Development of newly conceived biomimetic nano-structured biomaterials as scaffolds for bone and osteochondral regeneration

The present research thesis was focused on the development of new biomaterials and devices for application in regenerative medicine, particularly in the repair/regeneration of bone and osteochondral regions affected by degenerative diseases such as Osteoarthritis and Osteoporosis or serious traumas. More specifically, the work was focused on the synthesis and physico-chemical-morphological characterization of: i) a new superparamagnetic apatite phase; ii) new biomimetic superparamagnetic bone and osteochondral scaffolds; iii) new bioactive bone cements for regenerative vertebroplasty. The new bio-devices were designed to exhibit high biomimicry with hard human tissues and with functionality promoting faster tissue repair and improved texturing. In particular, recent trends in tissue regeneration indicate magnetism as a new tool to stimulate cells towards tissue formation and organization; in this perspective a new superparamagnetic apatite was synthesized by doping apatite lattice with di-and trivalent iron ions during synthesis. This finding was the pin to synthesize newly conceived superparamagnetic bone and osteochondral scaffolds by reproducing in laboratory the biological processes yielding the formation of new bone, i.e. the self-assembly/organization of collagen fibrils and heterogeneous nucleation of nanosized, ionically substituted apatite mimicking the mineral part of bone. The new scaffolds can be magnetically switched on/off and function as workstations guiding fast tissue regeneration by minimally invasive and more efficient approaches. Moreover, in the view of specific treatments for patients affected by osteoporosis or traumas involving vertebrae weakening or fracture, the present work was also dedicated to the development of new self-setting injectable pastes based on strontium-substituted calcium phosphates, able to harden in vivo and transform into strontium-substituted hydroxyapatite. The addition of strontium may provide an anti-osteoporotic effect, aiding to restore the physiologic bone turnover. The ceramic-based paste was also added with bio-polymers, able to be progressively resorbed thus creating additional porosity in the cement body that favour cell colonization and osseointegration.

Analytical methods for evaluating the quality and the genuineness of olive oils

This Ph.D. thesis focuses on the investigation of some chemical and sensorial analytical parameters linked to the quality and purity of different categories of oils obtained by olives: extra virgin olive oils, both those that are sold in the large retail trade (supermarkets and discounts) and those directly collected at some Italian mills, and lower-quality oils (refined, lampante and “repaso”). Concurrently with the adoption of traditional and well-known analytical procedures such as gas chromatography and high-performance liquid chromatography, I carried out a set-up of innovative, fast and environmentally-friend methods. For example, I developed some analytical approaches based on Fourier transform medium infrared spectroscopy (FT-MIR) and time domain reflectometry (TDR), coupled with a robust chemometric elaboration of the results. I investigated some other freshness and quality markers that are not included in official parameters (in Italian and European regulations): the adoption of such a full chemical and sensorial analytical plan allowed me to obtain interesting information about the degree of quality of the EVOOs, mostly within the Italian market. Here the range of quality of EVOOs resulted very wide, in terms of sensory attributes, price classes and chemical parameters. Thanks to the collaboration with other Italian and foreign research groups, I carried out several applicative studies, especially focusing on the shelf-life of oils obtained by olives and on the effects of thermal stresses on the quality of the products. I also studied some innovative technological treatments, such as the clarification by using inert gases, as an alternative to the traditional filtration. Moreover, during a three-and-a-half months research stay at the University of Applied Sciences in Zurich, I also carried out a study related to the application of statistical methods for the elaboration of sensory results, obtained thanks to the official Swiss Panel and to some consumer tests.

Continuous-Flow Magnetic Separation with Permanent Magnets for Water Treatment

More efficient water treatment technologies would decrease the water bodies’ pollution and the actual intake of water resource. The aim of this thesis is an in-depth analysis of the magnetic separation of pollutants from water by means of a continuous-flow magnetic filter subjected to a field gradient produced by permanent magnets. This technique has the potential to improve times and efficiencies of both urban wastewater treatment plants and drinking water treatment plants. It might also substitute industrial wastewater treatments. This technique combines a physico-chemical phase of adsorption and a magnetic phase of filtration, having the potential to bond magnetite with any conventional adsorbent powder. The removal of both Magnetic Activated Carbons (MACs) and zeolite-magnetite mix with the addition of a coagulant was investigated. Adsorption tests of different pollutants (surfactants, endocrine disruptors, Fe(III), Mn(II), Ca(II)) on these adsorbents were also performed achieving good results. The numerical results concerning the adsorbent removals well reproduced the experimental ones obtained from two different experimental setups. In real situations the treatable flow rates are up to 90 m3/h (2000 m3/d).

Milk and dairy products: evaluation of bioactive components by analytical techniques

Milk and dairy products are important source of bioactive compounds useful to satisfy the nutritional and physiological needs of any newborns of mammalian species and useful to guarantee adequate growth and development of infants as well as provide a complete nourishment of adults. Physico-chemical, nutritional and organoleptic properties of the main constituents and the “minor” components have a crucial role in the quality of milk and milk products. Although in the past decades dietary milk fat was often regarded as harmful for the human health, recent researches suggest that milk contains specific fatty acids with nutritional and physiological health benefits. For these reasons, a major attention is given to the quantity and quality of total fat intake. In the recent years, as a result of the new concept of multifunctional agriculture and the changing behaviours about diet, consumer demands in favor of high-quality, security and safety dairy products are increased. Moreover, milk proteins and milk-derived bioactive peptides are recognized to have a high nutritive value, several health-promoting functional activities and excellent technological properties. Accordingly, growing interest in the development of functional dairy products and preparation of infant formulae for babies who cannot be breast-fed, has been give in order to meet the specific consumer’s requests. This manuscript presents the main results obtained during my PhD research aimed to evaluate the main bioactive lipids and proteins in milk and dairy products using innovative analytical techniques. The experimental section of this manuscript is divided in two sections where are reported the main results obtained during my research activities on dairy products and human milks in order to characterize their bioactive compounds for functional food applications.

Global simulated soil biogenic nitric oxide (NO) emissions: Impact, improvement and innovation

rnNitric oxide (NO) is important for several chemical processes in the atmosphere. Together with nitrogen dioxide (NO2 ) it is better known as nitrogen oxide (NOx ). NOx is crucial for the production and destruction of ozone. In several reactions it catalyzes the oxidation of methane and volatile organic compounds (VOCs) and in this context it is involved in the cycling of the hydroxyl radical (OH). OH is a reactive radical, capable of oxidizing most organic species. Therefore, OH is also called the “detergent” of the atmosphere. Nitric oxide originates from several sources: fossil fuel combustion, biomass burning, lightning and soils. Fossil fuel combustion is the largest source. The others are, depending on the reviewed literature, generally comparable to each other. The individual sources show a different temporal and spatial pattern in their magnitude of emission. Fossil fuel combustion is important in densely populated places, where NO from other sources is less important. In contrast NO emissions from soils (hereafter SNOx) or biomass burning are the dominant source of NOx in remote regions.rnBy applying an atmospheric chemistry global climate model (AC-GCM) I demonstrate that SNOx is responsible for a significant part of NOx in the atmosphere. Furthermore, it increases the O3 and OH mixing ratio substantially, leading to a ∼10% increase in the oxidizing efficiency of the atmosphere. Interestingly, through reduced O3 and OH mixing ratios in simulations without SNOx, the lifetime of NOx increases in regions with other dominating sources of NOx

Geological and hydrogeological features affecting migration, multi-phase partitioning and degradation of chlorinated hydrocarbons through unconsolidated porous media.

Chlorinated solvents are the most ubiquitous organic contaminants found in groundwater since the last five decades. They generally reach groundwater as Dense Non-Aqueous Phase Liquid (DNAPL). This phase can migrate through aquifers, and also through aquitards, in ways that aqueous contaminants cannot. The complex phase partitioning to which chlorinated solvent DNAPLs can undergo (i.e. to the dissolved, vapor or sorbed phase), as well as their transformations (e.g. degradation), depend on the physico-chemical properties of the contaminants themselves and on features of the hydrogeological system. The main goal of the thesis is to provide new knowledge for the future investigations of sites contaminated by DNAPLs in alluvial settings, proposing innovative investigative approaches and emphasizing some of the key issues and main criticalities of this kind of contaminants in such a setting. To achieve this goal, the hydrogeologic setting below the city of Ferrara (Po plain, northern Italy), which is affected by scattered contamination by chlorinated solvents, has been investigated at different scales (regional and site specific), both from an intrinsic (i.e. groundwater flow systems) and specific (i.e. chlorinated solvent DNAPL behavior) point of view. Detailed investigations were carried out in particular in one selected test-site, known as “Caretti site”, where high-resolution vertical profiling of different kind of data were collected by means of multilevel monitoring systems and other innovative sampling and analytical techniques. This allowed to achieve a deep geological and hydrogeological knowledge of the system and to reconstruct in detail the architecture of contaminants in relationship to the features of the hosting porous medium. The results achieved in this thesis are useful not only at local scale, e.g. employable to interpret the origin of contamination in other sites of the Ferrara area, but also at global scale, in order to address future remediation and protection actions of similar hydrogeologic settings.

From carbazole-containing and pi-expanded triangular trinuclear porphyrinoids

Zusammenfassung:rnDie vorliegende Arbeit beschreibt das Design und die Synthese neuartiger Porphyrinoide anhand der Modifikation und der π-Systemausdehnung an der Peripherie des Porphyrin-Gerüsts. Die Darstellung künstlicher Porphyrine ist von Interesse, da neue physiko-chemischen Eigenschaften erhalten und untersucht werden können. Die in dieser Arbeit vorgestellten Porphyrinoide wurden mit Hilfe von modernen Synthesemethoden wie den metallkatalysierten Kreuzkupplungen und somit durch Aryl-Aryl Verknüpfungen aufgebaut.rnDer erste Teil dieser Arbeit befasst sich mit der Modifikation des Porphyrin-Gerüsts. Porphyrine bestehen aus jeweils zwei Pyrrol- und Pyrrolenin-Einheiten, welche systematisch ausgetauscht wurden. Die Pyrrol-Einheiten wurden durch Carbazol ersetzt, das sich formal vom Pyrrol durch Anfügen von zwei Benzogruppen ableitet und deshalb besonders gut geeignet ist. Die Pyrrolenin-Einheiten wurden aus folgenden Gründen durch andere Heterozyklen wie Pyridin, Pyrrol oder Triazol ersetzt: rn* Nachbildung des stabilen Porphyrin trans-NH-Tautomers (Carbazol und Pyridin)rn* Nachbildung von (NH)4-Liganden wie Calix[4]pyrrol (Carbazol und Pyrrol)rn* Vereinigung von N-H und C-H Wasserstoffbrücken-Donor-Einheiten in einem Makrozyklus (Carbazol und Triazol)rnDie Synthese eines drei-Zentren Porphyrinoids mit ausgedehntem π-System wird im zweiten Teil der vorliegenden Arbeit beschrieben. Dieses Thema basiert auf der aktuellen Entwicklung von nicht-Edelmetall basierten Katalysatoren für die Reduktion von Sauerstoff. Hier werden derzeit N4 makrozyklische Metallkomplexe, die mehrere katalytisch aktive Stellen aufweisen, untersucht. In diesem Zusammenhang, hat die Gruppe von Prof. Müllen einen neuartigen drei-Zentren-Komplex entwickelt. Ausgehend von diesen Erkenntnissen, dient diese Arbeit zur Verbesserung der katalytischen Aktivität des drei-Zentren-Komplex durch die Variation von verschieden Substituenten. Hierbei wurden zwei wesentliche Konzepte verfolgt:rn* Vernetzung durch die Bildung von Netzwerken oder durch Pyrolyse in der Mesophasern* Verbesserung des Katalysator-Trägermaterial-KontaktsrnNeben den Synthesen wurden die Eigenschaften und möglichen Anwendungen dieser neuartigen Materialen untersucht, wie z.B. als Liganden für Übergangsmetalle, als Anionenrezeptoren oder als Elektrokatalysatoren für die Reduktion von Sauerstoff. rn

Bone-implant interface. Evaluation of osteoblastic cells behavior on nanopatterned titanium surfaces: an in vitro analysis

Protein-adsorption occurs immediately following implantation of biomaterials. It is unknown at which extent protein-adsorption impacts the cellular events at bone-implant interface. To investigate this question, we compared the in-vitro outcome of osteoblastic cells grown onto titanium substrates and glass as control, by modulating the exposure to serum-derived proteins. Substrates consisted of 1) polished titanium disks; 2) polished disks nanotextured with H2SO4/H2O2; 3) glass. In the pre-adsorption phase, substrates were treated for 1h with αMEM alone (M-noFBS) or supplemented with 10%-foetal-bovine-serum (M-FBS). MC3T3-osteoblastic-cells were cultured on the pre-treated substrates for 3h and 24h, in M-noFBS and M-FBS. Subsequently, the culture medium was replaced with M-FBS and cultures maintained for 3 and 7days. Cell-number was evaluated by: Alamar-Blue and MTT assay. Mitotic- and osteogenic-activities were evaluated through fluorescence-optical-microscope by immunolabeling for Ki-67 nuclear-protein and Osteopontin. Cellular morphology was evaluated by SEM-imaging. Data were statistically analyzed using ANOVA-test, (p<0.05). At day3 and day7, the presence or absence of serum-derived proteins during the pre-adsorption phase had not significant effect on cell-number. Only the absence of FBS during 24h of culture significantly affected cell-number (p<0.0001). Titanium surfaces performed better than glass, (p<0.01). The growth rate of cells between day3 and 7 was not affected by the initial absence of FBS. Immunolabeling for Ki-67 and Osteopontin showed that the mitotic- and osteogenic- activity were ongoing at 72h. SEM-analysis revealed that the absence of FBS had no major influence on cell-shape. • Physico-chemical interactions without mediation by proteins are sufficient to sustain the initial phase of culture and guide osteogenic-cells toward differentiation. • The challenge is avoiding adsorption of ‘undesirables’ molecules that negatively impact on the cueing cells receive from surface. This may not be a problem in healthy patients, but may have an important role in medically-compromised-individuals in whom the composition of tissue-fluids is altered.

Speziation von Plutonium bei der Sorption und Diffusion in Opalinuston

Die vorliegende Arbeit wurde im Rahmen des BMWi-Verbundprojektes Wechselwirkung und Transport von Aktiniden im natürlichen Tongestein unter Berücksichtigung von Huminstoffen und Tonorganika – Wechselwirkung von Neptunium und Plutonium mit natürlichem Tongestein“ durchgeführt. Um die langfristige Sicherheit der nuklearen Endlager beurteilen zu können, muss eine mögliche Migration der radiotoxischen Abfälle in die Umwelt betrachtet werden. Wegen seiner langen Halbwertszeit (24000 a) leistet Pu-239 einen wesentlichen Beitrag zur Radiotoxizität abgebrannter Kernbrennstoffe in einem Endlager. Das redox-sensitive Pu tritt in Lösung unter umweltrelevanten Bedingungen in den Oxidationsstufen +III bis +VI auf und kann nebeneinander in bis zu vier Oxidationsstufen vorliegen. Tonsteinformationen werden als mögliches Wirtsgestein für Endlager hoch-radioaktiver Abfälle betrachtet. Deshalb sind ausführliche Informationen zur Mobilisierung und Immobilisierung des Pu durch/in das Grundwasser aus einem Endlager von besonderer Bedeutung. In dieser Arbeit wurden neue Erkenntnisse über die Wechselwirkung zwischen Pu und dem natürlichen Tongestein Opalinuston (OPA, Mont Terri, Schweiz) mit Hinblick auf die Endlagerung wärmeentwickelnder radioaktiver Abfälle in einem geologischen Tiefenlager gewonnen.rnDer Fokus der Arbeit lag dabei auf der Bestimmung der Speziation von Pu an der Mineraloberfläche nach Sorptions- und Diffusionsprozessen mittels verschiedener synchrotronbasierter Methoden (µ-XRF, µ-XANES/EXAFS, µ-XRD, XANES/EXAFS). rnDie Wechselwirkung zwischen Pu und OPA wurde zunächst in Batch- und Diffusionsexperimenten in Abhängigkeit verschiedener experimenteller Parameter (u.a. pH, Pu-Oxidationsstufe) untersucht. In Sorptionsexperimenten konnte gezeigt werden, dass einige Parameter (z.B. Temperatur, Huminsäure) einen deutlichen Einfluss auf die Sorption von Pu haben.rnDie Speziationsuntersuchungen wurden zum einen an Pulverproben aus Batchexperimenten und zum anderen an OPA-Dünnschliffen bzw. Diffusionsproben in Abhängigkeit verschiedener experimenteller Parameter durchgeführt. Die EXAFS-Messungen an der Pu LIII-Kante der Pulverproben ergaben, dass eine innersphäriche Sorption von Pu(IV) an Tongestein unabhängig von dem Ausgangsoxidationszustand des Plutoniums in Lösung stattgefunden hat. Durch die Kombination der ortsaufgelösten Methoden wurde erstmalig mittels μ-XRF die Verteilung von Pu und anderen in OPA enthaltenen Elementen bestimmt. µ-XANES-Spektren an Pu-Anreicherungen auf OPA-Dünnschliffen und in Diffusionsproben bestätigen, dass das weniger mobile Pu(IV) die dominierende Spezies nach den Sorptions- und Diffusionsprozessen ist. Darüber hinaus wurde zum ersten Mal ein Diffusionsprofil von Pu in OPA mittels µ-XRF gemessen. Die Speziationsuntersuchungen mittels μ-XANES zeigten, dass das eingesetzte Pu(V) entlang seines Diffusionspfades zunehmend zu Pu(IV) reduziert wird. Mit µ-XRD wurde Illit als dominierende Umgebung, in der Pu angereichert wurde, identifiziert und Siderit als eine redoxaktive Phase auftreten kann. Die Ergebnisse dieser Arbeit zeigen, dass die Sicherheit von OPA als Wirtsgestein eines Endlagers hoch-radioaktiver Abfälle positiv zu bewerten ist. rn

Complementary mass spectrometric techniques for the characterization of the organic fraction in atmospheric aerosols

Aerosol particles are strongly related to climate, air quality, visibility and human health issues. They contribute the largest uncertainty in the assessment of the Earth´s radiative budget, directly by scattering or absorbing solar radiation or indirectly by nucleating cloud droplets. The influence of aerosol particles on cloud related climatic effects essentially depends upon their number concentration, size and chemical composition. A major part of submicron aerosol consists of secondary organic aerosol (SOA) that is formed in the atmosphere by the oxidation of volatile organic compounds. SOA can comprise a highly diverse spectrum of compounds that undergo continuous chemical transformations in the atmosphere.rnThe aim of this work was to obtain insights into the complexity of ambient SOA by the application of advanced mass spectrometric techniques. Therefore, an atmospheric pressure chemical ionization ion trap mass spectrometer (APCI-IT-MS) was applied in the field, facilitating the measurement of ions of the intact molecular organic species. Furthermore, the high measurement frequency provided insights into SOA composition and chemical transformation processes on a high temporal resolution. Within different comprehensive field campaigns, online measurements of particular biogenic organic acids were achieved by combining an online aerosol concentrator with the APCI-IT-MS. A holistic picture of the ambient organic aerosol was obtained through the co-located application of other complementary MS techniques, such as aerosol mass spectrometry (AMS) or filter sampling for the analysis by liquid chromatography / ultrahigh resolution mass spectrometry (LC/UHRMS).rnIn particular, during a summertime field study at the pristine boreal forest station in Hyytiälä, Finland, the partitioning of organic acids between gas and particle phase was quantified, based on the online APCI-IT-MS and AMS measurements. It was found that low volatile compounds reside to a large extent in the gas phase. This observation can be interpreted as a consequence of large aerosol equilibration timescales, which build up due to the continuous production of low volatile compounds in the gas phase and/or a semi-solid phase state of the ambient aerosol. Furthermore, in-situ structural informations of particular compounds were achieved by using the MS/MS mode of the ion trap. The comparison to MS/MS spectra from laboratory generated SOA of specific monoterpene precursors indicated that laboratory SOA barely depicts the complexity of ambient SOA. Moreover, it was shown that the mass spectra of the laboratory SOA more closely resemble the ambient gas phase composition, indicating that the oxidation state of the ambient organic compounds in the particle phase is underestimated by the comparison to laboratory ozonolysis. These observations suggest that the micro-scale processes, such as the chemistry of aerosol aging or the gas-to-particle partitioning, need to be better understood in order to predict SOA concentrations more reliably.rnDuring a field study at the Mt. Kleiner Feldberg, Germany, a slightly different aerosol concentrator / APCI-IT-MS setup made the online analysis of new particle formation possible. During a particular nucleation event, the online mass spectra indicated that organic compounds of approximately 300 Da are main constituents of the bulk aerosol during ambient new particle formation. Co-located filter analysis by LC/UHRMS analysis supported these findings and furthermore allowed to determine the molecular formulas of the involved organic compounds. The unambiguous identification of several oxidized C 15 compounds indicated that oxidation products of sesquiterpenes can be important compounds for the initial formation and subsequent growth of atmospheric nanoparticles.rnThe LC/UHRMS analysis furthermore revealed that considerable amounts of organosulfates and nitrooxy organosulfates were detected on the filter samples. Indeed, it was found that several nitrooxy organosulfate related APCI-IT-MS mass traces were simultaneously enhanced. Concurrent particle phase ion chromatography and AMS measurements indicated a strong bias between inorganic sulfate and total sulfate concentrations, supporting the assumption that substantial amounts of sulfate was bonded to organic molecules.rnFinally, the comprehensive chemical analysis of the aerosol composition was compared to the hygroscopicity parameter kappa, which was derived from cloud condensation nuclei (CCN) measurements. Simultaneously, organic aerosol aging was observed by the evolution of a ratio between a second and a first generation biogenic oxidation product. It was found that this aging proxy positively correlates with increasing hygroscopicity. Moreover, it was observed that the bonding of sulfate to organic molecules leads to a significant reduction of kappa, compared to an internal mixture of the same mass fractions of purely inorganic sulfate and organic molecules. Concluding, it has been shown within this thesis that the application of modern mass spectrometric techniques allows for detailed insights into chemical and physico-chemical processes of atmospheric aerosols.rn

Pi-konjugierte Plasmapolymere als organische Halbleiter durch vorstrukturierte single-source-Precursoren

Die vorliegende Arbeit behandelt die Entwicklung einer neuartigen Synthesestrategie von π-konjugierten Plasmapolymeren durch die Anwendung von vorstrukturierten aromatischen Precursoren und gepulsten Niederdruckplasmen. Es gelang erstmals die strukturtreue Synthese von π-konjugierten organischen Plasmapolymeren mit dem vollkommenen Erhalt der aromatischen Funktionalität und der selektiven para-Verknüpfung aromatischer Einheiten durch geeignete Heteroatome. Hierbei kamen 1,4-Dithiophenol zur Synthese von Plasmapoly(p-phenylensulfid) und 4-Iodanilin zur Synthese von Plasmapolyanilin zur Anwendung. Die mit hoher Präzision abgeschiedenen Filme konnten sowohl postsynthetisch als auch in situ p-dotiert werden. Die chemischen Strukturen sowie deren physikalisch-chemischen Eigenschaften konnten vor allem mittels Röntgenphotoelektronen-, UV-VIS-NIR-, IR-, NMR-, ESR- und Impedanz-Spektroskopie aufgeklärt werden. Die synthetisierten dotierten Plasmapolymere zeigten eindeutig ohmsche Leistungsmechanismen, teilweise mit einer Leitfähigkeitserhöhung von bis zu 8 Dekaden gegenüber dem undotierten konventionellen Polymer.

Preparation and functional characterisation of recombinant silicatein-alpha from the sponge Suberites domuncula

The marine world is an immense source of biodiversity that provides substances with striking potentials in medicinal chemistry and biotechnology. Sponges (Porifera) are marine animals that represent the most impressive example of organisms possessing the ability to metabolise silica through a family of enzymes known as silicateins. Complex skeletal structures (spicules) made of pure biogenic silica (biosilica) are produced under physiological conditions. Biosilica is a natural material comprising inorganic and organic components with unique mechanical, optical, and physico-chemical properties, including promising potential to be used for development of therapeutic agents in regenerative medicine. Unravelling the intimate physiological mechanisms occurring in sponges during the construction of their siliceous spicules is an on-going project, and several questions have been addressed by the studies proposed by our working group. In this doctoral work, the recombinant DNA technology is exploited for functional and structural characterisation of silicatein. Its precursors are produced as fusion proteins with a chaperone tag (named TF-Ps), and a robust method for the overexpression of native soluble proteins in high concentrations has been developed. In addition, it is observed and proven experimentally that the maturation of silicatein is an autocatalytic event that: (i) can be modulated by rational use of protease inhibitors; (ii) is influenced by the temperature of the environment; (iii) only slightly depends on the pH. In the same experimental framework, observations on the dynamics in the maturation of silicateins allow a better understanding of how the axial filaments form during the early stages of spicule construction. In addition, the definition of new distinct properties of silicatein (termed “structure-guiding” and “structure-forming”) is introduced. By homology models and through comparisons with similar proteins (the cathepsins), domains with significant surface hydrophobicity are identified as potential self-assembly mediators. Moreover, a high-throughput screening showed that TF-Ps could generate crystals under certain conditions, becoming promising for further structural studies. With the goal of optimise the properties of the recombinant silicatein, implementation of new production systems are tried for the first time. Success in the expression of silicatein-type proteins in insect and yeast cells, constitute a promising basis for further development, towards the establishment of an efficient method for the production of a high-value pure and soluble protein.

Synthese und Charakterisierung von Polymer-Wirkstoff-Antikörper-Konjugaten zur Anwendung in der Krebsimmuntherapie

Polymere Wirkstoffsysteme gewinnen im Bereich der biomedizinischen Forschung immer größeres Interesse. Vielversprechende Systeme für die Entwicklung von neuartigen Krebs-immun¬therapien stellen insbesondere Polymer-Konjugate dar. Das ideale Polymer-Konjugat besitzt eine Größe zwischen 10 nm und 100 nm, ist nicht zytotoxisch und zeigt keine Aggregation in humanem Blutserum. In der vorliegenden Arbeit wurde die Synthese und Charakterisierung von Polymer-Wirkstoff-Konjugaten zur Anwendung in der Krebsimmuntherapie behandelt. Erstes Ziel der Arbeit war es, geeignete polymere Trägersysteme für die in vivo Anwendung zu finden. Hierzu wurde zunächst die Stabilität verschiedener potentieller polymerer Träger-systeme (Nanohydrogele, Succinyliertes-Poly-L-Lysin (Bürste), ELP-Bürsten und Poly(2-oxazolin)bürsten) in humanem Serum untersucht. Weiterhin wurde die unspezifische Zellaufnahme in murinen dendritischen Zellen (DCs) analysiert. Es konnte gezeigt werden, dass vor allem neutrale bzw. zwitterionische Partikel eine hohe Serumstabilität sowie keine unspezifische Zellaufnahme zeigen. Um eine gezielte Adressierung der DCs des Immunsystems zu erreichen und dadurch eine Immunantwort gegenüber einem bestimmten Krebs Zelltyp zu induzieren, wurden Biokonjugate - auf Basis der Succinylierten-Poly-L-Lysin-(Bürste) sowie der Azid-funktionalisierten Poly(2-oxazolin)bürste (POx) – entwickelt, da diese Polymerbürsten keine bzw. kaum unspezifische Aufnahme in DCs zeigen. Hierbei diente der Antikörper aDEC205 der gezielten Adressierung von CD8+ DCs. Die weiteren bioaktiven Komponenten waren das tumorassoziierte Antigen (TAA) mit der Kernsequenz SIINFEKL zur Induktion einer spezifischen Immunantwort sowie der immunaktivierende TLR9 Ligand, CpG1826. Die Komponenten wurden nacheinander an die Fluoreszenz-markierten Polymere kon¬jugiert. Die Konjugation des Antikörpers erfolgte nach vorangegangener DIBO-Modifizierung über kupferfreie Click-Chemie. Mit einer optimierten Aufarbeitungsmethodik gelang es, aggregat-freie, unimere DIBO-modifizierte aDEC205 Antikörper zu isolieren. Für die succinylierten Poly-L-Lysine konnten keine eindeutigen sowie reproduzierbaren Ergebnisse erhalten werden, sodass sich im weiteren Verlauf der Arbeit auf die POx konzentriert wurde. Die Konjugation von aDEC205 an POx wurde mittels verschiedener physiko-chemischer Methoden (UV-VIS, SDS-PAGE, FCS, GPC, CLSM und FACS) gezeigt. Mit Hilfe von „Specific-Hybridization-Internalisation-Sensor“ Experimenten konnte eine spezifische Aufnahme des Konjugats in CD8+ DCs nachgewiesen werden. rnDie Konjugation von Antigen und CpG erfolgte ebenso nach entsprechender Modifizierung über kupferfreie Click-Chemie. SDS-PAGE, UV-VIS und FCS bestätigten eine erfolgreiche Kopplung. T-Zell-Proliferationsversuche ergaben für Antigen enthaltende Polymer-Konjugate eine CD8+ T-Zell-Aktivierung. Des Weiteren zeigten die POx keine bemerkenswerte Toxizität und deren Konjugate keine Aggregation in humanem Serum. rnrnDarüber hinaus wurde der Einfluss verschiedener Polymertopologien auf ihre Biodistribution sowie Blutzirkulation untersucht. Für die nach GPC-Fraktionierung erhaltenen verschiedenen Polymerfraktionen - hochmolekulare wurmartige Polymerbürsten, ellipsoidartige Polymer-bürsten und niedermolekulare kugelförmige Moleküle - konnten vielversprechende Ergebnisse erhalten werden.

Visualization and validation of (Q)SAR models

Analyzing and modeling relationships between the structure of chemical compounds, their physico-chemical properties, and biological or toxic effects in chemical datasets is a challenging task for scientific researchers in the field of cheminformatics. Therefore, (Q)SAR model validation is essential to ensure future model predictivity on unseen compounds. Proper validation is also one of the requirements of regulatory authorities in order to approve its use in real-world scenarios as an alternative testing method. However, at the same time, the question of how to validate a (Q)SAR model is still under discussion. In this work, we empirically compare a k-fold cross-validation with external test set validation. The introduced workflow allows to apply the built and validated models to large amounts of unseen data, and to compare the performance of the different validation approaches. Our experimental results indicate that cross-validation produces (Q)SAR models with higher predictivity than external test set validation and reduces the variance of the results. Statistical validation is important to evaluate the performance of (Q)SAR models, but does not support the user in better understanding the properties of the model or the underlying correlations. We present the 3D molecular viewer CheS-Mapper (Chemical Space Mapper) that arranges compounds in 3D space, such that their spatial proximity reflects their similarity. The user can indirectly determine similarity, by selecting which features to employ in the process. The tool can use and calculate different kinds of features, like structural fragments as well as quantitative chemical descriptors. Comprehensive functionalities including clustering, alignment of compounds according to their 3D structure, and feature highlighting aid the chemist to better understand patterns and regularities and relate the observations to established scientific knowledge. Even though visualization tools for analyzing (Q)SAR information in small molecule datasets exist, integrated visualization methods that allows for the investigation of model validation results are still lacking. We propose visual validation, as an approach for the graphical inspection of (Q)SAR model validation results. New functionalities in CheS-Mapper 2.0 facilitate the analysis of (Q)SAR information and allow the visual validation of (Q)SAR models. The tool enables the comparison of model predictions to the actual activity in feature space. Our approach reveals if the endpoint is modeled too specific or too generic and highlights common properties of misclassified compounds. Moreover, the researcher can use CheS-Mapper to inspect how the (Q)SAR model predicts activity cliffs. The CheS-Mapper software is freely available at http://ches-mapper.org.

Kinetic assessment of persistent halogenated xenobiotics in cell culture models: comparison of mono- and poly-halogenated compounds

We evaluated the suitability of single and multiple cell type cultures as model systems to characterise cellular kinetics of highly lipophilic compounds with potential ecotoxicological impact. Confluent mono-layers of human skin fibroblasts, rat astrocytoma C6 cells, non-differentiated and differentiated mouse 3T3 cells were kept in culture medium supplemented with 10% foetal calf serum. For competitive uptake experiments up to four different cell types, grown on glass sectors, were exposed for 3h to (14)C-labelled model compounds, dissolved either in organic solvents or incorporated into unilamellar lecithin liposomes. Bromo-, or chloro-benzenes, decabromodiphenylether (DBP), and dichlorodiphenyl ethylene (DDE) were tested in rather high concentration of 20 microM. Cellular toxicity was low. Compound levels were related to protein, DNA, and triglyceride contents. Cellular uptake was fast and dependent on physico-chemical properties of the compounds (lipophilicity, molecular size), formulation, and cell type. Mono-halogenated benzenes showed low and similar uptake levels (=low accumulation compounds). DBP and DDE showed much higher cellular accumulations (=high accumulation compounds) except for DBP in 3T3 cells. Uptake from liposomal formulations was mostly higher than if compounds were dissolved in organic solvents. The extent of uptake correlated with the cellular content of triglycerides, except for DBP. Uptake competition between different cell types was studied in a sectorial multi-cell culture model. For low accumulation compounds negligible differences were found among C6 cells and fibroblasts. Uptake of DDE was slightly and that of DBP highly increased in fibroblasts. Well-defined cell culture systems, especially the sectorial model, are appropriate to screen for bioaccumulation and cytotoxicity of (unknown) chemical entities in vitro.