On the car sequencing problem: analysis and solution methods

This work deals with the car sequencing (CS) problem, a combinatorial optimization problem for sequencing mixed-model assembly lines. The aim is to find a production sequence for different variants of a common base product, such that work overload of the respective line operators is avoided or minimized. The variants are distinguished by certain options (e.g., sun roof yes/no) and, therefore, require different processing times at the stations of the line. CS introduces a so-called sequencing rule H:N for each option, which restricts the occurrence of this option to at most H in any N consecutive variants. It seeks for a sequence that leads to no or a minimum number of sequencing rule violations. In this work, CS’ suitability for workload-oriented sequencing is analyzed. Therefore, its solution quality is compared in experiments to the related mixed-model sequencing problem. A new sequencing rule generation approach as well as a new lower bound for the problem are presented. Different exact and heuristic solution methods for CS are developed and their efficiency is shown in experiments. Furthermore, CS is adjusted and applied to a resequencing problem with pull-off tables.

Assembly and characterization of supramolecular architectures for biosensor applications

The research has included the efforts in designing, assembling and structurally and functionally characterizing supramolecular biofunctional architectures for optical biosensing applications. In the first part of the study, a class of interfaces based on the biotin-NeutrAvidin binding matrix for the quantitative control of enzyme surface coverage and activity was developed. Genetically modified ß-lactamase was chosen as a model enzyme and attached to five different types of NeutrAvidin-functionalized chip surfaces through a biotinylated spacer. All matrices are suitable for achieving a controlled enzyme surface density. Data obtained by SPR are in excellent agreement with those derived from optical waveguide measurements. Among the various protein-binding strategies investigated in this study, it was found that stiffness and order between alkanethiol-based SAMs and PEGylated surfaces are very important. Matrix D based on a Nb2O5 coating showed a satisfactory regeneration possibility. The surface-immobilized enzymes were found to be stable and sufficiently active enough for a catalytic activity assay. Many factors, such as the steric crowding effect of surface-attached enzymes, the electrostatic interaction between the negatively charged substrate (Nitrocefin) and the polycationic PLL-g-PEG/PEG-Biotin polymer, mass transport effect, and enzyme orientation, are shown to influence the kinetic parameters of catalytic analysis. Furthermore, a home-built Surface Plasmon Resonance Spectrometer of SPR and a commercial miniature Fiber Optic Absorbance Spectrometer (FOAS), served as a combination set-up for affinity and catalytic biosensor, respectively. The parallel measurements offer the opportunity of on-line activity detection of surface attached enzymes. The immobilized enzyme does not have to be in contact with the catalytic biosensor. The SPR chip can easily be cleaned and used for recycling. Additionally, with regard to the application of FOAS, the integrated SPR technique allows for the quantitative control of the surface density of the enzyme, which is highly relevant for the enzymatic activity. Finally, the miniaturized portable FOAS devices can easily be combined as an add-on device with many other in situ interfacial detection techniques, such as optical waveguide lightmode spectroscopy (OWLS), the quartz crystal microbalance (QCM) measurements, or impedance spectroscopy (IS). Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) allows for an absolute determination of intrinsic rate constants describing the true parameters that control interfacial hybridization. Thus it also allows for a study of the difference of the surface coupling influences between OMCVD gold particles and planar metal films presented in the second part. The multilayer growth process was found to proceed similarly to the way it occurs on planar metal substrates. In contrast to planar bulk metal surfaces, metal colloids exhibit a narrow UV-vis absorption band. This absorption band is observed if the incident photon frequency is resonant with the collective oscillation of the conduction electrons and is known as the localized surface plasmon resonance (LSPR). LSPR excitation results in extremely large molar extinction coefficients, which are due to a combination of both absorption and scattering. When considering metal-enhanced fluorescence we expect the absorption to cause quenching and the scattering to cause enhancement. Our further study will focus on the developing of a detection platform with larger gold particles, which will display a dominant scattering component and enhance the fluorescence signal. Furthermore, the results of sequence-specific detection of DNA hybridization based on OMCVD gold particles provide an excellent application potential for this kind of cheap, simple, and mild preparation protocol applied in this gold fabrication method. In the final chapter, SPFS was used for the in-depth characterizations of the conformational changes of commercial carboxymethyl dextran (CMD) substrate induced by pH and ionic strength variations were studied using surface plasmon resonance spectroscopy. The pH response of CMD is due to the changes in the electrostatics of the system between its protonated and deprotonated forms, while the ionic strength response is attributed from the charge screening effect of the cations that shield the charge of the carboxyl groups and prevent an efficient electrostatic repulsion. Additional studies were performed using SPFS with the aim of fluorophore labeling the carboxymethyl groups. CMD matrices showed typical pH and ionic strength responses, such as high pH and low ionic strength swelling. Furthermore, the effects of the surface charge and the crosslink density of the CMD matrix on the extent of stimuli responses were investigated. The swelling/collapse ratio decreased with decreasing surface concentration of the carboxyl groups and increasing crosslink density. The study of the CMD responses to external and internal variables will provide valuable background information for practical applications.

Mixed finite-element methods for elliptic convection-dominated problems arising in semiconductor physics

In this work we develop and analyze an adaptive numerical scheme for simulating a class of macroscopic semiconductor models. At first the numerical modelling of semiconductors is reviewed in order to classify the Energy-Transport models for semiconductors that are later simulated in 2D. In this class of models the flow of charged particles, that are negatively charged electrons and so-called holes, which are quasi-particles of positive charge, as well as their energy distributions are described by a coupled system of nonlinear partial differential equations. A considerable difficulty in simulating these convection-dominated equations is posed by the nonlinear coupling as well as due to the fact that the local phenomena such as "hot electron effects" are only partially assessable through the given data. The primary variables that are used in the simulations are the particle density and the particle energy density. The user of these simulations is mostly interested in the current flow through parts of the domain boundary - the contacts. The numerical method considered here utilizes mixed finite-elements as trial functions for the discrete solution. The continuous discretization of the normal fluxes is the most important property of this discretization from the users perspective. It will be proven that under certain assumptions on the triangulation the particle density remains positive in the iterative solution algorithm. Connected to this result an a priori error estimate for the discrete solution of linear convection-diffusion equations is derived. The local charge transport phenomena will be resolved by an adaptive algorithm, which is based on a posteriori error estimators. At that stage a comparison of different estimations is performed. Additionally a method to effectively estimate the error in local quantities derived from the solution, so-called "functional outputs", is developed by transferring the dual weighted residual method to mixed finite elements. For a model problem we present how this method can deliver promising results even when standard error estimator fail completely to reduce the error in an iterative mesh refinement process.

Ion channels in mixed tethered bilayer lipid membranes

Mixed tethered bilayer lipid membranes (tBLMs) are described based on the self-assembly of a monolayer on template stripped gold, of an archea analogue thiolipid, 2,3-di-o-phytanyl-sn-glycerol-1-tetraethylene glycol-D,L--lipoic acid ester lipid (DPTL), and a newly designed dilution molecule, tetraethylene glycol-D,L--lipoic acid ester (TEGL). The usage of spacer and addition of extra dilution molecules between the substrate and the bilayer is that this architecture provides an ionic reservoir underneath the membrane, avoiding direct contact of the embedded membrane proteins with the gold electrodes and increasing the lateral diffusion of the bilayer, thus allowing for the incorporation of complex channels proteins which are failed in non-diluted systems. The tBLM is completed by fusion of liposomes made from a mixture of 1,2-diphythanolyl-sn-glycero-3-phosphocholine (DPhyPC), cholesterol, and 1,2-diphytanoyl-sn-Glycero-3-phosphate (DPhyPG) in a molar ratio of 6:3:1. Varying the mixing ratio, the optimum mixing ratio was obtained at a dilution factor of DPTL and TEGL at 90%:10%. Only under these conditions, the mixed tBLM showed electrical properties, as shown by EIS, which are comparable to a BLM. With higher dilution factors, a defect-free lipid bilayer was not formed. Formation of bilayers have been characterized by different techniques, such as surface plasmon resonance (SPR), electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM), and quartz crystal microbalance (QCM). Different proteins such as hemolysin, melittin, gramicidin, M2, Maxi-K, nAChR and bacteriohodopsin are incorporated into these tBLMs as shown by SPR and EIS studies. Ionic conductivity at 0 V vs. Ag|AgCl, 3M KCl were measured by EIS measurements. Our results indicate that these proteins have been successfully incorporated into a very stable tBLM environment in a functionally active form. Therefore, we conclude that the mixed tBLMs have been successfully designed as a general platform for biosensing and screening purposes of membrane proteins.

Fluorescent ionene-dye nanoparticles by electrostatic self-assembly

It is investigated that the association of linear cationic model polyelectrolytes with oppositely charged pyrenetetrasulfonate (PY) in aqueous solution. For this purpose water soluble ionenes were prepared via Menschutkin reaction from 1-4-diazabicyclo [2.2.2] octane and e.g. 1,6-dibromohexane and 1,4 dibromotransbuten. The complex formation between dye molecules PY and oppositely charged ionenes (PD4, PD6, PD4-2 and PD4coPD6) of different chemical structures in aqueous solution was studied by light scattering (LS), small angle neutron scattering (SANS), UV-Vis, fluorescence spectroscopy and atomic force microscopy (AFM). Spectrophotometric titration results revealed that PY molecules were bind to ionenes cooperative process due to π-π interaction. Cooperative binding constant KD was determined as 6.4 x 10^6 M^-1 (+ or - 10^5 M^-1). It was found that binding mode and geometry of PY is predominantly depending on inter-charge distances of corresponding ionenes. Resultant complexes have exhibited size and structure variation as a function of charge ratio (L), ionic strength, inter-charge distances. Spherical dye-ionene complexes of which radius of gyration ranging between (RG) 50 and 190 nm have been observed in PD4-PY system while this was not possible with a different ionene (PD6) or either case ionene excess. It was found that most of the PD4-PY complexes had RG / RH ~ 0.78. Based on the AFM and LS results, spherical complexes have certain colloidal stability and their size can effectively controlled by changing the L.

Untersuchung der Struktur und Assemblierung des Lichtsammelkomplexes II höherer Pflanzen mittels elektronenparamagnetischer Resonanz (EPR)

Der Haupt-Lichtsammelkomplex (LHCII) des Photosyntheseapparates höherer Pflanzen gehört zu den häufigsten Membranproteinen der Erde. Seine Kristallstruktur ist bekannt. Das Apoprotein kann rekombinant in Escherichia coli überexprimiert und somit molekularbiologisch vielfältig verändert werden. In Detergenzlösung besitzt das denaturierte Protein die erstaunliche Fähigkeit, sich spontan zu funktionalen Protein-Pigment-Komplexen zu organisieren, welche strukturell nahezu identisch sind mit nativem LHCII. Der Faltungsprozess findet in vitro im Zeitbereich von Sekunden bis Minuten statt und ist abhängig von der Bindung der Cofaktoren Chlorophyll a und b sowie verschiedenen Carotinoiden.rn Diese Eigenschaften machen LHCII besonders geeignet für Strukturuntersuchungen mittels der elektronenparamagnetischen Resonanz (EPR)-Spektrokopie. Diese setzt eine punktspezifische Spinmarkierung des LHCII voraus, die in dieser Arbeit zunächst optimiert wurde. Einschließlich der Beiträge Anderer stand eine breite Auswahl von über 40 spinmarkierten Mutanten des LHCII bereit, einen N-terminalen „Cys walk“ eingeschlossen. Weder der hierfür notwendige Austausch einzelner Aminosäuren noch die Anknüpfung des Spinmarkers beeinträchtigten die Funktion des LHCII. Zudem konnte ein Protokoll zur Präparation heterogen spinmarkierter LHCII-Trimere entwickelt werden, also von Trimeren, die jeweils nur ein Monomer mit einer Spinmarkierung enthalten.rn Spinmarkierte Proben des Detergenz-solubilisierten LHCII wurden unter Verwendung verschiedener EPR-Techniken strukturell analysiert. Als besonders aussagekräftig erwies sich die Messung der Wasserzugänglichkeit einzelner Aminosäurepositionen anhand der Electron Spin Echo Envelope Modulation (ESEEM). In Kombination mit der etablierten Double Electron-Electron Resonance (DEER)-Technik zur Detektion von Abständen zwischen zwei Spinmarkern wurde der membranständige Kernbereich des LHCII in Lösung eingehend untersucht und strukturell der Kristallstruktur für sehr ähnlich befunden. Die Vermessung kristallographisch nicht erfasster Bereiche nahe dem N-Terminus offenbarte die schon früher detektierte Strukturdynamik der Domäne in Abhängigkeit des Oligomerisierungsgrades. Der neue, noch zu vervollständigende Datensatz aus Abstandsverteilungen und ESEEM-Wasserzugänglichkeiten monomerer wie trimerer Proben sollte in naher Zukunft die sehr genaue Modellierung der N-terminalen Domäne des LHCII ermöglichen.rn In einem weiteren Abschnitt der Arbeit wurde die Faltung des LHCII-Apoproteins bei der LHCII-Assemblierung in vitro untersucht. Vorausgegangene fluoreszenzspektroskopi-sche Arbeiten hatten gezeigt, dass die Bindung von Chlorophyll a und b in aufeinanderfolgenden Schritten im Zeitbereich von weniger als einer Minute bzw. mehreren Minuten erfolgten. Sowohl die Wasserzugänglichkeit einzelner Aminosäurepositionen als auch Spin-Spin-Abstände änderten sich in ähnlichen Zeitbereichen. Die Daten deuten darauf hin, dass die Ausbildung der mittleren Transmembran-Helix mit der schnelleren Chlorophyll-a-Bindung einhergeht, während sich die Superhelix aus den beiden anderen Transmembranhelices erst im langsameren Schritt, zusammen mit der Chlorophyll-b-Bindung, ausbildet.rn

Structure and properties of nanostructures from polyelectrolytes and porphyrins: Self-Assembly in aqueous solution

In this work self-assembling model systems in aqueous solution were studied. The systems contained charged polymers, polyelectrolytes, that were combined with oppositely charged counterions to build up supramolecular structures. With imaging, scattering and spectroscopic techniques it was investigated how the structure of building units influences the structure of their assemblies. Polyelectrolytes with different chemical structure, molecular weight and morphology were investigated. In addition to linear polyelectrolytes, semi-flexible cylindrical bottle-brush polymers that possess a defined cross-section and a relatively high persistence along the backbone were studied. The polyelectrolytes were combined with structural organic counterions having charge numbers one to four. Especially the self-assembly of polyelectrolytes with different tetravalent water-soluble porphyrins was studied. Porphyrins have a rigid aromatic structure that has a structural effect on their self-assembly behavior and through which porphyrins are capable of self-aggregation via π-π interaction. The main focus of the thesis is the self-assembly of cylindrical bottle-brush polyelectrolytes with tetravalent porphyrins. It was shown that the addition of porphyrins to oppositely charged brush molecules induces a hierarchical formation of stable nanoscale brush-porphyrin networks. The networks can be disconnected by addition of salt and single porphyrin-decoratedrncylindrical brush polymers are obtained. These two new morphologies, brush-porphyrin networks and porphyrin-decorated brush polymers, may have potential as functional materials with interesting mechanical and optical properties.

Wet and dry model granulates under mechanical load : a confocal microscopy study

Granular matter, also known as bulk solids, consists of discrete particles with sizes between micrometers and meters. They are present in many industrial applications as well as daily life, like in food processing, pharmaceutics or in the oil and mining industry. When handling granular matter the bulk solids are stored, mixed, conveyed or filtered. These techniques are based on observations in macroscopic experiments, i.e. rheological examinations of the bulk properties. Despite the amply investigations of bulk mechanics, the relation between single particle motion and macroscopic behavior is still not well understood. For exploring the microscopic properties on a single particle level, 3D imaging techniques are required.rnThe objective of this work was the investigation of single particle motions in a bulk system in 3D under an external mechanical load, i.e. compression and shear. During the mechanical load the structural and dynamical properties of these systems were examined with confocal microscopy. Therefor new granular model systems in the wet and dry state were designed and prepared. As the particles are solid bodies, their motion is described by six degrees of freedom. To explore their entire motion with all degrees of freedom, a technique to visualize the rotation of spherical micrometer sized particles in 3D was developed. rnOne of the foci during this dissertation was a model system for dry cohesive granular matter. In such systems the particle motion during a compression of the granular matter was investigated. In general the rotation of single particles was the more sensitive parameter compared to the translation. In regions with large structural changes the rotation had an earlier onset than the translation. In granular systems under shear, shear dilatation and shear zone formation were observed. Globally the granular sediments showed a shear behavior, which was known already from classical shear experiments, for example with Jenike cells. Locally the shear zone formation was enhanced, when near the applied load a pre-diluted region existed. In regions with constant volume fraction a mixing between the different particle layers occurred. In particular an exchange of particles between the current flowing region and the non-flowing region was observed. rnThe second focus was on model systems for wet granular matter, where an additional binding liquid is added to the particle suspension. To examine the 3D structure of the binding liquid on the micrometer scale independently from the particles, a second illumination and detection beam path was implemented. In shear and compression experiments of wet clusters and bulk systems completely different dynamics compared to dry cohesive models systems occured. In a Pickering emulsion-like system large structural changes predominantly occurred in the local environment of binding liquid droplets. These large local structural changes were due to an energy interplay between the energy stored in the binding droplet during its deformation and the binding energy of particles at the droplet interface. rnConfocal microscopy in combination with nanoindentation gave new insights into the single particle motions and dynamics of granular systems under a mechanical load. These novel experimental results can help to improve the understanding of the relationship between bulk properties of granular matter, such as volume fraction or yield stress and the dynamics on a single particle level.rnrn