On the degradation mechanisms under the influence of pedological factors through the study of archeological bronze patina

This experimental thesis concerns the study of the long-term behaviour of ancient bronzes recently excavated from burial conditions. The scientific interest is to clarify the effect of soil parameters on the degradation mechanisms of ancient bronze alloy. The work took into consideration bronzes recovered from the archaeological sites in the region of Dobrudja, Romania. The first part of research work was dedicated to the characterization of bronze artefacts using non destructive (micro-FTIR, reflectance mode) and micro-destructive (based on sampling and analysis of a stratigraphical section by OM and SEM-EDX) methods. Burial soils were geologically classified and analyzed by chemical methods (pH, conductivity, anions content). Most of objects analyzed showed a coarse and inhomogeneous corroded structure, often made up of several corrosion layers. This has been explained by the silt nature of soils, which contain low amount of clay and are, therefore, quite accessible to water and air. The main cause of a high dissolution rate of bronze alloys is the alternate water saturation and instauration of the soil, for example on a seasonal scale. Moreover, due to the vicinity of the Black Sea, the detrimental effect of chlorine has been evidenced for few objects, which were affected by the bronze disease. A general classification of corrosion layers was achieved by comparing values of the ratio Cu/Sn in the alloy and in the patina. Decuprification is a general trend, and enrichment of copper within the corrosion layers, due to the formation of thick layers of cuprite (Cu2O), is pointed out as well. Uncommon corrosion products and degradation patterns were presented as well, and they are probably due to peculiar local conditions taking place during the burial time, such as anaerobic conditions or fluctuating environmental conditions. In order to acquire a better insight into the corrosion mechanisms, the second part of the thesis has regarded simulation experiments, which were conducted on commercial Cu-Sn alloys, whose composition resembles those of ancient artefacts one. Electrochemical measurements were conducted in natural electrolytes, such as solutions extracted from natural soil (sampled at the archaeological sites) and seawater. Cyclic potentiodynamic experiments allowed appreciating the mechanism of corrosion in both cases. Soil extract’s electrolyte has been evaluated being a non aggressive medium, while artificial solution prepared by increasing the concentration of anions caused the pitting corrosion of the alloy, which is demonstrated by optical observations. In particular, electrochemical impedance spectroscopy allows assessing qualitatively the nature of corroded structures formed in soil and seawater. A double-structured layer is proposed, which differ, in the two cases, for the nature of the internal passive layer, which result defectiveness and porous in case of seawater.

Archaeological Baltic amber: degradation mechanisms and conservation measures

The aim of this project was to achieve a deep understanding of the mechanisms by which Baltic amber degrades, in order to develop techniques for preventive conservation of archaeological amber objects belonging to the National Museum of Denmark’s collections. To examine deterioration of Baltic amber, a starting point was to identify and monitor surface and bulk properties which are affected during degradation. The way to operate consisted of the use of accelerated ageing to initiate degradation of raw Baltic amber samples in different conditions of relative humidity, oxygen exposure or pH and, successively, of the use of non/micro-destructive techniques to identify and quantify changes in visual, chemical and structural properties. A large piece of raw Baltic amber was used to prepare several test samples for two different kinds of accelerated ageing: thermal-ageing and photo-ageing. During the ageing, amber samples were regularly examined through several analytical techniques related to different information: appearance/colour change by visual examination, photography and colorimetry; chemical change by infrared spectroscopy, Raman spectroscopy and elemental analysis; rate of oxidation by oxygen measurement; qualitative analysis of released volatiles by gas chromatography – mass spectrometry. The obtained results were analysed through both critical evaluation and statistical study. After the interpretation of the achieved data, the main relations between amber and environmental factors during the degradation process became clearer and it was possible to identify the major pathways by which amber degrades, such as hydrolysis of esters into alcohols and carboxylic acids, thermal-oxidation and photo-oxidation of terpenoid components, depolymerisation and decomposition of the chemical structure. At the end it was possible to suggest a preventive conservation strategy based on the control of climatic, atmospheric and lighting parameters in the environment where Baltic amber objects are stored and displayed.

Surfactants and their aerobic degradation products

Die Ziele der vorliegenden Arbeit waren 1) die Entwicklung und Validierung von sensitiven und substanz-spezifischen Methoden für die quantitative Bestimmung von anionischen, nichtionischen und amphoteren Tensiden und deren Metaboliten in wässrigen Umweltproben unter Einsatz leistungsfähiger, massenspektrometrischer Analysengeräte,2) die Gewinnung von aeroben, polaren Abbauprodukten aus Tensiden in einem die realen Umweltbedingungen simulierenden Labor-Festbettbioreaktor (FBBR), dessen Biozönose oberflächenwasserbürtig war,3) zur Aufklärung des Abbaumechanismus von Tensiden neue, in 2) gewonnene Metabolite zu identifizieren und massenspektrometrisch zu charakterisieren ebenso wie den Primärabbau und den weiteren Abbau zu verfolgen,4) durch quantitative Untersuchungen von Tensiden und deren Abbauprodukten in Abwasser und Oberflächenwasser Informationen zu ihrem Eintrag und Verhalten bei unterschiedlichen hydrologischen und klimatischen Bedingungen zu erhalten,5) das Verhalten von persistenten Tensidmetaboliten in Wasserwerken, die belastetes Oberflächenwasser aufbereiten, zu untersuchen und deren Vorkommen im Trinkwasser zu bestimmen,6) mögliche Schadwirkungen von neu entdeckten Metabolite mittels ökotoxikologischer Biotests abzuschätzen,7) durch Vergleich der Felddaten mit den Ergebnissen der Laborversuche die Umweltrelevanz der Abbaustudien zu belegen. Die Auswahl der untersuchten Verbindungen erfolgte unter Berücksichtigung ihres Produktionsvolumens und der Neuheit auf dem Tensidmarkt. Sie umfasste die Waschmittelinhaltsstoffe lineare Alkylbenzol-sulfonate (LAS), welches das Tensid mit der höchsten Produktionsmenge darstellte, die beiden nichtionischen Tenside Alkylglucamide (AG) und Alkylpolyglucoside (APG), ebenso wie das amphotere Tensid Cocamidopropylbetain (CAPB). Außerdem wurde der polymere Farbübertragungsinhibitor Polyvinylpyrrolidon (PVP) untersucht.

Control of protein degradation pathways by BAG proteins and changes during aging

Many age-related neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis and polyglutamine disorders, including Huntington’s disease, are associated with the aberrant formation of protein aggregates. These protein aggregates and/or their precursors are believed to be causally linked to the pathogenesis of such protein conformation disorders, also referred to as proteinopathies. The accumulation of protein aggregates, frequently under conditions of an age-related increase in oxidative stress, implies the failure of protein quality control and the resulting proteome instability as an upstream event of proteinopathies. As aging is a main risk factor of many proteinopathies, potential alterations of protein quality control pathways that accompany the biological aging process could be a crucial factor for the onset of these disorders.rnrnThe focus of this dissertation lies on age-related alterations of protein quality control mechanisms that are regulated by the co-chaperones of the BAG (Bcl-2-associated athanogene) family. BAG proteins are thought to promote nucleotide exchange on Hsc/Hsp70 and to couple the release of chaperone-bound substrates to distinct down-stream cellular processes. The present study demonstrates that BAG1 and BAG3 are reciprocally regulated during aging leading to an increased BAG3 to BAG1 ratio in cellular models of replicative senescence as well as in neurons of the aging rodent brain. Furthermore, BAG1 and BAG3 were identified as key regulators of protein degradation pathways. BAG1 was found to be essential for effective degradation of polyubiquitinated proteins by the ubiquitin/proteasome system, possibly by promoting Hsc/Hsp70 substrate transfer to the 26S proteasome. In contrast, BAG3 was identified to stimulate the turnover of polyubiquitinated proteins by macroautophagy, a catabolic process mediated by lysosomal hydrolases. BAG3-regulated protein degradation was found to depend on the function of the ubiquitin-receptor protein SQSTM1 which is known to sequester polyubiquitinated proteins for macroautophagic degradation. It could be further demonstrated that SQSTM1 expression is tightly coupled to BAG3 expression and that BAG3 can physically interact with SQSTM1. Moreover, immunofluorescence-based microscopic analyses revealed that BAG3 co-localizes with SQSTM1 in protein sequestration structures suggesting a direct role of BAG3 in substrate delivery to SQSTM1 for macroautophagic degradation. Consistent with these findings, the age-related switch from BAG1 to BAG3 was found to determine that aged cells use the macroautophagic system more intensely for the turnover of polyubiquitinated proteins, in particular of insoluble, aggregated quality control substrates. Finally, in vivo expression analysis of macroautophagy markers in young and old mice as well as analysis of the lysosomal enzymatic activity strongly indicated that the macroautophagy pathway is also recruited in the nervous system during the organismal aging process.rnrnTogether these findings suggest that protein turnover by macroautophagy is gaining importance during the aging process as insoluble quality control substrates are increasingly produced that cannot be degraded by the proteasomal system. For this reason, a switch from the proteasome regulator BAG1 to the macroautophagy stimulator BAG3 occurs during cell aging. Hence, it can be concluded that the BAG3-mediated recruitment of the macroauto-phagy pathway is an important adaptation of the protein quality control system to maintain protein homeostasis in the presence of an enhanced pro-oxidant and aggregation-prone milieu characteristic of aging. Future studies will explore whether an impairment of this adaptation process may contribute to age-related proteinopathies.

Physical land degradation and loss of soil fertility: soil structural stability and bio-physical indicators

This study investigates the changes in soil fertility due to the different aggregate breakdown mechanisms and it analyses their relationships in different soil-plant systems, using physical aggregates behavior and organic matter (OM) changes as indicators. Three case studies were investigated: i) an organic agricultural soil, where a combined method, aimed to couple aggregate stability to nutrients loss, were tested; ii) a soil biosequence, where OM chemical characterisation and fractionation of aggregates on the basis of their physical behaviour were coupled and iii) a soils sequence in different phytoclimatic conditions, where isotopic C signature of separated aggregates was analysed. In agricultural soils the proposed combined method allows to identify that the severity of aggregate breakdown affected the quantity of nutrients lost more than nutrients availability, and that P, K and Mg were the most susceptible elements to water abrasion, while C and N were mainly susceptible to wetting. In the studied Chestnut-Douglas fir biosequence, OM chemical properties affected the relative importance of OM direct and indirect mechanisms (i.e., organic and organic-metallic cements, respectively) involved in aggregate stability and nutrient losses: under Douglas fir, high presence of carboxylate groups enhanced OM-metal interactions and stabilised aggregates; whereas under Chestnut, OM directly acted and fresh, more C-rich OM was preserved. OM direct mechanism seemed to be more efficient in C preservation in aggregates. The 13C natural abundance approach showed that, according to phytoclimatic conditions, stable macroaggregates can form both around partially decomposed OM and by organic-mineral interactions. In topsoils, aggregate resistance enhanced 13C-rich OM preservation, but in subsoils C preservation was due to other mechanisms, likely OM-mineral interactions. The proposed combined approach seems to be useful in the understanding of C and nutrients fate relates to water stresses, and in future research it could provide new insights into the complexity of soil biophysical processes.

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.

Studies of degradation of organic solar cell materials using electrical scanning probe microscopy

Intense research is being done in the field of organic photovoltaics in order to synthesize low band-gap organic molecules. These molecules are electron donors which feature in combination with acceptor molecules, typically fullerene derivarntives, forming an active blend. This active blend has phase separated bicontinuous morphology on a nanometer scale. The highest recorded power conversionrnefficiencies for such cells have been 10.6%. Organic semiconductors differ from inorganic ones due to the presence of tightly bonded excitons (electron-hole pairs)resulting from their low dielectric constant (εr ≈2-4). An additional driving force is required to separate such Frenkel excitons since their binding energy (0.3-1 eV) is too large to be dissociated by an electric field alone. This additional driving force arises from the energy difference between the lowest unoccupied molecular orbital (LUMO) of the donor and the acceptor materials. Moreover, the efficiency of the cells also depends on the difference between the highest occupied molecular orbital (HOMO) of the donor and LUMO of the acceptor. Therefore, a precise control and estimation of these energy levels are required. Furthermore any external influences that change the energy levels will cause a degradation of the power conversion efficiency of organic solar cell materials. In particular, the role of photo-induced degradation on the morphology and electrical performance is a major contribution to degradation and needs to be understood on a nanometer scale. Scanning Probe Microscopy (SPM) offers the resolution to image the nanometer scale bicontinuous morphology. In addition SPM can be operated to measure the local contact potential difference (CPD) of materials from which energy levels in the materials can be derived. Thus SPM is an unique method for the characterization of surface morphology, potential changes and conductivity changes under operating conditions. In the present work, I describe investigations of organic photovoltaic materials upon photo-oxidation which is one of the major causes of degradation of these solar cell materials. SPM, Nuclear Magnetic Resonance (NMR) and UV-Vis spectroscopy studies allowed me to identify the chemical reactions occurring inside the active layer upon photo-oxidation. From the measured data, it was possible to deduce the energy levels and explain the various shifts which gave a better understanding of the physics of the device. In addition, I was able to quantify the degradation by correlating the local changes in the CPD and conductivity to the device characteristics, i.e., open circuit voltage and short circuit current. Furthermore, time-resolved electrostatic force microscopy (tr-EFM) allowed us to probe dynamic processes like the charging rate of the individual donor and acceptor domains within the active blend. Upon photo-oxidation, it was observed, that the acceptor molecules got oxidized first preventing the donor polymer from degrading. Work functions of electrodes can be tailored by modifying the interface with monomolecular thin layers of molecules which are made by a chemical reaction in liquids. These modifications in the work function are particularly attractive for opto-electronic devices whose performance depends on the band alignment between the electrodes and the active material. In order to measure the shift in work function on a nanometer scale, I used KPFM in situ, which means in liquids, to follow changes in the work function of Au upon hexadecanethiol adsorption from decane. All the above investigations give us a better understanding of the photo-degradation processes of the active material at the nanoscale. Also, a method to compare various new materials used for organic solar cells for stability is proposed which eliminates the requirement to make fully functional devices saving time and additional engineering efforts.

Study of the genes involved in Naphthenic acids (NAs) degradation by Rhodococcus spp.

Naphthenic acids (NAs) are an important group of organic pollutants mainly found in hydrocarbon deposits. Although these compounds are toxic, recalcitrant, and persistent in the environment, we are just learning the diversity of microbial communities involved in NAs- degradation and the mechanisms by which NAs are biodegraded. Studies have shown that naphthenic acids are susceptible to biodegradation, which decreases their concentration and reduces toxicity. Nevertheless, little is still known about their biodegradability. The present PhD Thesis’s work is aimed to study the biodegradation of simple model NAs using bacteria strains belonging to the Rhodococcus genus. In particular, Rh. sp. BCP1 and Rh. opacus R7 were able to utilize NAs such as cyclohexane carboxylic acid and cyclopentane carboxylic acid as the sole carbon and energy sources, even at concentrations up to 1000 mg/L. The presence of either substituents or longer carboxylic acid chains attached to the cyclohexane ring negatively affected the growth by pure bacterial cultures. Moreover, BCP1 and R7 cells incubated in the presence of CHCA or CPCA show a general increase of saturated and methyl-substituted fatty acids in their membrane, while the cis-mono-unsaturated ones decrease, as compared to glucose-grown cells. The observed lipid molecules modification during the growth in the presence of NAs is suggested as a possible mechanism to decrease the fluidity of the cell membrane to counteract NAs toxicity. In order to further evaluate this toxic effect on cell features, the morphological changes of BCP1 and R7 cells were also assessed through Transmission Electron Microscopy (TEM), revealing interesting ultrastructural changes. The induction of putative genes, and the construction of a random transposon mutagenesis library were also carried out to reveal the mechanisms by which these Rhodococcus strains can degrade toxic compounds such as NAs.

3D-ultrastructure, functions and stress responses of rhogocytes from the freshwater gastropods Biomphalaria glabrata and Lymnaea stagnalis

Rhogocytes, also termed ‘pore cells’, exist free in the hemolymph or embedded in the connective tissue of different body parts of molluscs, notably gastropods. These unique cells can be round, elongated or irregularly shaped, and up to 30 μm in diameter. Their hallmark is the so-called slit apparatus: i.e. pocket-like invaginations of the plasma membrane creating extracellular lacunae, bridged by cytoplasmic bars. These bars form distinctive slits of ca. 20 nm width. A slit diaphragm composed of proteins establishes a molecular sieve with holes of 20 x 20 nm. Different functions have been assigned to this special molluscan cell type, notably biosynthesis of the hemolymph respiratory protein hemocyanin. It has further been proposed, but not proven, that in the case of red-blooded snail species rhogocytes might synthesize the hemoglobin. However, the secretion pathway of these hemolymph proteins, and the functional role of the enigmatic slit apparatus remained unclear. Additionally proposed functions of rhogocytes, such as heavy metal detoxification or hemolymph protein degradation, are also not well studied. This work provides more detailed electron microscopical, histological and immunobiochemical information on the structure and function of rhogocytes of the freshwater snails Biomphalaria glabrata and Lymnaea stagnalis. By in situ hybridization on mantle tissues, it proves that B. glabrata rhogocytes synthesize hemoglobin and L. stagnalis rhogocytes synthesize hemocyanin. Hemocyanin is present, in endoplasmic reticulum lacunae and in vesicles, as individual molecules or pseudo-crystalline arrays. The first 3D reconstructions of rhogocytes are provided by means of electron tomography and show unprecedented details of the slit apparatus. A highly dense material in the cytoplasmic bars close to the diaphragmatic slits was shown, by immunogold labeling, to contain actin. By immunofluorescence microscopy, the protein nephrin was localized at the periphery of rhogocytes. The presence of both proteins in the slit apparatus supports the previous hypothesis, hitherto solely based on similarities of the ultrastructure, that the molluscan rhogocytes are phylogenetically related to mammalian podocytes and insect nephrocytes. A possible secretion pathway of respiratory proteins that includes a transfer mechanism of vesicles through the diaphragmatic slits is proposed and discussed. We also studied, by electron microscopy, the reaction of rhogocytes in situ to two forms of animal stress: deprivation of food and cadmium contamination of the tank water. Significant cellular reactions to both stressors were observed and documented. Notably, the slit apparatus surface and the number of electron-dense cytoplasmic vesicles increased in response to cadmium stress. Food deprivation led to an increase in hemocyanin production. These observations are also discussed in the framework of using such animals as potential environmental biomarkers.

Effect of systemic tetracycline on the degradation of tetracycline-impregnated bilayered collagen membranes: an animal study

BACKGROUND: Premature collagen membrane degradation may compromise the outcome of osseous regenerative procedures. Tetracyclines (TTCs) inhibit the catalytic activities of human metalloproteinases. Preprocedural immersion of collagen membranes in TTC and systemic administration of TTC may be possible alternatives to reduce the biodegradation of native collagen membranes. AIM: To evaluate the in vivo degradation of collagen membranes treated by combined TTC immersion and systemic administration. MATERIALS AND METHODS: Seventy-eight bilayered porcine collagen membrane disks were divided into three groups and were immersed in 0, 50, or 100 mg/mL TTC solution. Three disks, one of each of the three groups, were implanted on the calvaria of each of 26 Wistar rats. Thirteen (study group) were administered with systemic TTC (10 mg/kg), while the remaining 13 received saline injections (control group). Calvarial tissues were retrieved after 3 weeks, and histological sections were analyzed by image analysis software. RESULTS: Percentage of remaining collagen area within nonimpregnated membranes was 52.26 ± 20.67% in the study group, and 32.74 ± 13.81% in the control group. Immersion of membranes in 100 mg/mL TTC increased the amount of residual collagen to 63.46 ± 18.19% and 42.82 ± 12.99% (study and control groups, respectively). Immersion in 50 mg/mL TTC yielded maximal residual collagen values: 80.75 ± 14.86% and 59.15 ± 8.01% (study and control groups, respectively). Differences between the TTC concentrations, and between the control and the study groups were statistically significant. CONCLUSIONS: Immersion of collagen membranes in TTC solution prior to their implantation and systemic administration of TTC significantly decreased the membranes' degradation.

A novel mode of action of the putative sphingosine kinase inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (SKI II): induction of lysosomal sphingosine kinase 1 degradation

Sphingosine kinase 1 (SK1) is a key enzyme in the generation of sphingosine 1-phosphate (S1P) which critically regulates a variety of important cell responses such as proliferation and migration. Therefore, inhibition of SK-1 has been suggested to be an attractive approach to treat tumor growth and metastasis formation.

Lysosomal degradation of RhoH protein upon antigen receptor activation in T but not B cells

RhoH is a member of the Rho (ras homologous) GTPase family, yet it lacks GTPase activity and thus remains in its active conformation. Unlike other Rho GTPases, the RhoH gene transcript is restricted to hematopoietic cells and RhoH was shown to be required for adequate T-cell activation through the TCR. Here, we demonstrate that both blood T and B cells, but not neutrophils or monocytes, express RhoH protein under physiological conditions. Upon TCR complex activation, RhoH was degraded in lysosomes of primary and Jurkat T cells. Pharmacologic activation of T cells distal to the TCR complex had no effect on RhoH protein levels suggesting that early events during T-cell activation are required for RhoH protein degradation. In contrast to T cells, activation of the BCR in blood B cells was not associated with changes in RhoH levels. These data suggest that RhoH function might be regulated by lysosomal degradation of RhoH protein following TCR complex but not BCR activation. This newly discovered regulatory pathway of RhoH expression might limit TCR signaling and subsequent T-cell activation upon Ag contact.