeigt eine solche Pumpe ein kompliziertes Strömungsbild in der aktiven Region, welches sich mit herkömmlichen Methoden wie Teilchenverfolgung nicht vermessen ließ. Hier wurde Zwei-Fokus-FCCS (2f-FCCS) genutzt, um das Strömungsbild in der Pumpe zu vermessen. Die hohe Genauigkeit der 2f-FCCS bei der Bestimmung von Fließgeschwindigkeiten auch mit kleinen fluoreszierenden Teilchen ermöglichte die Messung der Fließgeschwindigkeiten, aufgrund der auf das Lösungsmittel wirkenden elektrohydrodynamischen Kräfte, unter Ausschluss der auf größere Teilchen wirkenden dielektrophoretischen Kräfte. Die Analyse der Daten ergab, dass das Strömungsbild aus zwei entgegengesetzt rotierenden unterschiedlich großen Wirbeln besteht. Dieses Strömungsbild erklärt die komplizierten Teilchenbewegungsbahnen und die Anreicherung der Teilchen in klar abgegrenzten Bereichen über den Mikroelektroden. Im zweiten Teil dieser Arbeit wurde der RNAi-Mechanismus in lebenden Zellen mittels Zwei-Farben-FCCS untersucht. RNA Interferenz (RNAi) ist ein evolutionär erhaltener Geninaktivierungsmechanismus, der kurze doppelsträngige RNA Moleküle, so genannte kurze interferierende RNAs (siRNAs), als Effektormoleküle nutzt. Die Spezifi tät und Einfachheit der RNAi hat ihr ein weites Feld in der medikamentösen Therapie geöffnet. Zur Erweiterung dieses Feldes ist es nötig die Stabilität der siRNAs im Körper mittels chemischer Modi fikationen zu erhöhen. Einige dieser Modifikationen hemmen aber den RNAi-Mechanismus. Die hier vorgestellten FCCS Experimente sind sehr gut geeignet, um die einzelnen Schritte des Zusammenbaus der RNAi Maschinerie mit hoher Empfi ndlichkeit und Spezi fität in Echtzeit zu untersuchen und die Aktivität der RNAi Maschinerie zu studieren. Es konnte ein Zusammenhang zwischen der Aktivität der RNAi Maschinerie und den Ergebnissen der FCCS Messungen hergestellt werden. Der Einfluss von verschiedenen chemischen Modikationen auf den Zusammenbau und die Aktivität der RNAi Maschinerie wurde mit diesen neuartigen Methoden untersucht

G-Protein gekoppelte Rezeptoren (GPCR) sind Rezeptoren mit 7 Transmembrandomänen. Nach Bindung ihres Liganden werden über die Kopplung von G-Proteinen rezeptorspezifisch Signaltransduktionswege aktiviert. Ein bislang nicht ausreichend verstandener Prozess für die Funktion von GPCR ist deren Oligomerisierung. Für einige GPCR konnte gezeigt werden, dass die Oligomerisierung den Rezeptortransport und/oder die Dynamik der Rezeptoraktivierung moduliert. Dabei ist noch nicht aufgeklärt, ob die entsprechenden GPCR ausschließlich als Oligomere oder in einem bestimmten Monomer-Dimer Verhältnis (M/D) vorliegen und welcher Dynamik dieses Verhältnis unterliegt. In dieser Arbeit wurde die Homo-Oligomerisierung des Endothelin-B-Rezeptors (ETBR), des Vasopressin-V2-Rezeptors (V2R) und der Corticotropin-Releasing-Factor-Rezeptoren Typ 1 (CRF1R) und Typ 2(a) (CRF2(a)R) analysiert. Im Anschluss an diese Untersuchungen wurde das M/D der GPCR bestimmt. Zur Detektion der Protein-Protein Interaktionen wurden die biophysikalischen Methoden Fluoreszenz-Resonanz-Energie-Transfer (FRET) und Fluoreszenz-Kreuzkorrelations-Spektroskopie (FCCS) eingesetzt. Mit Hilfe der FCCS konnte das spezifische M/D der GPCR bestimmt und über FRET ein Unterschied in der Interaktions-Dynamik zwischen den GPCR der Familie 1 (am Bsp. des V2R) und der Familie 2 (am Bsp. des CRF1R) ermittelt werden. Des Weiteren lieferten die genutzten Methoden den Nachweis, dass der zum CRF1R homologe CRF2(a)R ausschließlich als Monomer vorliegt. Zusätzliche Untersuchungen an Signalpeptidmutanten des CRF1R und des CRF2(a)R weisen darauf hin, dass das Pseudosignalpeptid des CRF2(a)R, welches bislang einzigartig in der Superfamilie der GPCR ist, die Oligomerisierung des Rezeptors verhindert. Zusätzlich zu diesen neuen Daten konnte in dieser Arbeit erstmals ein Zusammenhang zwischen Rezeptorinteraktion und G-Protein Selektivität für den CRF1R und den CRF2(a)R festgestellt werden
The heptahelical G protein-coupled receptors (GPCRs) are important drug targets. Following activation by their ligands, they exert their function via the binding of G proteins and activation of specific signal transduction cascades. To date, the functional significance of the oligomerization of GPCRs is not completely understood. For some GPCRs it could be shown that the oligomerization modulates receptor transport and/or the dynamics of receptor activation. Most importantly, it is not clear whether the GPCRs exist exclusively as oligomers or in a certain monomer-dimer ratio (M/D) or whether a given ratio is dynamic. In this work, the homo-oligomerization of the endothelin-B-receptor (ETBR), the vasopressin-V2-receptor (V2R) and the corticotropin-releasing-factor-receptors type 1 (CRF1R) and type 2(a) (CRF2(a)R) was analysed. In addition, the M/D of these GPCRs was determined. For the detection of the protein-protein interactions, the following biophysical methods were established: fluorescence-resonance-energy-transfer (FRET) and fluorescence-crosscorrelation-spectroscopy (FCCS). With the help of FCCS, a specific M/D could be determined for each of the GPCRs. Using FRET, differences in the interaction dynamics between family 1 (V2R) and family 2 GPCRs (CRF1R) could be described. Moreover, it was experimentally verified that the CRF2(a)R is exclusively expressed as a monomer, in contrast to the other GPCRs and even the highly homologous CRF1R. Using signal peptide swap experiments, it could be demonstrated that the N-terminal pseudo signal peptide of the CRF2(a)R, which is so far unique in the superfamily of GPCRs, prevents oligomerization of the receptor. In addition, a relation of receptor oligomerization and G protein coupling selectivity was established for the CRF1R and the CRF2(a)R which is novel for the GPCR protein family.

碩士
國立臺灣大學
化學研究所
102
Type II topoisomerases are essential enzymes that are involved in chromosome segregation and structural modifications, and in regulating intracellular DNA supercoiling. In the past decades, the enzymatic function of topoisomerase II has been widely studied by biochemists. It acts by transiently breaking one DNA double strand (cleavage), allowing the second double strand to pass through the opened DNA gate, followed by rejoining the broken ends (religation) to close the gate. However, part of the mechanisms still remains unclear. Recently, optical techniques have been applied to investigate the kinetics of topoisomerase, especially single molecule methods. In this study, we describe three applications of confocal spectroscopy: fluorescence cross-correlation spectroscopy (FCCS), Forster resonance energy transfer (FRET) in solution, and single molecule FRET analysis in gel-based system. In our system, we use a unique oligonucleotide substrate with a pair of fluorophore straddling the topoisomerase II cleavage site to monitor the kinetic behavior of this enzyme. FCCS is a biophysical technique that measures fluorescence fluctuations in a confined focal volume to determine diffusion coefficient, and thus, the binding kinetics of DNA and topoisomerase II can be addressed. FRET monitors the distance between a pair of fluorophores on the DNA. The energy transfer efficiency depends on the DNA conformational change induced by the enzyme binding. Both solution and gel-based FRET system can be used to investigate the dynamics of DNA gate during the catalytic cycle. To sum up, our method provides an opportunity to study the individual steps in a Top II catalytic cycle: to measure the binding by FCCS and to monitor catalytic reactions by FRET.