Bioremediation of PAHs - Limitations and soultions

Introduction 1.1 Occurrence of polycyclic aromatic hydrocarbons (PAH) in the environment Worldwide industrial and agricultural developments have released a large number of natural and synthetic hazardous compounds into the environment due to careless waste disposal, illegal waste dumping and accidental spills. As a result, there are numerous sites in the world that require cleanup of soils and groundwater. Polycyclic aromatic hydrocarbons (PAHs) are one of the major groups of these contaminants (Da Silva et al., 2003). PAHs constitute a diverse class of organic compounds consisting of two or more aromatic rings with various structural configurations (Prabhu and Phale, 2003). Being a derivative of benzene, PAHs are thermodynamically stable. In addition, these chemicals tend to adhere to particle surfaces, such as soils, because of their low water solubility and strong hydrophobicity, and this results in greater persistence under natural conditions. This persistence coupled with their potential carcinogenicity makes PAHs problematic environmental contaminants (Cerniglia, 1992; Sutherland, 1992). PAHs are widely found in high concentrations at many industrial sites, particularly those associated with petroleum, gas production and wood preserving industries (Wilson and Jones, 1993). 1.2 Remediation technologies Conventional techniques used for the remediation of soil polluted with organic contaminants include excavation of the contaminated soil and disposal to a landfill or capping - containment - of the contaminated areas of a site. These methods have some drawbacks. The first method simply moves the contamination elsewhere and may create significant risks in the excavation, handling and transport of hazardous material. Additionally, it is very difficult and increasingly expensive to find new landfill sites for the final disposal of the material. The cap and containment method is only an interim solution since the contamination remains on site, requiring monitoring and maintenance of the isolation barriers long into the future, with all the associated costs and potential liability. A better approach than these traditional methods is to completely destroy the pollutants, if possible, or transform them into harmless substances. Some technologies that have been used are high-temperature incineration and various types of chemical decomposition (for example, base-catalyzed dechlorination, UV oxidation). However, these methods have significant disadvantages, principally their technological complexity, high cost , and the lack of public acceptance. Bioremediation, on the contrast, is a promising option for the complete removal and destruction of contaminants. 1.3 Bioremediation of PAH contaminated soil & groundwater Bioremediation is the use of living organisms, primarily microorganisms, to degrade or detoxify hazardous wastes into harmless substances such as carbon dioxide, water and cell biomass Most PAHs are biodegradable unter natural conditions (Da Silva et al., 2003; Meysami and Baheri, 2003) and bioremediation for cleanup of PAH wastes has been extensively studied at both laboratory and commercial levels- It has been implemented at a number of contaminated sites, including the cleanup of the Exxon Valdez oil spill in Prince William Sound, Alaska in 1989, the Mega Borg spill off the Texas coast in 1990 and the Burgan Oil Field, Kuwait in 1994 (Purwaningsih, 2002). Different strategies for PAH bioremediation, such as in situ , ex situ or on site bioremediation were developed in recent years. In situ bioremediation is a technique that is applied to soil and groundwater at the site without removing the contaminated soil or groundwater, based on the provision of optimum conditions for microbiological contaminant breakdown.. Ex situ bioremediation of PAHs, on the other hand, is a technique applied to soil and groundwater which has been removed from the site via excavation (soil) or pumping (water). Hazardous contaminants are converted in controlled bioreactors into harmless compounds in an efficient manner. 1.4 Bioavailability of PAH in the subsurface Frequently, PAH contamination in the environment is occurs as contaminants that are sorbed onto soilparticles rather than in phase (NAPL, non aqueous phase liquids). It is known that the biodegradation rate of most PAHs sorbed onto soil is far lower than rates measured in solution cultures of microorganisms with pure solid pollutants (Alexander and Scow, 1989; Hamaker, 1972). It is generally believed that only that fraction of PAHs dissolved in the solution can be metabolized by microorganisms in soil. The amount of contaminant that can be readily taken up and degraded by microorganisms is defined as bioavailability (Bosma et al., 1997; Maier, 2000). Two phenomena have been suggested to cause the low bioavailability of PAHs in soil (Danielsson, 2000). The first one is strong adsorption of the contaminants to the soil constituents which then leads to very slow release rates of contaminants to the aqueous phase. Sorption is often well correlated with soil organic matter content (Means, 1980) and significantly reduces biodegradation (Manilal and Alexander, 1991). The second phenomenon is slow mass transfer of pollutants, such as pore diffusion in the soil aggregates or diffusion in the organic matter in the soil. The complex set of these physical, chemical and biological processes is schematically illustrated in Figure 1. As shown in Figure 1, biodegradation processes are taking place in the soil solution while diffusion processes occur in the narrow pores in and between soil aggregates (Danielsson, 2000). Seemingly contradictory studies can be found in the literature that indicate the rate and final extent of metabolism may be either lower or higher for sorbed PAHs by soil than those for pure PAHs (Van Loosdrecht et al., 1990). These contrasting results demonstrate that the bioavailability of organic contaminants sorbed onto soil is far from being well understood. Besides bioavailability, there are several other factors influencing the rate and extent of biodegradation of PAHs in soil including microbial population characteristics, physical and chemical properties of PAHs and environmental factors (temperature, moisture, pH, degree of contamination). Figure 1: Schematic diagram showing possible rate-limiting processes during bioremediation of hydrophobic organic contaminants in a contaminated soil-water system (not to scale) (Danielsson, 2000). 1.5 Increasing the bioavailability of PAH in soil Attempts to improve the biodegradation of PAHs in soil by increasing their bioavailability include the use of surfactants , solvents or solubility enhancers.. However, introduction of synthetic surfactant may result in the addition of one more pollutant. (Wang and Brusseau, 1993).A study conducted by Mulder et al. showed that the introduction of hydropropyl-ß-cyclodextrin (HPCD), a well-known PAH solubility enhancer, significantly increased the solubilization of PAHs although it did not improve the biodegradation rate of PAHs (Mulder et al., 1998), indicating that further research is required in order to develop a feasible and efficient remediation method. Enhancing the extent of PAHs mass transfer from the soil phase to the liquid might prove an efficient and environmentally low-risk alternative way of addressing the problem of slow PAH biodegradation in soil.

Empirical and fundamental mechanical tests in the evaluation of dough and bread rheological properties

Bread dough and particularly wheat dough, due to its viscoelastic behaviour, is probably the most dynamic and complicated rheological system and its characteristics are very important since they highly affect final products’ textural and sensorial properties. The study of dough rheology has been a very challenging task for many researchers since it can provide numerous information about dough formulation, structure and processing. This explains why dough rheology has been a matter of investigation for several decades. In this research rheological assessment of doughs and breads was performed by using empirical and fundamental methods at both small and large deformation, in order to characterize different types of doughs and final products such as bread. In order to study the structural aspects of food products, image analysis techniques was used for the integration of the information coming from empirical and fundamental rheological measurements. Evaluation of dough properties was carried out by texture profile analysis (TPA), dough stickiness (Chen and Hoseney cell) and uniaxial extensibility determination (Kieffer test) by using a Texture Analyser; small deformation rheological measurements, were performed on a controlled stress–strain rheometer; moreover the structure of different doughs was observed by using the image analysis; while bread characteristics were studied by using texture profile analysis (TPA) and image analysis. The objective of this research was to understand if the different rheological measurements were able to characterize and differentiate the different samples analysed. This in order to investigate the effect of different formulation and processing conditions on dough and final product from a structural point of view. For this aim the following different materials were performed and analysed: - frozen dough realized without yeast; - frozen dough and bread made with frozen dough; - doughs obtained by using different fermentation method; - doughs made by Kamut® flour; - dough and bread realized with the addition of ginger powder; - final products coming from different bakeries. The influence of sub-zero storage time on non-fermented and fermented dough viscoelastic performance and on final product (bread) was evaluated by using small deformation and large deformation methods. In general, the longer the sub-zero storage time the lower the positive viscoelastic attributes. The effect of fermentation time and of different type of fermentation (straight-dough method; sponge-and-dough procedure and poolish method) on rheological properties of doughs were investigated using empirical and fundamental analysis and image analysis was used to integrate this information throughout the evaluation of the dough’s structure. The results of fundamental rheological test showed that the incorporation of sourdough (poolish method) provoked changes that were different from those seen in the others type of fermentation. The affirmative action of some ingredients (extra-virgin olive oil and a liposomic lecithin emulsifier) to improve rheological characteristics of Kamut® dough has been confirmed also when subjected to low temperatures (24 hours and 48 hours at 4°C). Small deformation oscillatory measurements and large deformation mechanical tests performed provided useful information on the rheological properties of samples realized by using different amounts of ginger powder, showing that the sample with the highest amount of ginger powder (6%) had worse rheological characteristics compared to the other samples. Moisture content, specific volume, texture and crumb grain characteristics are the major quality attributes of bread products. The different sample analyzed, “Coppia Ferrarese”, “Pane Comune Romagnolo” and “Filone Terra di San Marino”, showed a decrease of crumb moisture and an increase in hardness over the storage time. Parameters such as cohesiveness and springiness, evaluated by TPA that are indicator of quality of fresh bread, decreased during the storage. By using empirical rheological tests we found several differences among the samples, due to the different ingredients used in formulation and the different process adopted to prepare the sample, but since these products are handmade, the differences could be account as a surplus value. In conclusion small deformation (in fundamental units) and large deformation methods showed a significant role in monitoring the influence of different ingredients used in formulation, different processing and storage conditions on dough viscoelastic performance and on final product. Finally the knowledge of formulation, processing and storage conditions together with the evaluation of structural and rheological characteristics is fundamental for the study of complex matrices like bakery products, where numerous variable can influence their final quality (e.g. raw material, bread-making procedure, time and temperature of the fermentation and baking).

Influence of processing conditions on quality aspects of potato-based fresh pasta

Gnocchi is a typical Italian potato-based fresh pasta that can be either homemade or industrially manufactured. The homemade traditional product is consumed fresh on the day it is produced, whereas the industrially manufactured one is vacuum-packed in polyethylene and usually stored at refrigerated conditions. At industrial level, most kinds of gnocchi are usually produced by using some potato derivatives (i.e. flakes, dehydrated products or flour) to which soft wheat flour, salt, some emulsifiers and aromas are added. Recently, a novel type of gnocchi emerged on the Italian pasta market, since it would be as much similar as possible to the traditional homemade one. It is industrially produced from fresh potatoes as main ingredient and soft wheat flour, pasteurized liquid eggs and salt, moreover this product undergoes steam cooking and mashing industrial treatments. Neither preservatives nor emulsifiers are included in the recipe. The main aim of this work was to get inside the industrial manufacture of gnocchi, in order to improve the quality characteristics of the final product, by the study of the main steps of the production, starting from the raw and steam cooked tubers, through the semi-finished materials, such as the potato puree and the formulated dough. For this purpose the investigation of the enzymatic activity of the raw and steam cooked potatoes, the main characteristics of the puree (colour, texture and starch), the interaction among ingredients of differently formulated doughs and the basic quality aspects of the final product have been performed. Results obtained in this work indicated that steam cooking influenced the analysed enzymes (Pectin methylesterase and α- and β-amylases) in different tissues of the tuber. PME resulted still active in the cortex, it therefore may affect the texture of cooked potatoes to be used as main ingredient in the production of gnocchi. Starch degrading enzymes (α- and β-amylases) were inactivated both in the cortex and in the pith of the tuber. The study performed on the potato puree showed that, between the two analysed samples, the product which employed dual lower pressure treatments seemed to be the most suitable to the production of gnocchi, in terms of its better physicochemical and textural properties. It did not evidence aggregation phenomena responsible of hard lumps, which may occur in this kind of semi-finished product. The textural properties of gnocchi doughs were not influenced by the different formulation as expected. Among the ingredients involved in the preparation of the different samples, soft wheat flour seemed to be the most crucial in affecting the quality features of gnocchi doughs. As a consequence of the interactive effect of the ingredients on the physicochemical and textural characteristics of the different doughs, a uniform and well-defined split-up among samples was not obtained. In the comparison of different kinds of gnocchi, the optimal physicochemical and textural properties were detected in the sample made with fresh tubers. This was probably caused not only by the use of fresh steam cooked potatoes, but also by the pasteurized liquid eggs and by the absence of any kind of emulsifier, additive or preserving substance.

Untersuchung der Ordnung und Diffusion von Polymerdispersionen bei ihrer Verfilmung

Im Rahmen dieser Arbeit wurde die, für industrielle Applikationen sehr wichtige, Trocknung und Verfilmung von Latexdispersionen untersucht. Unter der Verfilmung wird in diesem Zusammenhang allgemein der Übergang einer Polymerdispersion in einen transparenten, mechanisch stabilen Polymerfilm während ihrer Trocknung verstanden. Für die Untersuchungen wurden schwerpunktmäßig Streumethoden verwendet. Die Untersuchungen haben gezeigt, daß die Streuung eine besonders geeignete Methode zur Untersuchung der Verfilmung ist, die in Abhängigkeit des beobachteten Streuvektorbereichs, der verwendeten Strahlung, der Probenpräparation und des resultierenden Kontrasts eine Vielzahl unterschiedlicher Informationen über die Verfilmung in ihren verschiedenen Phasen liefert. Von besonderem Interesse war es, den prinzipiellen Verlauf der Verfilmung bei den heterogen trocknenden Reinacrylatlatices zu untersuchen. Dazu wurde mit Hilfe der Röntgenultrakleinwinkelstreuung gezielt der Zustand der Partikel in den einzelnen Phasen der heterogen trocknenden Proben beobachtet. Mit Hilfe der Neutronenkleinwinkelstreuung konnte das Verhalten des Emulgators während der Verfilmung und dessen Verteilung im resultierenden Film genauer untersucht werden. Die Röntgenkleinwinkelstreuung erlaubte eine eingehende Untersuchung der Kristallisation des Emulgators im trockenen Film. Geeignete Kontrastierung durch gezielte Deuterierung ermöglichte die Untersuchung des Comonomereinflusses auf die Interdiffusion von Latexpartikeln mit Neutronenkleinwinkelstreuung. Aus den Meßergebnissen wurde ein Modell zur heterogenen Trocknung von Latexdispersionen entwickelt, das den Ablauf der Verfilmung in einem konsistenten Bild zusammenfaßt.

Food lipids: their effects on quality and oxidative stability of animal tissues and emulsions

Lipolysis and oxidation of lipids in foods are the major biochemical and chemical processes that cause food quality deterioration, leading to the characteristic, unpalatable odour and flavour called rancidity. In addition to unpalatability, rancidity may give rise to toxic levels of certain compounds like aldehydes, hydroperoxides, epoxides and cholesterol oxidation products. In this PhD study chromatographic and spectroscopic techniques were employed to determine the degree of lipid oxidation in different animal products and its relationship with technological parameters like feeding fat sources, packaging, processing and storage conditions. To achieve this goal capillary gas chromatography (CGC) was employed not only to determine the fatty acids profile but also, after solid phase extraction, the amount of sterols (cholesterol and phytosterols) and cholesterol oxidation products (COPs). To determine hydroperoxides, primary products of oxidation and quantify secondary products UV/VIS absorbance spectroscopy was applied. Beef and pork meat in this study were analysed. In actual fact, lipid oxidation is a major deterioration reaction in meat, meat products and results in adverse changes in the colour, flavour, texture of meat and develops different compounds which should be a risk to human health as oxysterols. On beef and pork meat, a study of lipid fraction during storage was carried out to evaluate its shelf-life and some nutritional features life saturated/unsaturated fatty acids ratio and sterols content, in according to the interest that has been growing around functional food in the last years. The last part of this research was focused on the study of lipid oxidation in emulsions. In oil-in-water emulsions antioxidant activity of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) was evaluated. The rates of lipid oxidation of 1.0% stripped soybean oil-in-water emulsions with DOPC were followed by monitoring lipid hydroperoxide and hexanal as indicators of primary and secondary oxidation products and the droplet surface charge or zeta potential (ζ) of the emulsions with varying concentrations of DOPC were tested. This manuscript reports the main results obtained in the three activities briefly summarized as follows: 1. study on effects of feeding composition on the photoxidative stability of lipids from beef meat, evaluated during storage under commercial retail conditions; 2. evaluation of effects of diets and storage conditions on the oxidative stability of pork meat lipids; 3. study on oxidative behavior of DOPC in stripped soybean oil-in-water emulsions stabilized by nonionic surfactant.

Untersuchung der Filmbildung aus Polymerdispersionen mit Hilfe der forcierten Rayleighstreuung

Der Filmbildungsprozess wässriger Polymerdispersionen wurde mit forcierter Rayleighstreuung untersucht.Diffusionskoeffizienten D von Farbstoffsonden sind damit zwischen 10^-21 und 10^-9 m^2s^-1 zugänglich. Das Diffusionsverhalten der hydrophoben FarbstoffsondeAberchrome in feuchten und trockenen Filmen aus Poly(n-butylmethacrylat-co-acrylsäure)-Dispersionen sowie bei der Wiederbefeuchtung trockener Filme wurde untersucht.Die Dynamik von Aberchrome weicht in feuchten Filmen von Fickscher Diffusion ab. Dies äußert sich in Abweichungen vomcharakteristischen q^2-Verhalten der Relaxationsrate tau^-1 (tau^-1 = Dq^2; q:Streuvektor) und im Auftreten gestrecktexponentieller Intensitätsrelaxationskurven. Diese Anomalie wurde mit einem apparenten, längenskalenabhängigen Diffusionskoeffizienten Dapp(Lambda) (Lambda= 2Pi/q) beschrieben, der für Lambda -> 0 Werte annimmt, die einem homogen hydroplastifizierten Polymermaterial entsprechen, während Dapp(Lambda) für Lambda -> Unendlich stark anwächst. Diese Anomalien verschwinden bei Wassergehalten entsprechend der Polymerwasserlöslichkeit. Weiteres Trocknen führt zum Absinken des Fickschen-Diffusionskoeffizienten auf einen Grenzwert für trockene Filme. Die Ergebnisse konnten mit einem Zwei-Zustands-Modell beschrieben werden: Die Sonde diffundiert Ficksch in einer hydrophilen Grenzflächenphase und einer langsamen Polymerphase. Austausch zwischendiesen Phasen ist ohne Einschränkung möglich. Das Modell erlaubt die Quantifizierung des Einflusses des Trocknungsprozesses auf Polymer- und Grenzflächenphaseneigenschaften.Dies wurde durch systematische Veränderungen der Grenzflächeneigenschaften demonstriert. Dies geschah durch Acrylsäuregehaltvariationen in den Poly(n-butylmethacrylat-co-acrylsäure)-Dispersionen und Emulgatorbedeckungsgradvariationen. In beiden Fällen reflektieren sich Dispersionsveränderungen in einer Veränderung der Modellparameter in der Grenzflächenphase.

Anisotrope Kolloide aus flüssigkristallinen Polymeren

In dieser Arbeit wird die Synthese, Charakterisierung und Manipulation anisotroper Kolloide aus flüssigkristallinen Polymeren beschrieben. Um Kolloide verschiedener Größe und aus verschiedenen Polymeren zu erhalten, wurden verschiedene Techniken verwendet. Einerseits wurden Kolloide aus nematischen und smektischen Polymeren mit Durchmessern meist im Bereich von 0,5 bis 3,5 Mikrometern hergestellt. Dazu wurden 16 verschiedene Acrylat- und Methacrylatmonomere synthetisiert und mittels Dispersionspolymerisation polymerisiert. Durch Variation der Polymerisationsbedingungen wurden Kolloide verschiedener Größe und Polydispersität erhalten. Durch Saatpolymerisation konnten zudem die Kugelgrößen bei gleichbleibend geringer Polydispersität erhöht werden. Polarisationsmikroskopie zeigt, dass die meisten Kolloide mit einer Größe zwischen ca. 2 bis 4 Mikrometern eine bipolare Direktorkonfiguration haben. Einige dieser Kolloide wurden mit einer optischen Pinzette mit zirkular polarisiertem Licht eingefangen und rotiert. Zum anderen wurden verschiedene flüssigkristalline Polymere (Polysiloxane, Hauptkettenpolymere und Polyacrylate) durch den Miniemulsionsprozess in Kolloide mit Durchmessern im Bereich von ca. 50 bis 300 nm überführt. Durch Variation der Emulgator- und Polymermenge sowie der Art des Emulgators konnte die Kugelgröße beeinflusst werden. Für die Polysiloxankolloide erfolgte die Aufklärung ihrer inneren Struktur mittels TEM und Kryo-TEM, da durch das Silizium im Polymerrückgrat ohne zusätzliches Anfärben ein Kontrast vorhanden ist. Die TEM-Aufnahmen zeigen deutlich die smektische Schichtstruktur innerhalb der Kolloide aus „verdünnten“ Copolysiloxanen und sind somit der erste direkte Beweis für die Mikrophasenseparation zwischen den Mesogenen und Polysiloxanketten, die bisher basierend auf Röntgenmessungen nur indirekt vorhergesagt wurde. Für die Copolysiloxane mit 2-Ring-Mesogenen wurden zwiebelartige Strukturen und für die Copolysiloxane mit 3-Ring-Mesogenen parallele Schichtstrukturen gefunden. Im ersten Fall folgt die smektische Schichtstruktur der Kugelsymmetrie des Kolloids, im zweiten Fall dominiert die Tendenz der smektischen Schichten, sich parallel anzuordnen.

Nicht-wässrige Emulsionspolymerisationen

Die vorliegende Arbeit beschäftigt sich mit der Darstellung von Latexpartikeln in nicht-wässrigen Emulsionssystemen. Hintergrund der Untersuchungen war die Frage, ob es durch die Anwendung von nicht-wässrigen Emulsionen ermöglicht werden kann, sowohl wassersensitive Monomere als auch feuchtigkeitsempfindliche Polymerisationen zur Darstellung von Polymer-Latexpartikeln und deren Primärdispersionen einzusetzen. Das Basiskonzept der Arbeit bestand darin, nicht-wässrige Emulsionen auf der Basis zweier nicht mischbarer organischer Lösungsmittel unterschiedlicher Polarität auszubilden und anschließend die dispergierte Phase der Emulsion zur Synthese der Latexpartikel auszunutzen. Hierzu wurden verschiedene nicht-wässrige Emulsionssysteme erarbeitet, welche als dispergierte Phase ein polares und als kontinuierliche Phase ein unpolares Lösungsmittel enthielten. Auf Basis dieser Ergebnisse wurde in den nachfolgenden Untersuchungen zunächst die Anwendbarkeit solcher Emulsionen zur Darstellung verschiedener Acrylat- und Methacrylatpolymerdispersionen mittels radikalischer Polymerisation studiert. Um zu zeigen, dass die hier entwickelten nicht-wässrigen Emulsionen auch zur Durchführung von Stufenwachstumsreaktionen geeignet sind, wurden ebenfalls Polyester-, Polyamid- und Polyurethan-Latexpartikel dargestellt. Die Molekulargewichte der erhaltenen Polymere lagen bei bis zu 40 000 g/mol, im Vergleich zu wässrigen Emulsions- und Miniemulsions¬polymerisationssystemen sind diese um den Faktor fünf bis 30 höher. Es kann davon ausgegangen werden, dass hauptsächlich zwei Faktoren für die hohen Molekulargewichte verantwortlich sind: Zum einen die wasserfreien Bedingungen, welche die Hydrolyse der reaktiven Gruppen verhindern, und zum anderen die teilweise erfüllten Schotten-Baumann-Bedingungen, welche an der Grenzfläche zwischen dispergierter und kontinuierlicher Phase eine durch Diffusion kontrollierte ausgeglichene Stöchiometrie der Reaktionspartner gewährleisten. Somit ist es erstmals möglich, hochmolekulare Polyester, -amide und -urethane in nur einem Syntheseschritt als Primär¬dispersion darzustellen. Die Variabilität der nicht-wässrigen Emulsionen wurde zudem in weiteren Beispielen durch die Synthese von verschiedenen elektrisch leitfähigen Latices, wie z.B. Polyacetylen-Latexpartikeln, aufgezeigt. In dieser Arbeit konnte gezeigt werden, dass die entwickelten nicht-wässrigen Emulsionen eine äußerst breite Anwendbarkeit zur Darstellung von Polymer-Latexpartikeln aufweisen. Durch die wasserfreien Bedingungen erlauben die beschriebenen Emulsionsprozesse, Latexpartikel und entsprechende nicht-wässrige Dispersionen nicht nur traditionell radikalisch, sondern auch mittels weiterer Polymerisationsmechanismen (katalytisch, oxidativ oder mittels Polykondensation bzw. -addition) darzustellen.

Neue Untersuchungen zum Verordnungsmonitoring sowie zur Kompatibilität und Stabilität applikationsfertiger Zytostatika-Zubereitungen

Applikationsfertige Zytostatikazubereitungen werden heute unter der Verantwortung eines Apothekers in zentralisierten Herstellungsbereichen hergestellt. Weil die Verordnung der Chemotherapie ein großes Fehlerrisiko birgt, ist konsequentes Verordnungsmonitoring ein wesentlicher Teilprozess der zentralen Zytostatikazubereitung. rnDie aktuelle Umsetzung und die Ergebnisse des Verordnungsmonitorings in den Universitätskliniken Deutschlands wurden im Rahmen dieser Arbeit in einer prospektiven Erhebung erfasst. Als häufigste Verordnungsirrtümer wurden Dosisberechnungsfehler (48%), welche als von hoher Relevanz (78%) für die Patientensicherheit angesehen wurden, genannt. Die Inzidenz der Verordnungsfehler betrug durchschnittlich 0,77% bei rund 1950 Verordnungen pro Tag. Das konsequente Verordnungsmonitoring von pharmazeutischer Seite erfolgt höchst effizient und leistet einen hohen Beitrag zur Patienten- und Arzneimitteltherapiesicherheit in der Onkologie.rnFür die Herstellung der applikationsfertiger Zytostatika-Zubereitungen sind fundierte Kenntnisse zu deren physikalisch-chemischen Stabilität erforderlich. Zu neu zugelassenen Zytostatika und insbesondere Biologicals, stehen häufig noch keine Daten zur Stabilität der applikationsfertigen Lösungen zur Verfügung. Die Bestimmung der physikalisch-chemischen Stabilität war daher Gegenstand dieser Arbeit. Die applikationsfertigen Infusionslösungen der Purin-Analoga Nelarabin und Clofarabin (RP-HPLC), sowie des monoklonalen Antiköpers Trastuzumab (SEC, UV-Spektroskopie, SDS-Page), erwiesen sich über einen Zeitraum von mindestens 28 Tagen als stabil. Die Stabilität zweier Camptothecin-Derivate (Topotecan und Irinotecan) beladen auf DC Beads™, wie auch die Ladungskapazität und Kompatibilität mit Kontrastmitteln, wurde ebenfalls bewiesen. rn

Nicht-wässrige Emulsionen zur Erzeugung neuer Nanopartikel

In der eingereichten Arbeit wurde die Nutzung von nicht-wässrigen Emulsionen, bestehend aus zwei organischen, aprotischen Lösungsmitteln, zur Erzeugung verschiedener polymerer Nanopartikel beschrieben. Diese Zweiphasenmischungen und die Verwendung maßgeschneiderter Emulgatoren bestehend aus Poly(isopren-block-methylmethacrylat) ermöglichten den Zugang zu einer Vielzahl an Reaktionen und Prozessen, welche in wässrigen Emulsionen bisher nicht oder nur schwer möglich waren. Die Generierung von Partikeln auf Basis katalytischer Polymerisationen erfolgte unter Verwendung der Ringöffnenden Metathese-Polymerisation (ROMP), der Acyclischen Dien-Metathese-Polymerisation (ADMET), der Cyclopolymerisation von α,ω-Diinen und der Ni-katalysierten Polymerisation von Isocyaniden. Mittels ROMP konnten stabile Dispersionen erzeugt werden, welche Partikel mit verschiedensten Molekulargewichten, Größen und Morphologien enthielten. Diese Eigenschaften konnten durch die Wahl des Monomers, die Katalysatorkonzentration oder den Emulgatortyp beeinflusst werden. Des Weiteren wurden Partikel mit komplexen Morphologien wie Kern-Schale-Strukturen synthetisiert. Dazu erfolgte die Generierung von Partikeln aus Poly(urethan) oder Poly(norbornenderivaten), welche in situ und ohne intermediäre Aufarbeitung mit einer Schale aus Poly(methacrylat) versehen wurden. Der Nachweis dieser Strukturen gelang mittels verschiedener Schwermetall-Markierungsverfahren in der Transmissionselektronenmikroskopie. Schlussendlich erfolgte die Herstellung von hochvernetzten und molekular geprägten Poly(acrylsäure)-Partikeln. Hierbei wurden unterschiedliche pharmakologische Wirkstoffe und Farbstoffe in die Partikel eingebracht, um deren Migrationsverhalten und Wiederanbindung an die Partikel zu untersuchen. Weiterhin wurden die Partikel erfolgreich in Zellaufnahmeexperimenten eingesetzt.

Microemulsion: prediction of the phase diagram with a modified Helfrich free energy

Microemulsions are thermodynamically stable, macroscopically homogeneous but microscopically heterogeneous, mixtures of water and oil stabilised by surfactant molecules. They have unique properties like ultralow interfacial tension, large interfacial area and the ability to solubilise other immiscible liquids. Depending on the temperature and concentration, non-ionic surfactants self assemble to micelles, flat lamellar, hexagonal and sponge like bicontinuous morphologies. Microemulsions have three different macroscopic phases (a) 1phase- microemulsion (isotropic), (b) 2phase-microemulsion coexisting with either expelled water or oil and (c) 3phase- microemulsion coexisting with expelled water and oil.rnrnOne of the most important fundamental questions in this field is the relation between the properties of the surfactant monolayer at water-oil interface and those of microemulsion. This monolayer forms an extended interface whose local curvature determines the structure of the microemulsion. The main part of my thesis deals with the quantitative measurements of the temperature induced phase transitions of water-oil-nonionic microemulsions and their interpretation using the temperature dependent spontaneous curvature [c0(T)] of the surfactant monolayer. In a 1phase- region, conservation of the components determines the droplet (domain) size (R) whereas in 2phase-region, it is determined by the temperature dependence of c0(T). The Helfrich bending free energy density includes the dependence of the droplet size on c0(T) as

Expanding the scope of poly(ethylene glycol)s for bioconjugation to squaric acid-mediated PEGylation and pH-sensitivity

Poly(ethylene glycol) (PEG) is used in a broad range of applications due to its unique combination of properties and is approved use in formulations for body-care products, edibles and medicine. This thesis aims at the synthesis and characterization of novel heterofunctional PEG structures and the establishment of diethyl squarate as a suitable linker for the covalent attachment to proteins. Chapter 1 is an introduction on the properties and applications of PEG as well as the fascinating chemistry of squaric acid derivatives. In Chapter 1.1, the synthesis and properties of PEG are described, and the versatile applications of PEG derivatives in everyday products are emphasized with a focus on PEG-based pharmaceuticals and nonionic surfactants. This chapter is written in German, as it was published in the German Journal Chemie in unserer Zeit. Chapter 1.2 deals with PEGs major drawbacks, its non-biodegradability, which impedes parenteral administration of PEG conjugates with polyethers exceeding the renal excretion limit, although these would improve blood circulation times and passive tumor targeting. This section gives a comprehensive overview of the cleavable groups that have been implemented in the polyether backbone to tackle this issue as well as the synthetic strategies employed to accomplish this task. Chapter 1.3 briefly summarizes the chemical properties of alkyl squarates and the advantages in protein conjugation chemistry that can be taken from its use as a coupling agent. In Chapter 2, the application of diethyl squarate as a coupling agent in the PEGylation of proteins is illustrated. Chapter 2.1 describes the straightforward synthesis and characterization of squaric acid ethyl ester amido PEGs with terminal hydroxyl functions or methoxy groups. The reactivity and selectivity of theses activated PEGs are explored in kinetic studies on the reactions with different lysine and other amino acid derivatives, followed by 1H NMR spectroscopy. Further, the efficient attachment of the novel PEGs to a model protein, i.e., bovine serum albumin (BSA), demonstrates the usefulness of the new linker for the PEGylation with heterofunctional PEGs. In Chapter 2.3 initial studies on the biocompatibility of polyether/BSA conjugates synthesized by the squaric acid mediated PEGylation are presented. No cytotoxic effects on human umbilical vein endothelial cells exposed to various concentrations of the conjugates were observed in a WST-1 assay. A cell adhesion molecule - enzyme immunosorbent assay did not reveal the expression of E-selectin or ICAM-1, cell adhesion molecules involved in inflammation processes. The focus of Chapter 3 lies on the syntheses of novel heterofunctional PEG structures which are suitable candidates for the squaric acid mediated PEGylation and exhibit superior features compared to established PEGs applied in bioconjugation. Chapter 3.1 describes the synthetic route to well-defined, linear heterobifunctional PEGs carrying a single acid-sensitive moiety either at the initiation site or at a tunable position in the polyether backbone. A universal concept for the implementation of acetal moieties into initiators for the anionic ring-opening polymerization (AROP) of epoxides is presented and proven to grant access to the degradable PEG structures aimed at. The hydrolysis of the heterofunctional PEG with the acetal moiety at the initiating site is followed by 1H NMR spectroscopy in deuterium oxide at different pH. In an exploratory study, the same polymer is attached to BSA via the squarate acid coupling and subsequently cleaved from the conjugate under acidic conditions. Furthermore, the concept for the generation of acetal-modified AROP initiators is demonstrated to be suitable for cholesterol, and the respective amphiphilic cholesteryl-PEG is cleaved at lowered pH. In Chapter 3.2, the straightforward synthesis of α-amino ω2-dihydroxyl star-shaped three-arm PEGs is described. To assure a symmetric length of the hydroxyl-terminated PEG arms, a novel AROP initiator is presented, who’s primary and secondary hydroxyl groups are separated by an acetal moiety. Upon polymerization of ethylene oxide for these functionalities and subsequent cleavage of the acid-labile unit no difference in the degree of polymerization is seen for both polyether fragments.

Darstellung von fluorierten Amphiphilen zur Modifikation von Grenzflächeneigenschaften

Die vorliegende Arbeit behandelt die Darstellung von neuen fluorierten Amphiphilen in Hinblick auf deren spätere Anwendung zur Modifikation von flüssig-gasförmigen, flüssig-flüssigen und flüssig-festen Grenzflächen. Jedes der drei Themengebiete richtete sich an eine andere Grenzfläche. Die erste Aufgabe wurde mit der Darstellung und Charakterisierung von Fluortensiden kurzen Perfluoralkyl-Ketten erfüllt, welche als möglichen umweltfreundlichen Ersatz für die Derivate der langkettigen Perfluoroktancarbonsäure und Perfluoroktansulfonsäure Anwendung finden. Es wurden Fluortenside entwickelt, welche die Oberflächenspannung von Wasser auf bis zu 18 mN/m absenken können. Der zweite Fokus lag auf der Stabilisierung einer fluorierten Emulsion, die sich für die carbo-kationische Polymerisationen eignete. Im Rahmen der Arbeit wurde das neue Blockcopolymer Poly(isobutylen-b-pentafluorstyrol) dargestellt. Neben der Homopolymerisation von Isobutylen zur PIB-Partikelsynthese wurde die Eignung des Stabilisator-Emulsionssystems durch die Darstellung von hart-weichen PS-PIB-Hybridpartikeln erfolgreich verfolgt. Dazu wurde die Synthese von quervernetzten PS-Kernpartikeln und der anschließenden Umhüllung durch PIB in einem weiteren Schritt. Im dritten Teil wurde die Adsorption von amphiphilen Fluorpolymeren zur Solvophobierung von rauen anorganischen Oberflächen untersucht. Es konnten sowohl superhydrophobe als auch superoleophobe Eigenschaften erzielt werden.

Monoclonal antibody directed against Neospora caninum tachyzoite carbohydrate epitope reacts specifically with apical complex-associated sialylated beta tubulin

Monoclonal antibodies (mabs) were generated against whole sonicated Neospora caninum tachyzoites as immunogen. Initial ELISA screening of the reactivity of hybridoma culture supernatants using the same antigen and antigen treated with sodium periodate prior to antibody binding resulted in the identification of 8 supernatants with reactivity against putative carbohydrate epitopes. Following immunoblotting, mab6D12 (IgG1), binding a 52/48-kDa doublet, and mab6C6 (IgM), binding a 190/180-kDa doublet, were selected for further studies. Immunofluorescence of tachyzoite-infected cultures localized the corresponding epitopes not to the surface, but to interior epitopes at the apical part of N. caninum tachyzoites. During in vitro tachyzoite to bradyzoite stage conversion, mab6C6 labeling translocated toward the cyst periphery, while for mab6D12 no changes in localization were noted. Upon extraction of tachyzoites with the nonionic detergent Triton-X-100, the 52-kDa band recognized by mab6D12 was present exclusively in the insoluble, cytoskeletal fraction of both N. caninum and Toxoplasma gondii tachyzoites. Tandem mass spectrometry analysis identified this protein as N. caninum beta tubulin. The 48-kDa band labeled by mab6D12 was a Vero cell protein contamination. The protein(s) reacting with mab6C6 could not be conclusively identified by mass spectrometry. Immunofluorescence consistently failed to label T. gondii tachyzoites, indicating that beta tubulin in T. gondii and N. caninum could be differentially modified or that the reactive epitope in T. gondii is masked. Immunogold TEM of isolated apical cytoskeletal preparations and dual immunofluorescence with antibody to tubulin confirmed that mab6D12 binds to the anterior part of apical complex-associated microtubules. The sodium periodate sensitivity of the beta tubulin associated epitope was confirmed by immunoblotting and ELISA, and treatment of N. caninum cytoskeletal proteins with sialidase prior to mab6D12 labeling resulted in a profound loss of antibody binding, suggesting that mab6D12 reacts with sialylated beta tubulin.

Modulation of Immune Response in Lambs

Experiments using different types of antigen-adjuvant preparations were conducted in outbred sheep to compare effects of adjuvants on immune responses. Trinitrophenyl-ovalbumin (TNP-ovalbumin) incorporated in a preparation with nonionic block copolymers elicited high antibody titers to both ovalbumin and TNP. Different humoral immune responses were observed when Pasteurella haemolytica lipopolysaccharide (LPS) was added to the preparations. Responses to ovalbumin and TNP were reduced when Pasteurella haemolytica LPS was added to copolymer L121. The antibody titers to ovalbumin or TNP were not affected by the addition of LPS to the preparation containing copolymer L180.5. Lymphocyte proliferation assays demonstrated high stimulation indices at day 17 to ovalbumin by lymphocytes from lambs receiving preparations containing copolymers without Pasteurella haemolytica LPS.