Ionization of Atomic Hydrogen in Strong Infrared Laser Fields

Advisor: Klaus BartschatWe have used the matrix iteration method of Nurhuda and Faisal (Phys. Rev. A 60 (1999) 3125) to treat ionization of atomic hydrogen by a strong laser pulse. After testing our predictions against a variety of previous calculations, we present ejected-electron spectra as well as angular distributions for few-cycle infrared laser pulses with peak intensities of up to 1015W/cm2. It is shown that the convergence of the results with the number of partial waves is a serious issue, which can be managed in a satisfactory way by using the velocity form of the electric dipole operator in connection with an efficient time-propagation scheme.Drake University, Department of Physics and Astronom

Characterisation of a modular and protein-based contrast agent for Magnetic Resonance Imaging

Die vorliegende Promotionsarbeit befasst sich mit der biochemischen und biophysikalischen Charakterisierung des Proteins Zarvin, welches als erster Ansatz zur Entwicklung eines variablen (modularen) Gadolinium(III)-basierten "T" _"1" -Kontrastmittels zur Detektion von Metastasen mit Hilfe der Magnetresonanztomographie dienen soll. Ein in der Literatur bisher wenig untersuchter Ansatz der direkten Kopplung des Gadolinium(III) an ein Protein sollte dabei weiter evaluiert werden, da dieser Ansatz eine hohe Effizienz des Kontrastmittels bei verhältnismäßig niedrigem Molekulargewicht erlauben würde. Zarvin wurde am Institut für Bioinformatik der Universität Duisburg-Essen entwickelt und besteht zur Erfüllung dieser Aufgaben aus zwei Domänen, die durch einen flexiblen, aus zehn Glycinen bestehenden, Linker voneinander getrennt sind. Die sich N terminal befindende Domäne ist eine Mutante der sog. B Domäne des Proteins A aus dem Organismus Staphylococcus aureus und wird in der Literatur als Z Domäne bezeichnet. Letztere besitzt die Eigenschaft, selektiv an den "F" _"c" -Bereich humaner (z. T. auch anderer Säugetiere) Antikörper zu binden. Diese Eigenschaft sollte hier dazu genutzt werden, dem Kontrastmittel in Kombination mit kommerziell verfügbaren therapeutischen Antikörpern eine schnell variierbare Zielführung zu unterschiedlichen Zielstrukturen zu ermöglichen. Als C-terminale Domäne des Zarvins wurde zunächst eine hoch affin Ca2+-bindende Mutante eines Parvalbumin-Proteins aus der Ratte, das S55D/E59D α Parvalbumin, gewählt. Diese Domäne sollte dazu genutzt werden, zwei Gadolinium(III)-Ionen zu binden, welche dem Protein die benötigten Kontrast gebenden Eigenschaften eines "T" _"1" -Kontrastmittels verleihen. Für die Charakterisierung von Zarvin wurden dessen Nukleotidsequenz sowie die Nukleotidsequenz der einzelnen Domänen mittels rekombinanter DNA-Technologie in einen Expressionsvektor kloniert und die entsprechend translatierten Proteine gereinigt. Mit Hilfe von CD- sowie 2D-NMR-Spektroskopie konnte eine korrekte Faltung beider Domänen innerhalb des Proteins Zarvin bestätigt werden. Die Funktionalität der Z-Domäne wurde weiterhin mittels Fluoreszenzmarkierung von Zarvin bzw. analog produzierter Cystein-Mutanten untersucht. Es konnte nachgewiesen werden, dass das Protein in vitro mit ähnlicher Affinität wie die separate Z Domäne an einen IgG-Antikörper bindet sowie in Kombination mit letzerem in der Lage ist, an Zellen mit entsprechenden Zielstrukturen in der Zellmembran zu binden. Die Funktionalität der Parvalbumin-Domäne in Bezug auf die Bindung von Lanthanoiden wie Gadolinium(III) wurde mit Hilfe der Etablierung eines Terbium(III)-Lumineszenz-Systems untersucht. Es konnte nachgewiesen werden, dass Zarvin Lanthanoide wie Tb3+ und Gd3+ mit subpicomolarer Affinität bindet. Die mittels CD-Spektroskopie untersuchte thermische Stabilität der Faltung dieser Holoform des Proteins erwies sich als sehr hoch. Ebenso war das Protein zumindest in vitro in fötalem Kälberserum nicht suszeptibel gegenüber proteolytischem Abbau. Beide Eigenschaften zeichnen das Protein als gutes Gerüst für die Entwicklung eines proteinbasierten Kontrastmittels aus. Zu lösende Problematiken, die in Verbindung mit Zarvin herausgestellt wurden, sind die unter simulierten in vivo Bedingungen sehr niedrigen kinetischen Stabilitäten der Interaktion zwischen der Parvalbumin-Domäne und Lanthanoiden sowie zwischen der Z-Domäne und einem Antikörper. Die Charakterisierung der relaxometrischen Eigenschaften des Zarvin:(Gd3+)2 zeigte, dass eine entsprechende Optimierung dieser kinetischen Eigenschaften lohnend wäre. Die in Form der "T" _"1" -Relaxivität "r" _"1" quantifizierte Effizienz des Zarvin:(Gd3+)2 als "T" _"1" Kontrastmittel erwies sich im Bereich von Feldstärken um 1,5 Tesla und auch noch bei 3 Tesla als sehr hoch. Das in vitro bei diesen Feldstärken geschätzte Detektionslimit liegt im niedrigen mikromolaren Bereich und befindet sich damit in der Größenordnung hoch exprimierter Zielstrukturen auf Tumorzellen. Die in dieser Arbeit experimentell erhaltenen und diskutierten Ergebnisse bilden daher eine Grundlage für Ansätze zur Entwicklung eines verbesserten proteinbasierten "T" _"1" -Kontrastmittels.The present thesis deals with the biochemical and biophysical characterisation of the protein Zarvin, which was designed as a first approach for the development of a variable (modular) Gadolinium(III)-based "T" _"1" contrast agent capable of detecting metastases employing Magnetic Resonance Imaging. For that, a so far hardly investigated approach of directly coupling the Gadolinium(III) to a protein should be further evaluated, because in this way a highly efficient contrast agent based on a relatively low molecular weight could be developed. Zarvin was designed at the Department of Bioinformatics at the University of Duisburg-Essen. To fulfil both of its tasks, modularity as well as contrast producing properties, Zarvin consists of two domains separated by a flexible decaglycine linker. The N terminal domain is a mutant of the so called B-domain of protein A from Staphylococcus aureus and is designated as Z domain in literature. The latter domain of Zarvin is able to bind selectively to the constant region ("F" _"c" ) of human (in part also of other mammals) IgG antibodies. This property was intended to be used in combination with commercially available therapeutic antibodies to allow for a quick and variable targeting of the contrast agent to different physiological target structures. For the C terminal domain of Zarvin the highly affine Ca2+-binding S55D/E59D rat α Parvalbumin mutant was chosen in a first step. This domain was intended to be used for binding of two Gadolinium(III) ions and thus for providing the necessary contrast producing properties of a "T" _"1" contrast agent. In order to be able to characterise Zarvin, the nucleotide sequences of Zarvin as well as of the single domains were cloned into an expression vector. After purification of the expressed proteins, the proper folding of both domains within the two-domain protein Zarvin was confirmed employing CD and 2D-NMR spectroscopy. Further, the functionality of the Z domain was investigated using fluorescently labelled cystein mutants of Zarvin. It could be shown that Zarvin in vitro binds to an IgG antibody with an affinity similar to the separate Z domain and that in combination with this antibody Zarvin is able to target cells expressing the respective target structures in the cell membrane. The functionality of the Parvalbumin-domain concerning the binding of lanthanides like Gadolinium(III) was investigated by establishing a Terbium(III) luminescence system. It could be shown that Zarvin binds lanthanides like Tb3+ and Gd3+ with subpicomolar affinity. The thermal stability of this holoform of the protein was investigated using CD spectroscopy and proved to be very high. Further, the protein was in vitro not susceptible towards proteolytic degradation in fetal calf serum. Both properties characterise Zarvin as a good scaffold for the development of a protein-based contrast agent. Remaining problems to be solved, which were pointed out in combination with Zarvin by simulating in vivo conditions, are the low kinetic stabilities of the interaction between the Parvalbumin-domain and lanthanides as well as between the Z domain and an antibody. The relaxometric characterisation of the Zarvin:(Gd3+)2 complex however showed that an optimisation of the respective kinetic properties would be worthwhile. The quantified efficiency of Zarvin:(Gd3+)2 in terms of the "T" _"1" relaxivity "r" _"1" proved to be very high at field strengths around 1,5 Tesla and also as clearly higher than "r" _"1" values of small molecular weight contrast agents at 3 Tesla. Concerning the detection limit of the contrast agent at these field strengths, low micromolar concentrations were estimated, which are in the range of concentrations of highly expressed target structures in the cell membrane of tumour cells. The experimentally obtained and discussed results of this thesis therefore provide a basis for further approaches to develop an improved protein-based "T" _"1" contrast agent

The Identification of Five Seedlings Hyper-responsive to Light (SHL), and Characterization of SHL7

Light is one of the major environmental factors that controls plant development, through a process known as photomorphogenesis. Plants perceive light via photoreceptors, and the information used to direct a myriad of developmental responses. Analysis of mutants defective in photomorphogenic responses elucidates the complex interactions between light and plants. Previous genetic screens have yielded a class of mutants which exhibit exaggerated responses to ambient light, designated shl (seedling hyper-responsive to light). The following work encompasses the identification of five new shl mutants, a detailed examination of one of these mutants (shl7), and of the SHL7 gene. The mutants were isolated in a low-white light screen of seedlings derived from T-DNA mutagenesis. Each of the mutants exhibits a heritable hyper-responsive phenotype in low-white light, but displays minimal effects in darkness. For each, a putative site of T-DNA insertion has been located. In addition to a low-white light phenotype, the shl7 mutant exhibits a mild hyper-responsive phenotype to 670 nm red and 735 nm far-red light, but significant hyper-responses to 420 nm blue light. SHL7 encodes a small, unique, and previously undescribed protein annotated as At4g04925. GFP protein fusion analysis indicates that the protein is localized to mitochondria

Labetalol infusion for refractory hypertension causing severe hypotension and bradycardia: an issue of patient safety

Incremental doses of intravenous labetalol are safe and effective and, at times, such therapy may need to be augmented by a continuous infusion of labetalol to control severe hypertension. Continuous infusions of labetalol may exceed the recommended maximum daily dose of 300 mg on occasion. We report a case in which hypertension occurring after an abdominal aortic aneurysm repair, initially responsive to intermittent intravenous beta-blockade, became resistant to this therapy leading to the choice of an intravenous labetalol infusion as the therapeutic option. The labetalol infusion resulted in a profound cardiovascular compromise in this postoperative critically ill patient. While infusions of labetalol have successfully been used, prolonged administration in the intensive care unit requires vigilance and the establishment of a therapeutic rationale/policy for interventions, such as the ready availability of glucagon, β-agonists, phosphodiesterase inhibitors, insulin, and vasopressin when severe cardiovascular depression occurs

Fibroblast heterogeneity in the cancer wound

Fibroblasts regulate the structure and function of healthy tissues, participate transiently in tissue repair after acute inflammation, and assume an aberrant stimulatory role during chronic inflammatory states including cancer. Such cancer-associated fibroblasts (CAFs) modulate the tumor microenvironment and influence the behavior of neoplastic cells in either a tumor-promoting or tumor-inhibiting manner. These pleiotropic functions highlight the inherent plasticity of fibroblasts and may provide new avenues to understand and therapeutically intervene in malignancies. We discuss the emerging themes of CAF biology in the context of tumorigenesis and therapy

Human PAPS Synthase Isoforms Are Dynamically Regulated Enzymes with Access to Nucleus and Cytoplasm

In higher eukaryotes, PAPS synthases are the only enzymes producing the essential sulphate-donor 3′-phospho-adenosine-5′-phosphosulphate (PAPS). Recently, PAPS synthases have been associated with several genetic diseases and retroviral infection. To improve our understanding of their pathobiological functions, we analysed the intracellular localisation of the two human PAPS synthases, PAPSS1 and PAPSS2. For both enzymes, we observed pronounced heterogeneity in their subcellular localisation. PAPSS1 was predominantly nuclear, whereas PAPSS2 localised mainly within the cytoplasm. Treatment with the nuclear export inhibitor leptomycin B had little effect on their localisation. However, a mutagenesis screen revealed an Arg-Arg motif at the kinase interface exhibiting export activity. Notably, both isoforms contain a conserved N-terminal basic Lys-Lys-Xaa-Lys motif indispensable for their nuclear localisation. This nuclear localisation signal was more efficient in PAPSS1 than in PAPSS2. The activities of the identified localisation signals were confirmed by microinjection studies. Collectively, we describe unusual localisation signals of both PAPS synthase isoforms, mobile enzymes capable of executing their function in the cytoplasm as well as in the nucleus

Estudio de los resultados de 155 resecciones hepáticas

The authors analyse a series of 155 patients thatunderwent hepatic resections between 1972 and 1991.The diagnostica/ elements of the different etiologies,surgica/ indications and results as regards complicationsand mortality are considered. The authors a/so makeconsiderations on different changes in criteria as regardsresectability and mortality causes, emphasizing hepaticfailure as the main one. They a/so point out the need topreserve hepatic parenchyma provided that oncologicalcriteria are kept to.Se analizan 155 pacientes sometidos a reseccioneshepáticas entre 1972 y 1991. Se consideran elementosdiagnósticos de las diferentes etiologías, las indicacionesquirúrgicas, los resultados en cuanto a complicaciones ymortalidad. Se consideran los diferentes cambios decriterio en cuanto a la resecabilidad y a las causas demortalidad, destacando la insuficiencia hepática comocausa de las mismas. Asimismo, la necesidad depreservar masa hepática siempre y cuando se conservenlos criterios oncológico

Consideraciones técnico tácticas de la cirugía hepática

As regards 155 hepatic resections, technico-tacticalaspects are considered. These include mainly thedetermination of risk factors for resection, the election ofpatients and functional and anatomica/counterindications. Considerations are made on patientpreparation as well as on details of surgical technique;intraoperative echography stands out indecision-making. Special emphasis is made on clampingand its consequences, pointing out the advantages ofsectorial vascular isolation of the /iver.A propósito de 155 resecciones hepáticas se consideranaspectos técnico tácticos. Ellos incluyenfundamentalmente la determinación de los elementos deriesgo para una resección, la elección de los pacientes ylas contraindicaciones funcionales y anatómicas. Sehacen consideraciones de la preparación del enfermo asícomo detalles de la técnica quirúrgica, destacando laecografía intraoperatoria en la toma de decisiones. Sehace especial hincapié en los clampeos y susrepercusiones, destacando las ventajas del aislamientovascular sectorial del hígado. Se indican protocolos deseguimiento preoperatorio y la necesidad de contar conequipos multidisciplinarios para llevar con buenosresultados esta cirugía