Der Gebrauch spezies-spezifischer ELISAs und Bioassays für die Detektion pyrogener Verunreinigungen

Deckblatt-Impressum Zitat Contents/Glossary Introduction/Literature Materials and Methods Results Discussion Summary Zusammenfassung References Appendix Danksagung SelbständigkeitserklärungIn this study the reaction of the blood monocytes of rabbits and humans towards pyrogens (fever-inducing substances) was compared. Pyrogens in intravenously applied parenterals are being recognized by blood monocytes and induce the production of cytokines which cause a fever reaction in the organism. The fever reaction of rabbits has been used since 1942 for testing drugs for pyrogenic contaminations. A new in vitro pyrogen test (IPT) which measures the cytokine production of human monocytes in vitro in an ELISA system was recently validated and introduced. ELISAs for the rabbit were developed in order to compare the reaction of both species in vitro. The first question that was addressed was whether the cytokine production of the monocytes of the rabbit corresponded to the in vivo fever reaction. This was the case. The required amount of pyrogen in vivo corresponded exactly to the amount in vitro. Having that established, the in vitro pyrogen test using human whole blood was compared to the reaction of rabbit whole blood. The human and the rabbit whole blood test were performed in parallel, whenever possible. A comparison of these two assays revealed a very high similarity in the reactivity between both species, but the rabbit appeared less sensitive towards 3 stimuli. The humans had a more uniform response and sensitivity towards all stimuli used than had the rabbits, the different donors as well as the different endpoints, IL-, IL-6, TNF- and IL-8. The amount of pyrogens that was necessary to evoke a cytokine production varied between picograms, nanograms and even micrograms depending on the stimulus. Since the dog as well had once been discussed as an animal for pyrogen testing, a bioassay for canine blood was established and successfully compared to the corresponding bioassay using human blood. Testing for pyrogenic contaminations is a major issue in human medicine. The in vitro pyrogen tests reliably detect pyrogenic contaminations in parenterals. From our perspective, the already established readouts, and, if necessary, further developments of species-specific ELISAs and bioassays could prove valuable for ensuring drug safety in the veterinary field.In dieser Arbeit wurde die Reaktion der Blutmonozyten von Kaninchen und Mensch auf Pyrogene (fiebererzeugende Substanzen) verglichen. Pyrogene in intravenös applizierten Parenteralia werden von Blutmonozyten erkannt und führen zur Ausschüttung von Zytokinen, die im Organismus Fieber verursachen. Die Fieberreaktion von Kaninchen wird seit 1942 genutzt, um Medikamente auf pyrogene Kontaminationen zu prüfen. Ein neuer in vitro Pyrogentest (IPT), der die Zytokinausschüttung humaner Monozyten in vitro im ELISA misst, wurde kürzlich erfolgreich validiert und eingeführt. ELISAs für das Kaninchen wurden entwickelt, um die Reaktion beider Spezies in vitro zu vergleichen. Die erste Frage, die sich stellte war, ob die Zytokinausschüttung der Blutmonozyten des Kaninchens im in vitro pyrogen test mit der in vivo Fieberreaktion des Gesamt- organismus zusammenhing. Dies konnte bestätigt werden. Die notwendige Menge an Pyrogen in vivo stimmte mit der in vitro genau überein. Mit diesem Ergebnis wurde dann der neu entwickelte in vitro Pyrogentest mit Humanblut mit dem in vitro Pyrogentest mit Kaninchenblut verglichen. Beide Tests wurden, wann immer möglich, parallel durchge-führt. Ein Vergleich der beiden Assays ergab eine sehr hohe Ähnlichkeit bezüglich der Sensitivität beider Spezies, allerdings erschien das Kaninchen bei drei Stimuli weniger sensitiv. Die Humanspender hatten eine einheitlichere Reaktion und Sensitivität allen Stimuli gegenüber als die Kaninchen, und zwar galt sowohl für die verschiedenen Spender untereinander als auch für die verschiedenen gemessenen Zytokine, IL-1, IL-6, TNF- und IL-8. Die Menge an Pyrogenen, die eingesetzt werden musste, um eine Zytokinausschüttung hervorzurufen, bewegte sich je nach Stimulus im Pikogramm-, Nanogramm- oder sogar Mikrogrammbereich. Da auch der Hund als Versuchstier zur Pyrogentestung diskutiert wurde, wurde noch ein Bioassay für Hundeblut etabliert und erfolgreich mit dem entsprechenden Assay mit Humanblut verglichen. Die Testung auf pyrogene Kontaminationen ist ein großes Thema in der Humanmedizin. Die in vitro Pyrogentests entdecken verlässlich pyrogene Kontaminationen in Parenteralia. Aus unserer Sicht könnten sich die bereits etablierten Endpunkte und, falls nötig, weitere Entwicklungen spezies- spezifischer ELISAs und Bioassays als nützlich erweisen, um die Sicherheit von Parenteralia im Veterinärbereich zu gewährleisten

Forming the link between strategic performance and operational success: the development of a balanced scorecard at Lisbon Consulting Group

Directed internshipThe purpose of this research is to develop a Balanced Scorecard for Lisbon Consulting Group (LCG) that forms the link between Strategic Performance and Operational Success. Therefore, strategic internal and external analyses, such as SWOT, PEST and Customer Value Proposition Analyses were conducted in order to adapt the Balanced Scorecard to the company’s reality. Furthermore, this paper examines the market environment of consulting companies. For this purpose, a value chain for consulting companies was established and the industries’ best practices were explored through a Benchmark Approach that followed a qualitative research method. As a result, the Balanced Scorecard for LCG is both, a reflection of the company’s reality and the consulting market. Thus, this work serves as well as a general Balanced Scorecard framework for consulting companies. The implementation will contribute to the future value of LCG as it measures the company’s performance and sets and communicates targets and initiatives in a short-term and long-term perspective

Existence of similarity profiles for diffusion equations and systems

We study the existence of self-similar profiles for diffusion equations and reaction-diffusion systems on the real line, where different nontrivial limits are imposed at both sides of infinity. The theses profiles solve a coupled system of nonlinear ODEs that can be treated by monotone operator theory

Convergence to self-similar profiles in reaction-diffusion systems

We study a reaction-diffusion system on the real line, where the reactions of the species are given by one reversible reaction pair satisfying the mass-action law. We describe different positive limits at both sides of infinityand investigate the long-time behavior. Rescaling space and time according to the parabolic scaling, we show that solutions converge exponentially to a similarity profile when the scaled time goes to infinity. In the original variables, these profiles correspond to asymptotically self-similar behavior describing the phenomenon of diffusive mixing of the different states at infinity.Our method provides global exponential convergence for all initial states with finite relative entropy. For the case with equal stoichiometric coefficients, we can allow for self-similar profiles with arbitrary equilibrated states,while in the other case we need to assume that the two states atinfinity are sufficiently close such that the self-similar profile is relative flat

Self-similar pattern in coupled parabolic systems as non-equilibrium steady states

We consider reaction-diffusion systems and other related dissipative systems on unbounded domains which would have a Liapunov function (and gradient structure) when posed on a finite domain. In this situation, the system may reach local equilibrium on a rather fast time scale but the infinite amount of mass or energy leads to persistent mass or energy flow for all times. In suitably rescaled variables the system converges to a steady state that corresponds to asymptotically self-similar behavior in the original system

Convergence to self-similar profiles in reaction-diffusion systems

We study a reaction-diffusion system on the real line, where the reactions of the species are given by one reversible reaction according to the mass-action law. We describe different positive limits at both sides of infinity and investigate the long-time behavior. Rescaling space and time according to the parabolic scaling, we show that solutions converge exponentially to a constant profile. In the original variables these profiles correspond to asymptotically self-similar behavior describing the diffusive mixing or equilibration of the different states at infinity. Our method provides global exponential convergence for all initial states with finite relative entropy

Gradient flows for bounded linear evolution equations

We study linear evolution equations in separable Hilbert spaces defined by a bounded linear operator. We answer the question which of these equations can be written as a gradient flow, namely those for which the operator is real diagonalisable. The proof is constructive, from which we also derive geodesic lambda-convexity

Beiträge zu Verbreitung und Analytik des subtilen alternativen Spleißens

Das alternative Spleißen (AS) ist ein Hauptakteur der Diversifizierung von Transkriptom und Proteom eines eukaryotischen Organismus. Die Studien dieser Dissertationsschrift thematisieren das erst kürzlich entdeckte subtile AS, das die Einführung kleiner Variationen im Transkript und in vielen Fällen auch im kodierten Protein bewirkt. Im Arabidopsis thaliana-Genom konnte eine häufige Präsenz von NAGNAG Tandem-Motiven nachgewiesen werden, die in den Spleißfaktor-kodierenden SR-Protein-Genen überrepräsentiert sind. Ausgewählte experimentell analysierte Fälle zeigten ähnliche organ- und bedingungsspezifische Änderungen der Splei߬varianten-Verhältnisse. Im Mensch wurde ein völlig neuer, seltener Typus des subtilen AS entdeckt. Eine Population von 36 Introns verwendet TG Dinukleotide als alternative 3’ Splei߬stellen und widerspricht damit den etablierten Spleißregeln. TG-3‘-Splei߬stellen wurden ausschließlich im Kontext einer AG Splei߬stelle mit einer maximalen Distanz von 28 nt gefunden. In deren orthologen 3’ Splei߬stellen sind TG-Dinukleotid und flankierende Intronsequenz zwischen Säuge¬tieren auffällig stark konserviert. Deren Verwendungshäufigkeit steigt mit der Konservierung von Splei߬stelle und flankierender Intronsequenz und wird höchstwahrscheinlich durch cis- und/oder trans-Elemente vermittelt. Zur quantitativen Ermittlung der Splei߬variantenverhältnisse wurden die Pyrosequenzierung und die Fluoreszenz-basierte Kapillarelektro¬phorese verwendet. Beide Methoden wurden hinsichtlich Reproduzierbarkeit und Genauigkeit, Experimentaufbau und Datenanalyse im Vergleich zur häufig verwendeten Polyacrylamid-Gelelektrophorese mit Ethidiumbromid-vermittelter Densitometrie analysiert. CE-LIF erzielte dabei die höchste Genauigkeit und Reproduzierbarkeit und stellte gleichzeitig die arbeits- und zeiteffizienteste Methode dar

Comparative analysis of sequence features involved in the recognition of tandem splice sites

<p>Abstract</p> <p>Background</p> <p>The splicing of pre-mRNAs is conspicuously often variable and produces multiple alternatively spliced (AS) isoforms that encode different messages from one gene locus. Computational studies uncovered a class of highly similar isoforms, which were related to tandem 5'-splice sites (5'ss) and 3'-splice sites (3'ss), yet with very sparse anecdotal evidence in experimental studies. To compare the types and levels of alternative tandem splice site exons occurring in different human organ systems and cell types, and to study known sequence features involved in the recognition and distinction of neighboring splice sites, we performed large-scale, stringent alignments of cDNA sequences and ESTs to the human and mouse genomes, followed by experimental validation.</p> <p>Results</p> <p>We analyzed alternative 5'ss exons (A5Es) and alternative 3'ss exons (A3Es), derived from transcript sequences that were aligned to assembled genome sequences to infer patterns of AS occurring in several thousands of genes. Comparing the levels of overlapping (tandem) and non-overlapping (competitive) A5Es and A3Es, a clear preference of isoforms was seen for tandem acceptors and donors, with four nucleotides and three to six nucleotides long exon extensions, respectively. A subset of inferred A5E tandem exons was selected and experimentally validated. With the focus on A5Es, we investigated their transcript coverage, sequence conservation and base-paring to U1 snRNA, proximal and distal splice site classification, candidate motifs for <it>cis</it>-regulatory activity, and compared A5Es with A3Es, constitutive and pseudo-exons, in <it>H. sapiens </it>and <it>M. musculus</it>. The results reveal a small but authentic enriched set of tandem splice site preference, with specific distances between proximal and distal 5'ss (3'ss), which showed a marked dichotomy between the levels of in- and out-of-frame splicing for A5Es and A3Es, respectively, identified a number of candidate NMD targets, and allowed a rough estimation of a number of undetected tandem donors based on splice site information.</p> <p>Conclusion</p> <p>This comparative study distinguishes tandem 5'ss and 3'ss, with three to six nucleotides long extensions, as having unusually high proportions of AS, experimentally validates tandem donors in a panel of different human tissues, highlights the dichotomy in the types of AS occurring at tandem splice sites, and elucidates that human alternative exons spliced at overlapping 5'ss posses features of typical splice variants that could well be beneficial for the cell.</p