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Monitoring Oxidation Reactions by Photochemical Techniques
No full textThis doctoral thesis is devoted to monitor oxidation reactions processes by photochemical techniques. Oxidation reactions play a crucial role in both environmental and biological systems. Therefore, my thesis started with the study of aryl–substituted benzyl radicals. In the second part of my thesis, the effect of the solvent on H atom abstractions from C–H donors by oxygen–centered radicals.
In the last part, I developed a fluorescence–based supramolecular tandem assay to monitor a biologically important oxidation process, namely an enzymatic oxidation. When searching for a macrocyclic receptor (required for tandem assays) for the substrate of peroxidases, namely iodide, I found a novel interaction (halogen bonding) in supramolecular host-guest complexes associated with cucurbiturils, which is based on the encapsulation of molecular iodine inside the nanocavity of CB6
Antioxidant Activity Measured by Fluorescence Investigation of Antioxidant and Probe Structure as well as their Mobility and Position
No full textThis doctoral thesis is concerned with determining antioxidant mobility by fluorescence, especially in lipid membranes. It expands on previously determined data regarding antioxidant diffusion by employing different lipids, synthetic antioxidants, and varying pH. The probes used are derivatives of 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO), which are known for their exceptionally long lifetimes in aerated aqueous media (325 ns in H2O under air). Their n,π* singlet-excited state is efficiently quenched by an aborted hydrogen abstraction mechanism, which makes them ideally suited to probe antioxidant diffusion.
During the course of my Ph. D. thesis I was able to expand the toolbox of available DBO derivatives with a very reactive one, Fluorazophore-R, and a more acid-stable lipophilic derivative, Fluorazophore-LE.
I was also able to determine the mutual lateral diffusion coefficient of a synthetic vitamin E derivative with single-photon counting measurements. This N-tocopherol displays higher mutual lateral diffusion coefficients than α-tocopherol (5.4 × 10–7 cm–1 s–1 vs. 3.73 × 10–7 cm–1 s–1 at 40°C, respectively) with a very similar activation energy of 43 ± 6 kJ/mol (compared to 47±5 kJ/mol, which is indicative of a higher antioxidant potency.
Expanding on previous experiments I was also able to probe the differing behavior of α-tocopherol at the phase transition threshold of liposomes composed of a high melting fully saturated lipid, DPPC. Above the phase transition temperature, mutual lateral diffusion coefficients of 10–7 cm2 s–1 were found, similar to the ones in POPC, while above the pre-transition temperature the coefficients were 10–8 cm2 s–1. This confirms the high accuracy of our measurement principle.
Furthermore, the first steps have been taken in investigating the pH dependence of the synthetic antioxidant L-ascorbyl-6-palmitate, which is only a decent lipophilic antioxidant in low pH environments.
Additionally, I have worked on a third party funded research project for a company analyzing urine and blood samples, the results of which I am not at liberty to discuss
Synthesis and Analysis of the Dietary Relevant Isomers of Chlorogenic Acids, Their Derivatives and Hydroxycinnamates
No full textAbstract Chlorogenic acids (CGAs) are phenolic natural products, widely present in food materials and plants. Coffee is one of the richest sources of CGAs and one of the most consumed beverages in the world. The LC-MSn methods have been used to detect and characterize CGAs in green Robusta and Arabica coffee beans. During this study, 40 samples of green Robusta and Arabica coffee beans discriminated by their origin and type were investigated for their chlorogenic acid contents. Twenty-one novel CGAs and eight novel classes of CGAs were reported in green coffee beans. An LC-MSn hierarchical scheme was developed for the identification, structure elucidation, and assignment of regiochemistry of triacyl CGAs. The high resolution LC-MS-TOF data of chlorogenic acids was used to discriminate between the Arabica and Robusta coffee beans by the principal component analysis (PCA) approach. In another study on roasted coffee 20 samples of Robusta and Arabica coffee were analyzed by LC-MSn for their content of CGAs, lactones, and shikimates. Fifteen novel CGAs completely absent in green coffee samples were identified in roasted coffee samples. The CGAs of maté were investigated by LC-MSn. Forty-two CGAs were detected and characterized, twenty-four of them for the first time from this source. This was the first time when an LC-MSn method was used for the identification and structure assignment of shikimates. By these LC-MSn methods it became easy to discriminate between the shikimates and CGA lactones. Methyl quinates of CGAs are present in many food materials (including maté) and they are the most common artifacts during the methanolic extraction of chlorogenic acids. In this study an LC-MSn method was developed for their identification and characterization. This is the first time when an LC-MSn method was developed for the identification of hydroxycinnamates. Arnica (Arnica montana) flowers and Burdock (Arctium lappa) roots from the Asteraceae family and Gardeniae Fructus (Gardeniae jasminoids fruit) from the Rubiaceae family were analyzed for their chlorogenic acid contents and chlorogenic acids containing alkoyl residues such as fumaroyl, maloyl, methoxyoxaloyl, hydroxyl-methylglutaroyl, and succinoyl were identified. These chlorogenic acids show different tandem mass spectral behavior compared to previous studies. An LC-MSn method was also developed to discriminate between the feruloylquinic acids and isoferuloylquinic acids. Both classes of polyphenols are possibly present in food materials (nature) and as metabolites. A new protecting group strategy was developed by using allyl ether for the selective synthesis of CGAs and epimers of CGAs. A number of chlorogenic acids and epimers of chlorogenic acids containing muco-quinic acid moiety were synthesized by this approach. This was the first time when caffeoyl- and feruloyl-muco-quinic acids were synthesized efficiently in excellent yield. The epimers of CGAs showed promising enzyme inhibition activity against DnMT3a enzyme
Asymmetric Michael Addition of Carbonyl Compounds to Nitro-olefins Catalyzed by Simple Organocatalysts
No full textAsymmetric Michael addition of carbonyl compounds to nitro-olefins is one of the important carbon carbon bond forming reactions and especially so when quaternary stereogenic carbons are formed. Over the past decade a large number of organocatalysts have been developed for the asymmetric Michael addition of carbonyl compounds to nitro-olefins. Impressive results have been obtained but many parameters require improvement. For example, stereo inductions, catalyst loading, stoichiometry of starting materials, reaction time, limited substrate scope, etc. I focused on improving these parameters by designing new organocatalysts that incorporated old concepts from outside the field and by introducing new templates based on known concepts from within the field. This manifested itself in a two-prong approach: 1) tertiary-primary diamines and 2) assembled catalysts. The assembled catalysts were found to be superior for the intended goal
Rapid and Direct Food Investigation Using Raman Spectroscopy
No full textRaman spectroscopy, a vibrational technique based on an inelastic scattering of incident light, provides content-relevant information about molecular vibrations with well defined and resolved spectral features in various sample categories including liquids, solids, and gases. Moreover; the major advantages of Raman spectroscopy are that it requires no sample preparation; it is a rapid and nondestructive tool. All these advantages make this technique ideally suited for online industrial processing, since the direct contact with the sample is not necessarily required to obtain spectra. Although Raman spectroscopy has become a widely used tool for analytical purposes in scientific research, it is still considered a new emerging technique in the field of food analysis and few researches have implemented this spectroscopic method in applications concerned with food chemistry. As a matter of fact, extensive research is still to be achieved on the authentication of the potential of Raman spectroscopy technique in the field of food investigation. In this thesis the Raman spectroscopy technique demonstrates its potential in the discrimination between different edible oils, quantitative detection of the adulteration of extra virgin olive oil (EVOO) and determination of free fatty acid in EVOO. In addition, Raman spectroscopy has been applied to evaluate the effect of microwave and conventional heating on EVOO antioxidant. Moreover, this technique has been successfully applied for the discrimination between the two main green coffee species (Arabica and Robusta). On the other hand, the fat milk content and its unsaturation level have been quantitatively determined using Raman spectroscopy as a fast and nondestructive technique
Mind and Virtue A Cross-Cultural Analysis of Beliefs about Learning
No full textInternational mobility has become a central dimension of academic life. The numbers of students from diverse cultural backgrounds entering European institutions of higher edcuation are increasing annually. The current project is aimed at understanding the concepts of learning that both students and members of faculty from different cultural backgrounds endorse. The theory that forms the main theoretical base of this project is the mind - virtue orientation theory by Li (2003; 2005). Li found that Western students have a "mind oriented" approach to learning and Asian students have a "virtue oriented" approach. At the core of the mind orientation is the belief that learning is a cognitive endeavor. Learning is primarily a process of developing ones thinking skills. In the virtue orientation, learning is perceived as the development of the person as a whole, including moral and social domains. The dissertation presents four studies that were conducted on the basis of this framework. First, an extensive theoretical analysis was conducted to assess whether previously segregated findings in the literature could be integrated into the framework of the mind - virtue orientation. The following two chapters present the findings of survey studies that assessed the beliefs about learning of German and Chinese students and faculty, but also of students from two Eastern European countries; Poland and Romania. Findings indicate a cross-cultural difference between the Western and Asian context for both students and members of faculty, but not between the Western and Eastern European contexts. Lastly, an experimental study is reported that measured the mind and virtue orientation implicitly in German and Chinese students. In conclusion, the conducted studies provide evidence for the hypothesis that Western and East-Asian academics place a relatively different value on certain beliefs about learning over others. These beliefs can be meaningfully integrated into the framework of the mind and virtue orientation
Quaternary structure and regulation of mammalian DNA methyltransferase 3a (Dnmt3a)
No full textThe Dnmt3 family includes two catalytically active de novo methyltransferases (Dnmt3a and Dnmt3b) and one catalytically inactive regulatory factor called Dnmt3L. The N-terminal part of Dnmt3a consists of an ADD and a PWWP domain, we have shown that the ADD domain interacts with H3K4 unmethylated tails and the PWWP domain interacts with H3K36me3 marks. These interactions are responsible for the regulation of Dnmt3a's activity as well as for targeting to heterochromatin. We have identified that Dnmt3a and Dnmt3L form a heterotetramer that methylates two CpG sites on opposite DNA strands in a distance of 8-10 bps. We have shown that Dnmt3a forms a linear multimer and it binds to many DNA molecules oriented in parallel. This process is required for the heterochromatic localization of Dnmt3a in cells. The regulator protein Dnmt3L reorganizes the quaternary structures of Dnmt3a, thereby prevents multimerization. In addition to formation of protein multimers, Dnmt3a complexes bind cooperatively to DNA forming protein-DNA filaments. This might be required for generation of 8-10 bp periodicity patterns at imprinted genes and tight packing of Dnmt3a at the heterochromatic region. In addition, we have identified that the enzymatic activity Dnmt3a is regulated by phosphorylation. Casein kinase 2 mediated phosphorylation of the Dnmt3a negatively regulates its enzymatic activity in cells. Adding to existing evidence, our data uncovered novel mechanism of regulation of Dnmt3a activity, which are required for the proper DNA methylation patterns in cells. Also, using a newly developed high throughput assay, we have identified novel inhibitors to Dnmt3a from chemical libraries and identified new compounds purified from the dietary black tea and coffee polyphenols, which inhibit the Dnmt3a activity moderately
Data integration in microbial genomics Contextualizing sequence data in aid of biological knowledge
No full textDeoxyribonucleic acid (DNA) is the primary structure that carries the genetic information of organisms in genomes. The introduction of the first DNA sequencing methods in 1977 marked a major breakthrough in life sciences. Today, these methods are widely applied and grant insight into the 'blueprints' of organisms from all domains of life. The analysis of environmental microbial sequence data is becoming increasingly important in times of global climate change, because microbes are central catalysts in nutrient cycles such as the carbon cycle that profoundly a ffects Earth's climate. Microbes perform almost all metabolic processes that are thermodynamically possible. DNA sequencing is carried out around the globe and the resulting data is submitted to the public repositories of the International Nucleotide Sequence Database Collaboration (INSDC). Data in the INSDC is accumulating exponentially. This trend shows the need for efficient data processing strategies in order to gain knowledge out of this ever increasing amount of sequence data. For this, it is important to annotate sequence data with as much contextual data as possible. Contextual data are data about the environmental context and the processing steps that were applied. These can range from data about the geographic location, sampling time, habitat, or about experimental procedures used to obtain the sequences up to video data recorded during sampling. Especially data about the geographic location (x, y, z) and the point in time (t), when samples are taken from the environment are essential. Comparability and interpretability are preserved. Ample analysis approaches become possible, when contextual and sequence data are integrated. In this doctoral thesis, data integration is promoted in three ways: Firstly, through the development of contextual data capture, submission and integration tools. Secondly, through the development of standards for contextual data and thirdly, through demonstration of in silico hypothesis generation for a large metagenomic data set
Cloning, characterization and directed evolution of pyranose oxidase from Trametes versicolor for biofuel cell applications
No full textBiofuel cells convert chemical energy to electric energy using an enzyme or a whole cell. Glucose oxidase (GOx) has become the prime enzyme used in the anodic compartment of biofuel cells. Miniaturized implanted biofuel cells have been reported losing 6 % of its power per day due to blood conditions which are suboptimal for GOxs. Prolonging the life span for such a battery is a main goal in developing these devices. This could be addressed by tailoring GOx or, alternatively, by using pyranose oxidase (POx): POxs show a broader substrate profile, a higher affinity towards glucose and no anomeric preference. POxs optimum pH is around 7 and at values superior to 7, kinetics is barely affected by buffer composition. However, the catalytic efficiency needs to be improved. Moreover, POxs are specially challenging since in contrast to GOxs there is little knowledge about POx structure, their structure-function relationships, electrochemical properties, and enzymatic stabilities. In this work, POx from Trametes versicolor was cloned from a cDNA library and was expressed in Escherichia coli as an intracellular active recombinant enzyme. Directed evolution via error-prone PCR random mutagenesis was used to generate mutant libraries, which were tested for improved catalytic efficiency via a high-throughput ABTS assay. Further cloning in a mutator E. coli strain led to a C-terminally truncated POx variant (trPOx), that exhibited a ̃14-fold increased kcat and a ̃78-fold improved catalytic efficiency. trPOx is the smallest active POx yet described. Results of this study proved the directed evolution strategy to be successful at optimizing catalytic activities of POxs. Simultaneously, a novel HTS assay based in the reaction of phenylhydrazine with 2-keto-glucose, the enzymatic product of POxs over D-glucose, was developed. This assay will allow assessing catalytic activity of O2-independent POxs. The ferrocenemethanol assay could assist tailoring POxs for O2-independency
Images of aging are related to the plasticity of adult development
No full textImages of Aging Are Related to the Plasticity of Adult Development Catherine Bowen 150 images of aging, age stereotypes, lifespan psychology, adult development, work and aging Empirical studies have demonstrated that images of aging influence acute functionality as well as long-term development. In this dissertation I (a) contribute to building a more complete theoretical framework for understanding how images of aging affect adults' acute functionality and long-term development and (b) empirically investigate images of aging in the work context and their relationship with developmental and work-related outcomes. In particular, I investigate the construct of older worker meta-stereotypes, that is, what people think other people think about older workers, in a given work context. Drawing on the lifespan framework of development, a broader aim of the dissertation is to demonstrate that images of aging are related to the plasticity of adult development. After a review of the images of aging literature, I present a theoretical framework based on the tenets of lifespan psychology to describe how images of aging affect both acute functionality and long-term development. In a theoretical analysis, I critically discuss the effects of age stereotype activation experiments and the inconsistent results in this literature. I present an integrated model of the mechanisms by which age stereotypes affect functioning and suggest a number of personal factors that may moderate the effects of age stereotype activation. Moreover, I discuss the age-trends of these moderating factors. I present the results of three empirical studies on a sample of working adults (N =337). I demonstrate the meaningfulness of the older worker meta-stereotype construct. Furthermore, I empirically demonstrate that images of aging (in the work context) are related to (a) promotion regulatory focus and (b) turnover intentions. I interpret the results from a developmental as well as an organizational perspective. I conclude with a discussion of the results of the dissertation as a whole as well as whether images of aging can be changed and make suggestions for future research