Magnetosome-specific expression of chimeric proteins in Magnetospirillum gryphiswaldense for applications in cell biology and biotechnology

Magnetosomes are magnetic nanoparticles that are formed by magnetotactic bacteria (MTB) by a complex, genetically controlled biomineralization process. Magnetosomes from the model organism Magnetospirillum gryphiswaldense consist of single-magnetic-domain sized nanocrystals of chemically pure magnetite, which are formed intracellularly within specialized membranous compartments. The natural coating by the biological membrane and the defined physico-chemical properties designate magnetosomes as a biogenic material with high bio- and nanotechnological potential. In addition, there is a great interest in the cell biology of magnetosome formation in MTB. The development of these true bacterial organelles involves the invagination of distinctly sized membrane vesicles and the assembly of magnetosome vesicles in chain-like arrangements along novel cytoskeletal structures. The first part of this thesis focussed on the development of genetic tools for the functionalization and expression of modified magnetosome proteins. The identification of proteins that are specifically and efficiently inserted into the magnetosome membrane (MM) was facilitated by analysis of green fluorescent protein (GFP) fusions of different magnetosome membrane proteins (MMP). After optimization of cultivation conditions for the utilization of GFP in MTB, it has been demonstrated that fusions of the proteins MamC, MamF and MamG are specifically targeted to the MM. In particular, the MamC-GFP fusion protein was stably integrated and highly abundant in the MM. Therefore, MamC represents an ideal anchor protein for the immobilization of functional proteins in the MM. To address the question, if a specific signal sequence determines the magnetosome specific targeting of MamC-GFP, the localization of truncated MamC derivatives was studied. These experiments have shown that, except for the last nine C-terminal amino acids, the entire sequence is required for the correct targeting and membrane insertion of MamC. Stability of MamC-GFP is greatly reduced if larger parts are missing or if the N-terminus is deleted. MamC-GFP localized at the expected position of the magnetosome chain irrespective of cultivation conditions that impeded magnetite formation. This shows that MMP targeting, magnetosome vesicle formation and magnetosome chain assembly are not dependent on the prevalence of magnetite inducing conditions or the presence of magnetite crystals. In contrast, the localization of MamC-GFP was altered in the magnetic mamK as well as in the non-magnetic MSR-1B, mamB, mamM, mamJKL mutants in comparison to the wild type. This indicates that the interaction with specific proteins in the magnetosome vesicle is required for the correct localization of MamC. The spotted MamC-GFP signals in the mamJ mutant, which are congruent with the position of magnetosomes in this strain, indicate that MamJ is not required for the magnetosome-specific targeting of MamC-GFP. It has also been demonstrated that the native MamC protein and other proteins encoded by the mamGFDC operon are not required for the magnetosome-directed targeting of MamC, as the localization patterns of MamC-GFP in the mamC and mamGFDC mutants were similar to the localization of MamC-GFP in the wild type and congruent with the position of the magnetosomes. The comparison of different promoters from E. coli and M. gryphiswaldense by fluorometry and flow cytometry with a GFP-reporter system revealed that the magnetosomal promoter, PmamDC, is highly efficient in M. gryphiswaldense. The applicability of this promoter for the functionalization of magnetosomes has been demonstrated by expression of a fusion protein of MamC and the antibody binding ‘ZZ’ protein in the MM to generate antibody-binding magnetosomes. In addition, the E. coli Ptet promoter has been identified as the first inducible promoter for regulated gene expression in MTB. The expression was tightly regulated in the absence of an inducer and a ten-fold increase of the proportion of fluorescent cells was observed in the presence of the inducer anhydrotetracycline. Therefore, the Ptet promoter is an important addition to the M. gryphiswaldense genetic toolbox. In the second part of this thesis, magnetosomes were tested for their use in biomedical and biotechnological applications. To this end, large scale procedures for the purification of intact magnetosomes were developed. In collaboration with the groups of Prof. Dr. C. M. Niemeyer (Universität Dortmund) and Dr. R. Wacker (Chimera Biotec), streptavidin-biotin chemistry was employed to develop a modular system for the production of DNA- and antibody-coated magnetosomes. The modified magnetosomes were used in DNA- and protein detection systems, and an automatable magnetosome-based Magneto-Immuno-PCR procedure was developed for the sensitive detection of antigens. With collaborators from the groups of Dr. T. Hieronymus (RWTH Aachen) and Dr. I. Hilger (Universität Jena), it has been shown that magnetosomes can be used as specific magnetic resonance imaging (MRI) contrast agents for phagocytotic cells such as macrophages and dendritic cells to study cell migration. Fluorescently labelled magnetosomes were successfully used as bimodal contrast agents for the visualization of labelled cells by MRI and fluorescence imaging

Quantitative Analysis of Relaxation Rate Dependence on Interecho Time in MagA-expressing, Iron-labeled Cells

Reporter gene-based methods of labeling cells with iron is an emerging method of providing magnetic resonance imaging (MRI) contrast for long-term cell tracking and monitoring of cellular activities. This thesis investigates 9.4 T NMR properties of mammalian cells over-expressing a magnetotactic bacterial putative iron transport gene, MagA, and the associated untransfected parental cells. Cells were cultured in medium alone or supplemented with 250 μM ferric nitrate. Using the Carr-Purcell-Meiboom-Gill sequence, the relationship between R2 and interecho time was analyzed for each of the cell types using a model based on water diffusion in weak magnetic field inhomogeneities (Jensen and Chandra, 2000) as well as a fast-exchange model (Luz and Meiboom, 1963). Iron levels were assessed with inductively-coupled plasma mass spectrometry. As expected from previous work, the iron content in iron-supplemented, MagA-expressing cells was higher than the unsupplemented or parental cell lines. With regard to NMR, increases in R2 with increasing interecho time were typically greatest in the cells containing higher iron content. The dependence of R2 on interecho time in iron-supplemented, MagA-expressing cells was better represented by the Jensen-Chandra model compared to the Luz-Meiboom model, which is consistent with comparisons of these models in iron-containing tissues. On the other hand, the Luz-Meiboom model performed better than the Jensen-Chandra model for the remaining cell types. These findings provide insight into the high field relaxation mechanisms present in cells expressing a candidate MR reporter gene, which should be valuable for optimizing MRI contrast for long-term cell tracking and monitoring of cellular activities

Magnetosome-specific expression of chimeric proteins in Magnetospirillum gryphiswaldense for applications in cell biology and biotechnology

Magnetosomes are magnetic nanoparticles that are formed by magnetotactic bacteria (MTB) by a complex, genetically controlled biomineralization process. Magnetosomes from the model organism Magnetospirillum gryphiswaldense consist of single-magnetic-domain sized nanocrystals of chemically pure magnetite, which are formed intracellularly within specialized membranous compartments. The natural coating by the biological membrane and the defined physico-chemical properties designate magnetosomes as a biogenic material with high bio- and nanotechnological potential. In addition, there is a great interest in the cell biology of magnetosome formation in MTB. The development of these true bacterial organelles involves the invagination of distinctly sized membrane vesicles and the assembly of magnetosome vesicles in chain-like arrangements along novel cytoskeletal structures. The first part of this thesis focussed on the development of genetic tools for the functionalization and expression of modified magnetosome proteins. The identification of proteins that are specifically and efficiently inserted into the magnetosome membrane (MM) was facilitated by analysis of green fluorescent protein (GFP) fusions of different magnetosome membrane proteins (MMP). After optimization of cultivation conditions for the utilization of GFP in MTB, it has been demonstrated that fusions of the proteins MamC, MamF and MamG are specifically targeted to the MM. In particular, the MamC-GFP fusion protein was stably integrated and highly abundant in the MM. Therefore, MamC represents an ideal anchor protein for the immobilization of functional proteins in the MM. To address the question, if a specific signal sequence determines the magnetosome specific targeting of MamC-GFP, the localization of truncated MamC derivatives was studied. These experiments have shown that, except for the last nine C-terminal amino acids, the entire sequence is required for the correct targeting and membrane insertion of MamC. Stability of MamC-GFP is greatly reduced if larger parts are missing or if the N-terminus is deleted. MamC-GFP localized at the expected position of the magnetosome chain irrespective of cultivation conditions that impeded magnetite formation. This shows that MMP targeting, magnetosome vesicle formation and magnetosome chain assembly are not dependent on the prevalence of magnetite inducing conditions or the presence of magnetite crystals. In contrast, the localization of MamC-GFP was altered in the magnetic mamK as well as in the non-magnetic MSR-1B, mamB, mamM, mamJKL mutants in comparison to the wild type. This indicates that the interaction with specific proteins in the magnetosome vesicle is required for the correct localization of MamC. The spotted MamC-GFP signals in the mamJ mutant, which are congruent with the position of magnetosomes in this strain, indicate that MamJ is not required for the magnetosome-specific targeting of MamC-GFP. It has also been demonstrated that the native MamC protein and other proteins encoded by the mamGFDC operon are not required for the magnetosome-directed targeting of MamC, as the localization patterns of MamC-GFP in the mamC and mamGFDC mutants were similar to the localization of MamC-GFP in the wild type and congruent with the position of the magnetosomes. The comparison of different promoters from E. coli and M. gryphiswaldense by fluorometry and flow cytometry with a GFP-reporter system revealed that the magnetosomal promoter, PmamDC, is highly efficient in M. gryphiswaldense. The applicability of this promoter for the functionalization of magnetosomes has been demonstrated by expression of a fusion protein of MamC and the antibody binding ‘ZZ’ protein in the MM to generate antibody-binding magnetosomes. In addition, the E. coli Ptet promoter has been identified as the first inducible promoter for regulated gene expression in MTB. The expression was tightly regulated in the absence of an inducer and a ten-fold increase of the proportion of fluorescent cells was observed in the presence of the inducer anhydrotetracycline. Therefore, the Ptet promoter is an important addition to the M. gryphiswaldense genetic toolbox. In the second part of this thesis, magnetosomes were tested for their use in biomedical and biotechnological applications. To this end, large scale procedures for the purification of intact magnetosomes were developed. In collaboration with the groups of Prof. Dr. C. M. Niemeyer (Universität Dortmund) and Dr. R. Wacker (Chimera Biotec), streptavidin-biotin chemistry was employed to develop a modular system for the production of DNA- and antibody-coated magnetosomes. The modified magnetosomes were used in DNA- and protein detection systems, and an automatable magnetosome-based Magneto-Immuno-PCR procedure was developed for the sensitive detection of antigens. With collaborators from the groups of Dr. T. Hieronymus (RWTH Aachen) and Dr. I. Hilger (Universität Jena), it has been shown that magnetosomes can be used as specific magnetic resonance imaging (MRI) contrast agents for phagocytotic cells such as macrophages and dendritic cells to study cell migration. Fluorescently labelled magnetosomes were successfully used as bimodal contrast agents for the visualization of labelled cells by MRI and fluorescence imaging

DEVELOPMENT OF A LINE-FIELD MAGNETO-MOTIVE OPTICAL COHERENCE TOMOGRAPHY SYSTEM

The mechanism by which certain species of animals are able to detect the Earth’s magnetic field has remained a mystery for as long as we have known that they exhibit geomagnetic navigation. Certain species of bacteria are known to contain single chains of magnetite crystals, each with a diameter of ~50 nm, that are used to orient the bacteria. Searching for similar magnetoreceptors in larger animals requires a high-speed, high-resolution imaging system with the ability to detect single magnetic nanoparticles. Optical coherence tomography (OCT) is a biomedical imaging modality that produces 2D, cross-sectional images of optically turbid media with a resolution on the order of 1-10 µm. Magneto-motive OCT (MMOCT) is a functional form of OCT that can detect the sub-resolution displacement of magnetic nano- or micro-particles embedded in weakly diamagnetic, optically scattering, elastic media (such as human and animal tissues) subject to a sinusoidally-varying magnetic gradient force. This dissertation describes the design and implementation of an MMOCT system composed of a novel combination of a line-field configuration with a supercontinuum light source and a faster MMOCT imaging scheme. The combination of the line illumination with a high-speed 2D camera and the low-noise, high-power supercontinuum light source produces the best combination of axial resolution, optical SNR, and imaging speed of any line-field-OCT (LFOCT) system to date. The performance of the LF-OCT system combined with the faster magnet modulation scheme results in a LF-MMOCT system with a volumetric imaging speed comparable to that of the highest speed MMOCT system to date. High volumetric imaging speed is essential for the problem of endogenous magnetite detection, as is high magnetic sensitivity. The LF-MMOCT system is optimized to produce the best possible magnetic SNR at kilohertz framerates. We then demonstrate the detection of single magnetic point particles, measure the vibration amplitude produced by an external magnetic gradient force on each point particle, and compare that vibration amplitude to a theoretical value. The ability to image a single magnetic point particle with a high-resolution, high-sensitivity, and high-speed LF-MMOCT system provides a key proof of concept that this system may be used for endogenous magnetite detection.Doctor of Philosoph

Quantitative Interpretation of Magnetic Measurements in Archaeological Prospecting

The non-destructive investigation of archaeological sites with magnetic gradiometry is of great importance for archaeological research since the layout of the complete site can be mapped. However, in most case studies the interpretation solely consists of an image interpretation. This does not exploit the full potential of the method since no quantitative model of the magnetic source bodies, i.e. the archaeological features, is derived. Therefore, we have developed site-specific inversion approaches for a quantitative interpretation of magnetic measurements. The inversion approaches need to be customized to the characteristics of the features to reduce ambiguity and consequently be able to yield suitable results. This thesis targets two archaeological sites with each a specific inversion approach. The site Maidanetske (Ukraine; ~ 3950 - 3650 BCE) belongs to the Chalcolithic Cucuteni-Tripolye culture. The site comprises remains of approx. 3000 houses, that are mostly burned. Forward calculations of documented finds identify the burned clay (daub) as source of the magnetic anomalies. The daub is concentrated in a distinct depth range and the characteristics of this layer are used as a priori information in the inversion computations. We neglect the magnetization of the surrounding material under the assumption of a much smaller magnetization than in the burned layer. Moreover, we restrict the depth range of the magnetized layer to the depth range of the daub layer. Via inversion computations the magnetization of this layer is calculated. The magnetization distribution of three excavated buildings is compared to the mass distribution of daub to infer a magnetization-mass-relation. The quantitative interpretation of not excavated buildings then comprises two steps: the calculation of the magnetization distribution; and the application of magnetization-mass-relation to infer the total mass. The evaluation of total masses of 45 not excavated buildings indicated two different sets of buildings, possibly related to different construction types. At the Linearbandkeramik site Vráble (Slovakia; ~ 5250 - 4950 cal BCE), the remains of the houses are accumulations of pits forming a longpit at each side of the former building. The pits were dug into the Loess and are filled with material related to the use of the house. Multi-method geophysical measurements were conducted during an excavation as part of the documentation. The joint interpretation of ground penetrating radar and electromagnetic induction measurements enabled us to image the bottom of the pits with their distinct microtopography related to their evolution. The geophysical measurements show that the bottom of the pits is in greater depth than expected due to the archaeological excavation. Moreover, we show that the observed magnetic anomalies can not be explained solely by induced magnetization. This conclusion is derived from forward calculations of two-dimensional susceptibility distributions that were measured downhole alongprofiles of densely spaced drillings crossing the pits. We derive the remanent magnetization with an inversion approach based on the susceptibility distribution. The remanent magnetization is described by the Koenigsberger ratio. For the six coring profiles, the mean Koenigsberger ratio varies between 1.8 and 7.0 with most values smaller than 4.0. Considering geoarchaeological data, the source of the remanent magnetization is determined as magnetotactic bacteria that increase the amount of ferrimagnetic iron compounds in the pit filling.Die zerstörungsfreie Erkundung archäologischer Fundorte mit magnetischen Gradiometermessungen ist von großer Bedeutung für die Archäologie, da diese ermöglichen einen Gesamtplan des Fundorts zu erstellen. Bislang beschränkt sich in vielen Fallbeispielen die Auswertung auf die Bildinterpretation der kartierten Messwerte. Dies schöpft nicht das volle Potential der Magnetik aus. Es fehlt die Bestimmung eines quantitativen Modells der magnetischen Störkörper, d.h. der archäologischen Befunde. Die hier erarbeiteten Inversionskonzepte, angepasst an den jeweiligen Fundort, zur quantitativen Auswertung der Messungen nutzen dieses vernachlässigte Potential. Die Inversionsrechnungen müssen die Charakteristika der jeweiligen archäologischen Überreste beachten, um die Mehrdeutigkeit magnetischer Messungen einzuschränken. Diese Arbeit untersucht zwei archäologische Fundorte mit unterschiedlichen Befunden. Der Fundort Maidanetske (Ukraine; ~ 3950 - 3650 BCE) gehört zur kupferzeitlichen Cucuteni-Tripolye Kultur. Er umfasst die Überreste von etwa 3000, meist verbrannten, Häusern. Mittels Vorwärtsrechnungen der dokumentierten Funde wurde der Brandlehm als Quelle der magnetischen Anomalien bestimmt. Der Brandlehm befindet sich in einem diskreten Tiefenbereich und die Charakteristika dieser Schicht werden als a priori Informationen für die Inversion verwendet. Auf Grund der Annahme, dass die Magnetisierung des umgebenden Materials wesentlich geringer ist als diejenige der Brandlehmschicht, wird erstere vernachlässigt. Der Tiefenbereich der magnetisierten Schicht wird auf denjenigen der Brandlehmschicht begrenzt. Über Inversion wird die Magnetisierungsverteilung dieser Schicht bestimmt. Der Vergleich der Magnetisierungsverteilung dreier ausgegrabener Häuser mit der Massenverteilung des Brandlehms liefert eine empirische Beziehung zwischen diesen Größen. Zur quantitativen Interpretation nicht ausgegrabener Häuser wird zunächst deren Magnetisierungsverteilung berechnet und dann über die Magnetisierungs-Massen-Beziehung die Gesamtmasse bestimmt. Die Auswertung der Gesamtmassen von 45 nicht ausgegrabenen Häusern lässt auf zwei unterschiedliche Gruppen schließen, die möglicherweise auf zwei unterschiedliche Bauweisen hindeuten. Die Überreste der Häuser der linearbandkeramischen Siedlung Vráble (Slowakische Republik; ~ 5250 - 4950 cal BCE), bestehen aus Ansammlungen von Gruben, die sich zu Längsgruben entlang beider Seiten der ehemaligen Gebäude zusammensetzen. Die Gruben wurden in den Löss gegraben und sind nun mit Material verfüllt, das in Bezug zum jeweiligen Haus steht. Als Teil der Grabungsdokumentation wurden Multimethodenmessungen in der Ausgrabung durchgeführt. Die gemeinsame Interpretationvon Bodenradar- und elektromagnetischen Induktionsmessungen ermöglicht die Abbildung der Unterkante der Gruben inklusive deren Mikrotopographie, die die schrittweise Entstehung der Längsgruben belegt. Die geophysikalischen Messungen bestimmen die Unterkante der Gruben in einer größeren Tiefe als in der Ausgrabung erwartet. Außerdem zeigt sich, dass die gemessenen Magnetikanomalien nicht durch ausschließlich induzierte Magnetisierung erklärt werden können. Diese Schlussfolgerung resultiert aus Vorwärtsrechnungen basierend auf zweidimensionalen Suszeptibilitätsverteilungen, die in Bohrlöchern entlang von Profilen aus dicht platzierten Bohrpunkten durch die Gruben gemessen wurden. Die remanente Magnetisierung wird daraufhin über eine Inversion basierend auf der Suszeptibilitätsverteilung bestimmt und durch das Königsberger Verhältnis beschrieben. Für die sechs untersuchten Bohrprofile ergeben sich Werte des mittleren Königsbergerverhältnis zwischen 1.8 und 7.0, wobei die meisten Werte kleiner als 4.0 sind. Mittels geoarchäologischer Daten werden magnetotaktische Bakterien in der Grubenfüllung als Ursache der Remanenz bestimmt, da sie den Anteil von ferrimagnetischen Eisenverbindung erhöhen

V Jornadas de Investigación de la Facultad de Ciencia y Tecnología. 2016

171 p.I. Abstracts. Ahozko komunikazioak / Comunicaciones orales: 1. Biozientziak: Alderdi Molekularrak / Biociencias: Aspectos moleculares. 2. Biozientziak: Ingurune Alderdiak / Biociencias: Aspectos Ambientales. 3. Fisika eta Ingenieritza Elektronika / Física e Ingeniería Electrónica. 4. Geología / Geología. 5. Matematika / Matemáticas. 6. Kimika / Química. 7. Ingenieritza Kimikoa eta Kimika / Ingeniería Química y Química. II. Abstracts. Idatzizko Komunikazioak (Posterrak) / Comunicaciones escritas (Pósters): 1. Biozientziak / Biociencias. 2. Fisika eta Ingenieritza Elektronika / Física e Ingeniería Electrónica. 3. Geologia / Geologia. 4. Matematika / Matemáticas. 5. Kimika / Química. 6. Ingenieritza Kimikoa / Ingeniería Química

Observing magnetic objects in fluids

Observation of the motion of particles in fluids give valuable information about the particles, the environment and the interaction between them. Two distinct particle-fluid systems were studied in this framework. The first system considers centimetre-sized magnetic particles suspended in an upward water flow to create neutral buoyancy as well as a source of turbulence. This macroscopic reactor acts as an analogue simulator for microscopic self-assembly processes. From observation of the trajectories of single and two-particle systems we found that in terms of velocity, diffusion and particle interaction the laws of thermodynamics describe the macroscopic system with surprising accuracy. We have shown that we can control the amount of disturbing energy by changing the asymmetry of the water inflow, but that this method affects the particle behaviour differently in separate spatial dimensions. We found that the method used to generate disturbing energy is not that critical; also when the particles are mechanically shaken on a table in 2D, rather than in a turbulent flow in 3D, the velocity and diffusion still obey the laws of thermodynamics. The macroscopic reactor was used to study self-assembly of 3D-printed objects with embedded magnets. A system of four spheres was analysed by both humans and neural networks. Although yielding very similar results, they significantly deviate from theoretical predictions, likely due to underestimation of the disturbing energy. When using objects with anisotropic shape, we found that the geometry and aspect ratio highly define the nature of resulting structures. The particle shape for instance controls the dimensionality (1D, 2D, 3D) and flexibility (straight versus flexible angles) of the resulting assemblies. The second system involves the study of the motion of magnetotactic bacteria (MTB) under influence of varying magnetic fields. From microscopy observations of the trajectories of individual MTB we found that their response to magnetic fields accurately follows a simple model based on the ratio between magnetic and drag torque. We characterised the properties of MTB and interaction with the environment. An optical density based method was developed to monitor the properties of entire colonies of MTB with high temporal resolution. We were able to monitor four distinct parameters corresponding to growth and magnetic growth of MTB and found that these types of growth are decoupled. Although magnetic objects studied in this thesis are seemingly very distinct, with various sizes and shapes, their analysis has strong similarity. The most important aspects for fluid-particle interaction are the interplay between magnetic torque and the drag force as well as the interplay between magnetic potential energy and (equivalent) thermal energy. The parameters underpinning the models based on these concepts can be determined through observation of the motion of the particles.Beobachtung der Bewegung von Partikeln in Flüssigkeiten bringt wertvolle Informationen über die Partikel, die Umgebung als auch die Interaktion von beidem. Zwei verschiedene Partikel-Flüssigkeitssysteme wurden in dieser Studie näher untersucht. Das erste System setzte sich zusammen aus zentimeter-großen magnetischen Partikeln, ausgebracht in einem aufwärtsgerichteten Wasserstrom, welcher einen neutralen statischen Auftrieb erzeugte als auch den Ursprung von Turbulenzen darstellt. Dieser makroskopische Reaktor wurde betrieben als analoge Simulation für mikroskopische Selbstassemblierungsprozesse. Durch das Beobachten der Trajektorien von Ein- sowie Zwei-Partikelsystemen wurde festgestellt, dass die Gesetze der Thermodynamik überraschend genau das System charakterisieren, vor allem in Bezug auf Geschwindigkeit, Diffusion und Partikel-Interaktion. Wir konnten zeigen, dass wir die Stärke der Störenergie kontrollieren können durch Änderung der Asymmetrie des Wassereinlasses, aber auch das diese Methode die Partikel unterschiedlich beeinflusst, je Lage im dreidimensionalen Raum. Es konnte nachgewiesen werden, dass die Methode zur Erzeugung der Störenergie kein kritischer Einflussfaktor ist, da auch beim mechanischen Schütteln von Partikeln auf einem Tisch in 2D, im Gegensatz zu einer turbulenten Flussrate in 3D, Geschwindigkeit und Diffusion weiterhin den Gesetzen der Thermodynamik unterliegen. Der makroskopische Reaktor wurde zur Untersuchung von Selbstassemblierungsprozessen von 3D-gedruckten Objekten mit eingeschlossenen Magneten verwendet. Ein System aus vier Kugeln wurde sowohl durch Probanden als auch durch Neurale Computernetzwerk analysiert. Trotz der sehr ähnlichen Ergebnisse konnte ein signifikanter Unterschied zu den theoretischen Vorhersagen festgestellt werden, welcher höchstwahrscheinlich in der Unterschätzung der Störenergie begründet war. Bei der Benutzung von Objekten mit anisotropen Formen konnten wir zeigen, dass die Geometrie sowie das Seitenverhältnis starken Einfluss nehmen auf die entstehenden Strukturen. Die Form der Partikel hat beispielsweise entscheidenden Einfluss auf die Dimensionalität (1D, 2D, 3D) und Flexibilität (Grade vs. Flexible Winkel) der entstehenden Verbindun- gen. Das zweite System umfasste die Analyse der Bewegung von magnetotaktischen Bakterien (MTB) unter Einfluss von wechselnden Magnetfeldern. Durch mikroskopische Beobachtung der Bewegungsbahnen von einzelnen MTB konnten wir nachweisen, dass deren Bewegungsantwort auf magnetische Felder exakt einem einfachen Modell folgen, basierend auf dem Verhältnis zwischen magnetischem Drehmoment und Dreh-Strömungswiderstandes. Hierzu wurden die Eigenschaften der MTB und deren Interaktion mit der Umgebung charakterisiert. Eine Methode, basierend auf optischer Dichte-Messung, wurde entwickelt um Eigenschaften von ganzen Kolonien von MTBs mit hoher zeitlicher Auflösung zu untersuchen. Es war uns möglich vier verschiedene Parameter bezüglich Wachstum und Wachstum der magnetischen Partikel zu überwachen um festzustellen, dass diese Typen des Wachstums sich als entkoppelt darstellen. Obwohl die in dieser Doktorarbeit verwendeten magnetischen Objekte stark unterschiedlich in Bezug auf Größe und Form waren zeigte deren Auswertung hohe Ähnlichkeiten. Der wichtigste Aspekt der Partikel-Flüssigkeitsinteraktion stellt das Zusammenspiel von magnetischem Drehmoment und des Dreh-Strö- mungswiderstandes dar, als auch das Zusammenspiel der potenziellen magnetischen Energie und der (äquivalenten) thermalen Energie. Diese Parameter der Modelle konnten durch Beobachtung der Bewegung der Partikel untermauern werden, auf welchen die Konzepte des magnetischen Moments und des Strömungswiderstandes basieren

Computational studies of magnetite Fe₃O₄ and related spinel-structured materials

This thesis presents the results of ab initio based simulation studies of magnetite (Fe₃O₄) and related FeM₂X₄ (thio)spinels with M = Cr, Mn, Fe, Co and Ni and X = O and S. Using density functional theory with long-range dispersion correction and on-site Coulomb interactions (DFT + U – D2), we have investigated a number of properties of these materials. Firstly, we present a study of the inversion degree and its relevance in the electronic structure and magnetic properties of the spin filter candidates FeM₂X₄, which are one of the key devices in spintronic applications. We also analyze the role played by the size of the ions and by the crystal field stabilization effects in determining the equilibrium inversion degree. Secondly, we present the calculations of the elastic constants and other macroscopic mechanical properties by applying elastic strains on the unit cell of Fe₃O₄, which is the main component in different types of catalysts used in myriad of industrial processes. Thirdly, we calculate the geometries and surface free energies of a number of Fe₃O₄ surfaces at different compositions, including the non-dipolar stoichiometric plane, and those with a deficiency or excess of oxygen atoms. We propose a morphology in thermodynamic equilibrium conditions for the nanocrystals of this compound. We also present the simulated scanning tunnelling microscopy images of the different terminations of the surfaces shown on the Fe₃O₄ morphology. Finally, we investigate the initial oxidation stages of the greigite (Fe₃S₄) (001) surface induced by water. Fe₃S₄ is a mineral widely identified in anoxic aquatic environments and certain soils, which can be oxidised by these environments producing and extremely acid solution of sulfur-rich wastewater called acid mine drainage (AMD). We propose a number of mechanisms involving one or two water molecules and one OH group to explain the replacement of one sulfur by one oxygen atom in this mineral. The findings presented in this thesis provides a theoretical insight into various bulk and surface properties of this group of compounds

CMOS and MEMS Based Microsystems for Manipulation and Detection of Magnetic Beads for Biomedical Applications

RÉSUMÉ Les micro et nano billes magnétiques dédiées à l'étiquetage des bio-particules attirent de plus en plus d'intérêt dans de nombreuses applications environnementales et sanitaires, tels que l'analyse de gènes, le transport des médicaments, la purification et l'immunologie. Les dimensions réduites et la haute sensibilité des billes magnétiques rendent leurs manipulations à haute précision possibles. Leur simplicité de suivi dans le milieu biologique et leur biocompatibilité permettent d’effectuer des détections rapides et à haute sensibilité pour des applications in vivo et in vitro. L'utilisation traditionnelle des billes magnétiques prend place dans un laboratoire se servant du matériel encombrant et dispendieux. Avec le développement de la technologie de microfabrication, des billes magnétiques peuvent être traitées dans un microsystème, plus précisément, dans une structure laboratoire sur puce (LoC). La combinaison microfluidique et microélectronique offre des possibilités d’autoévaluation, ce qui peut augmenter l'efficacité du travail. Cette thèse est orientée vers de nouvelles approches pour la manipulation et la détection de bio-particules se servant de la technologie de microsystèmes basées sur des structures microelectroniques et microfluidiques et en utilisant des marqueurs de billes magnétiques. Basé sur un réseau de microbobines à la fois comme une source de champ magnétique et un capteur inductif, le microsystème proposé est réalisé grâce à l'efficacité de fabrication de structures CMOS-MEMS, ainsi que des circuits intégrés dédiés CMOS de haute performance afin d'obtenir un rendement élevé de manipulation et de détection de billes magnétiques. Plusieurs défis ont été analysés dans la mise en œuvre de ces microsystèmes et des solutions correspondantes fournies. Plus précisément, la conception et la mise en œuvre d'une plate-forme contrôlée en température en format portable sont d'abord présentées, dans un effort réalisé pour résoudre la question de la chaleur par effet Joule lors de l'application du réseau de microbobines comme une source de champ magnétique dédié à la manipulation de billes magnétiques. Une plateforme similaire à cette dernière a été améliorée pour effectuer une analyse magnétique immunologique, en ajoutant des circuits de détection par des billes magnétiques. De plus, des IgG et anti-IgG de souris ont été utilisés dans des expériences pour vérifier les performances de détection de la plateforme de microsystème proposé.----------ABSTRACT Magnetic micro/nano beads as labels of bio-particles have been attracting more and more interest in many environmental and health applications, such as gene and drug delivery, purification, and immunoassay. The miniature size and high sensitivity of magnetic bead allow accurate manipulation, whereas its high distinguishability from biological background and biocompatibility make fast and high sensitivity detection possible for in vitro and in vivo applications. Traditional employment of magnetic beads is done in laboratory environment with the assist of bulky and expensive equipment. Thanks to the development of microfabrication technology, magnetic beads therefore can be handled on a microsystem, more specifically, a Lab-on-Chip (LoC). The combination of microfluidics with microelectronics offers the possibility of automatic analyses, which can liberate the labor and increase the efficiency.This thesis focuses on new approaches for bio-particles manipulation and detection on microelectronic/microfluidic hybrid microsystems using magnetic beads as labels. Based on planar microcoil array as both magnetic field source and the front-end inductive sensor, the proposed microsystems can take advantage of the massive producible CMOS/MEMS fabrication process, as well as the customized high performance CMOS circuits, to achieve a high efficient magnetic beads manipulation and a quantitative detection. Several challenges in implementing such microsystems are analyzed and corresponding solutions are provided. Specifically, the design and implementation of a temperature controllable LoC platform in portable format is firstly presented, for the sake of resolving the Joule heat issue when applying microcoil array as magnetic field source in magnetic beads manipulation. The similar platform is then improved to be used for magnetic immunoassay, by adding magnetic beads sensing circuits. Mouse IgG and anti-mouse IgG are employed in experiments to verify the detection performance of the proposed microsystem platform. Additionally, a fully integrated silicon substrate MEMS chip which integrates both microfluidic channel and microcoil array on a single chip is designed and fabricated following the Finite Element Analysis (FEA) simulation results and tested using bio-particles attached magnetic beads. This monolithic chip has the potential to be applied for in vivo applications

Imaging and Control of Engineered Cells using Magnetic Fields

Making cells magnetic is a long-standing goal of synthetic biology, aiming to enable the separation of cells from complex biological samples and their non-invasive visualization in vivo using Magnetic Resonance Imaging (MRI). Previous efforts towards this goal, focused on engineering cells to biomineralize superparamagnetic or ferromagnetic iron oxides, have largely been unsuccessful due to the stringent required chemical conditions. In this thesis, we introduce an alternative approach to making cells magnetic, focusing on biochemically maximizing cellular paramagnetism. Here, we show that a novel genetic construct combining the functions of ferroxidation and iron-chelation enables engineered bacteria to accumulate iron in 'ultraparamagnetic' macromolecular complexes, which subsequently allows for these cells to be trapped using strong magnetic field gradients and imaged using MRI in vitro and in vivo. We characterize the properties of these cells and complexes using magnetometry, an array of spectroscopic techniques, biochemical assays, and computational modeling to elucidate the unique mechanisms and implications of this 'ultraparamagnetic' concept. In addition to making cells magnetic, remote control of cellular localization in deep tissue is another long-standing goal of synthetic biology. Such an ability to non-invasively direct cells to sites of interest will not only improve therapeutic outcomes by minimizing off-target activity, but more broadly enable new research on complex cellular communities, such as the gut microbiome, in living animals. Given their deep penetrance through tissues, magnetic fields are ideally suited for facilitating non-invasive targeting of cells; however, the rapid decay of magnetic flux density from its source currently limits the depths to which magnetic targeting can be employed to within 1-2 mm from the surface. Here, we demonstrate a new approach wherein the retention of orally-administered and synthetically magnetized cell-like-particles is selectively enhanced within the murine intestinal tract to depths of up to 13 mm from the surface. Our cellular localization assisted by magnetic particles (CLAMP) strategy can potentially be generalized to any cell (bacterial, mammalian) or drug-containing nanoparticle of interest, and can be combined with existing non-invasive imaging modalities thereby facilitating remote environmental sensing at sites of interest. Finally, while magnetic fields in MRI scanners are widely used today to safely and non-invasively image anatomical structures in living animals, much of the image contrast in MRI is the result of microscale magnetic-field variations in tissues. However, the connection between these microscopic patterns and the appearance of macroscopic MR images has not been the subject of direct experimental studies due to a lack of methods to map microscopic fields in biological samples under ambient conditions. Here, we optically probed magnetic fields in mammalian cells and tissues with submicron resolution and nanotesla sensitivity using nitrogen-vacancy (NV) diamond magnetometry and combined these measurements with simulations of nuclear-spin precession to predict the corresponding MRI contrast. Additionally, we demonstrate the broad utility of this technology for imaging an in vitro model of cellular iron uptake, as well as imaging histological samples from a mouse model of hepatic iron overload. Taken together, our approach bridges a fundamental intellectual gap between a macroscopic MRI voxel and its microscopic constituents.</p