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Imitation paradigms are used in various domains of developmental psychological research to assess various cognitive processes such as memory (deferred imitation), action perception and action understanding (mainly direct imitation), as well as categorization and learning about objects (deferred imitation with a change in target objects and generalized imitation). Although these processes are most likely not independent from each other, their relations are still largely unclear. On the one hand, deferred imitation studies have shown that infants' performance improves with increasing age, resulting in the reproduction of more target actions after longer delay intervals. On the other hand, imitation studies focusing on infants' action understanding have found that infants do not necessarily imitate the model's exact actions – actions or action steps that seem to be irrational or irrelevant are omitted by infants under certain circumstances (selective imitation). Additionally, findings of imitation studies that require a transfer of the target actions to novel objects have demonstrated that infants do not only learn about actions, but also about objects, when they engage in imitation.
The present dissertation aims at integrating different perspectives of imitation research by testing 12- and 18-month-old infants in deferred imitation tests consisting of functional vs. arbitrary target actions, and by combining deferred imitation with eye tracking in half of the experiments. A deferred imitation paradigm was chosen to assess memory performance. Systematic variation of target action characteristics enabled the assessment of infants' imitation pattern, i.e., if they would imitate one kind of target actions more frequently than the other. Functionality was chosen as the action characteristic in focus because function is an object's most important property, thus this variation might shed some light on infants' learning about objects in the context of an imitation test. The main goal of the eye tracking experiments was to tackle the relations between infants' visual attention to, and deferred imitation of, different kinds of target actions.
The behavioral experiments revealed that both 12- and 18-month-olds imitated significantly more functional than arbitrary target actions after a delay of 30 minutes. In addition, while 12-month-olds showed a memory effect only for functional actions, 18-month-olds showed a memory effect for both kinds of actions. Thus, 12-month-olds imitated strictly selectively, and 18-month-olds imitated more exactly. This shows that the well established memory effect is modulated by target action functionality, which affects 12- and 18-month-olds' imitation differently. Furthermore, when retested after a two weeks delay, 18-month-olds' performance rates of functional and arbitrary target actions decreased parallel. This suggests that selective imitation is not affected by the duration of the retention interval, and that selection of target actions takes place at an earlier stage of action perception and memory processes.
In the eye tracking experiments, both 12- and 18-month-olds' imitation patterns replicated the findings of the behavioral experiments, showing consistently higher imitation rates of functional than arbitrary target actions. Contrary to this, infants' fixation times to the target actions were not affected by target action functionality. This contrast was supported by statistical analyses that found no clear correspondence between visual attention to and deferred imitation of target actions. This suggests that selective imitation cannot be explained by selective visual attention. Nevertheless, finer-grained analyses of gaze and imitation data in the 18 months old group suggested that infants' increased attention to the social-communicative context of the imitation task was related to more exact imitation, i.e. imitation of not only functional, but also arbitrary target actions.
The findings are discussed against the background of imitation theories, with regard to the relations between different cognitive processes underlying infants' imitation, such as memory, action perception and learning about objects.
In the past sixty years, excessive water consumption and dam construction have significantly influenced natural flow regimes and surface freshwater ecosystems throughout China, and thus resulted in serious environmental problems. In order to balance the competing water demands between human and environment and provide knowledge on sustainable water management, assessments on anthropogenic flow alterations and their impacts on aquatic and riparian ecosystems in China are needed.
In this study, the first evaluation on quantitative relationships between anthropogenic flow alterations and ecological responses in eleven river basins and watersheds in China was performed based on the data that could be obtained from published case studies. Quantitative relationships between changes in average annual discharge, seasonal low flow and seasonal high flow and changes in ecological indicators (fish diversity, fish catch and vegetation cover, etc.) were analyzed. The results showed that changes in riparian vegetation cover as well as changes in fish diversity and fish catch were strongly correlated with the changes in flow magnitude (r = 0.77, 0.66), especially with changes in average annual river discharge. In addition, more than half of the variations in vegetation cover could be explained by changes in average annual river discharge (r² = 0.63) and roughly 50 % changes in fish catch in arid and semi-arid region and 60% changes of fish catch in humid region could be related to alterations in average annual river discharge (r² = 0.53, 0.58).
In a supplementary analysis of this study, the first estimation on quantitative relationships between decreases in native fish species richness and anthropogenic flow alterations in 34 river basins and sub-basins in China was conducted. Linear relationships between losses of native fish species and five ecologically relevant flow indicators were analyzed by single and multiple regression models. For the single regression analysis, significant linear relationships were detected for the indicators of long-term average annual discharge (ILTA) and statistical low flow Q90 (IQ90). For the multiple regressions, no indicator other than ILTA has significant relationships with changes in number of fish species mainly due to collinearity. Two conclusions emerged from the analysis: 1) losses of fish species were positively correlated with changes in ILTA in China and 2) indicator of ILTA was dominant over other flow indicators included in this research for the given dataset. These results provide a guideline for the sustainable water resources management in rivers with high risk of fish extinction in China.
Cryo-electron tomography (CET) is a unique technique to visualize biological objects under near-to-native conditions at near-atomic resolution. CET provides three-dimensional (3D) snapshots of the cellular proteome, in which the spatial relations between macromolecular complexes in their near native cellular context can be explored. Due to the limitation of the electron dose applicable on biological samples, the achievable resolution of a tomogram is restricted to a few nanometers, higher resolution can be achieved by averaging of structures occurring in multiples. For this purpose, computational techniques such as template matching, sub-tomogram averaging and classification are essential for a meaningful processing of CET data.
This thesis introduces the techniques of template matching and sub-tomogram averaging and their applications on real biological data sets. Subsequently, the problem of reference bias, which restricts the applicability of those techniques, is addressed. Two methods that estimate the reference bias in Fourier and real space are demonstrated. The real space method, which we have named the “M-free” score, provides a reliable estimation of the reference bias, which gives access to the reliability of the template matching or sub-tomogram averaging process. Thus, the “M-free” score makes those approaches more applicable to structural biology. Furthermore, a classification algorithm based on Neural Networks (NN) called “KerDenSOM3D” is introduced, which is implemented in 3D and compensates for the missing-wedge. This approach helps extracting different structural states of macromolecular complexes or increasing the class purity of data sets by eliminating outliers. A comprehensive comparison with other classification methods shows superior performance of KerDenSOM3D.
Cancer is a disease characterized by uncontrolled cell growth and the capacity to disseminate to distant organs. The properties of cancers are caused by genetic and epigenetic alterations when compared to their normal counterparts. Genetic mutations occur in oncogenes and tumor suppressor genes and are the initial drivers of cellular transformation (Lengauer et al., 1998; Vogelstein and Kinzler, 2004). In addition, epigenetic alterations, which influence the expression of oncogenes and tumor suppressor genes independently from sequence alterations, are also involved in the transformation process (Esteller and Herman, 2001; Sharma et al., 2010). Genetic alterations and epigenetic regulatory signals cooperate in tumor etiology. Glioblastoma multiforme (GBM) is a frequent and aggressive malignant brain tumor in humans. The median survival of GBM patients is about 15 months after diagnosis. Like in other cancers, genetic and epigenetic alterations can be detected in GBM. Genetic alterations in GBM affect cell growth, apoptosis, angiogenesis, and invasion; however, epigenetic alterations in GBM also affect the expression of oncogenes or tumor suppresser genes that increase tumor malignancy (Nagarajan and Costello, 2009).
Reprogramming is a cellular process in which somatic cells can be induced to assume the properties of less differentiated stem cells. This process can be mediated through epigenetic modifications of the genome of somatic cells by the action of four defined transcription factors (Oct4, Sox2, Klf4 and Myc) or by the action of the miR 302/367 cluster (Anokye-Danso et al., 2011; Takahashi and Yamanaka, 2006; Takahashi et al., 2007) and result in the generation of induced pluripotent stem cells (iPS cells). Reprogramming of somatic cells by the miR 302/367 cluster can generate nontumorigenic iPS cells through the inhibition of the epithelial to mesenchymal transition (EMT), cell cycle regulatory genes and epigenetic modifiers (Lin and Ying, 2013).
Heme-copper oxidases (HCOs) are the terminal enzymes of the aerobic respiratory chain in the inner mitochondrial membrane or the plasma membrane in many prokaryotes. These multi-subunit membrane protein complexes catalyze the reduction of oxygen to water, coupling this exothermic reaction to the establishment of an electrochemical proton gradient across the membrane in which they are embedded. The energy stored in the electrochemical proton gradient is used e.g. by the FOF1-ATP synthase to generate ATP from ADP and inorganic phosphate. The superfamily of HCOs is phylogenetically classified into three major families: A, B and C. The A-family HCOs, represented by the well-studied aa3-type cytochrome c oxidases (aa3-CcOs), are found in mitochondria and many bacteria. The B-family of HCOs contains a number of bacterial and archaeal oxidases. The C-family comprises only the cbb3-type cytochrome c oxidase (cbb3-CcO) and is most distantly related to the mitochondrial respiratory oxidases.
In this thesis, a novel 257 kHz chopper device was numerically developed, technically designed and experimentally commissioned; a 4-solenoid, low-energy ion beam transport line was numerically investigated, installed and experimentally commissioned; and a novel massless beam-separation system was numerically developed.
The chopper combines a pulsed electric field with a static magnetic field in an ExB or Wien-filter type field configuration. Chopped beam pulses with a 257 kHz repetition rate and rise times of 110 ns were experimentally achieved using a 14 keV helium beam.
Due to the achieved results, the complete LEBT line for the future Frankfurt Neutron Source FRANZ is ready to deliver a dc or a pulsed beam. At the same time, the LEBT section represents an attractive test stand for the study of low-energy ion beams. It combines magnetic lenses, which allow space-charge compensated beam transport, and a chopper system capable of producing short beam pulses in the hundred nanosecond range. Since these beam pulses are transported onwards, their longitudinal and transverse properties can be analyzed. The pulse duration and time of flight are well below the rise time for the space-charge compensation through residual gas ionization. This opens the possibility for dedicated investigations of the transport of short, low-energy beam pulses including longitudinal and transverse space-charge effects and of relevant issues like the dynamics of space-charge compensation and electron effects in short pulses.
Immune cells are key players in several physiological and pathophysiological events such as acute and chronic inflammation, atherosclerosis and cancer. Especially in acute inflammation, macrophages are indispensable for the switch from the acute inflammatory phase to the resolution phase. Not only the phagocytosis of apoptotic cells, but especially the surrounding cytokines and mediators are able to switch macrophage polarization from inflammatory- to anti-inflammatory phenotypes. Within this cytokine environment, sphingosine-1-phosphate (S1P) plays an important role for immune cell activation, polarization and migration.
The Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes high fever, rash, and recurrent arthritis in humans. The majority of symptoms disappear after about one week. However, arthritis can last for months or even years (in about 30% of cases), which makes people unable to work during this period. The virus is endemic in Sub-Saharan Africa, the Indian Ocean islands, India, and Southeast Asia. It has additionally caused several large outbreaks in the last few years, affecting millions of people. The mortality rate is very low (0.1%), but the infection rates are high (sometimes 30%) and the number of asymptomatic cases is rare (about 15%). The first CHIKV outbreak in a country with a moderate climate was detected in Italy in 2007. Furthermore, the virus has spread to the Caribbean in late 2013. Due to climate change, globalization, and vector switching, the virus will most likely continue to cause new worldwide outbreaks. Additionally, more temperate regions of the world like Europe or the USA, which have recently reported their first cases, will likely become targets. Alarmingly, there is no specific treatment or vaccination against CHIKV available so far.
The cell entry process of CHIKV is also not understood in detail, and was thusly the focus of study for this project. The E2 envelope protein is responsible for cell attachment and entry. It consists of the domain C, located close to the viral membrane, domain A, in the center of the protein, and domain B, at the distal end, prominently exposed on the viral surface.
In this work, the important role of cell surface glycosaminoglycans (GAGs) for CHIKV cell attachment was uncovered. GAGs consist of long linear chains of heavily sulfated disaccharide units and can be covalently linked to membrane associated proteins. They play an important role in different cell signaling pathways. So far, solely cell culture passage has revealed an increased GAG-dependency of CHIKV due to mutations in E2 domain A, which was associated with virus attenuation in vivo. However, in this work it could be shown that cell surface GAGs promote CHIKV entry using non-cell culture adapted CHIKV envelope (Env) proteins. Transduction and infection of cell surface GAG-deficient pgsA-745 cells with CHIKV Env pseudotyped vector particles (VPs) and with wild-type CHIKV revealed decreased transduction and replication rates. Furthermore, cell entry and transduction rates of GAG-containing cells were also dose-dependently decreased in the presence of soluble GAGs. In contrast, transduction of pgsA-745 cells with CHIKV Env pseudotyped VPs was enhanced by the addition of soluble GAGs. This data suggests a mechanism by which GAGs activate CHIKV particles for subsequent binding to a cellular receptor. However, at least one GAG-independent entry pathway might exist, as CHIKV entry could not be totally inhibited by soluble GAGs and entry into pgsA-745 was, albeit at a lower rate, still possible. Further binding experiments using recombinant CHIKV E2 domains A, B, and C suggest that domain B is responsible for the GAG binding, domain A possibly for receptor binding, and domain C is not involved in cell binding. These results are in line with the geometry of CHIKV Env on the viral surface. They altogether reveal that GAG binding promotes viral cell entry and that the E2 domain B plays a central role for this mechanism.
As no vaccine against CHIKV has been approved so far, another goal of this project was to test new vaccination approaches. It has been published that a single linear epitope of E2 is the target of the majority of early neutralizing antibodies against CHIKV in patients. Artificial E2-derived proteins were created, expressed in E.coli, and successfully purified. They consisted of 5 repeats of the mentioned linear epitope (L), the surface exposed regions of domain A linked by glycine-serine linkers (sA), the whole domain B plus a part of the β-ribbon connector (B+), or a combination of these 3 modules. Vaccination experiments revealed that B+ was necessary and sufficient to induce a neutralizing immune response in mice, with the protein sAB+ yielding the best results. sAB+, as a protein vaccine, efficiently and significantly reduced viral titers in mice upon CHIKV challenge, which was not the case for recombinant Modified Vaccinia virus Ankara (MVA; MVA-CHIKV-sAB+), as a vaccine platform expressing the same protein. These experiments show that a small rationally designed CHIKV Env derived protein might, after optimization of some vaccination parameters, be sufficient as a safe, easy-to-produce, and cheap CHIKV vaccine.
Epigallocatechin-3-gallate (EGCG) is a catechin found in green tea and was, in this work, found to inhibit the CHIKV life cycle at the entry state in in vitro experiments using CHIKV Env VPs and wild-type virus. EGCG was recently published to inhibit attachment of several viruses to cell surface GAGs, which is in line with the role for GAGs in CHIKV entry revealed in this work. EGCG might serve as a lead compound for the development of a small molecule treatment against CHIKV.
In the interest of understanding the development of a multicellular organism, subcellular events must be seen in the context of the entire three-dimensional tissue. In addition, events that occur within a short period of time can be of great importance for the relatively long developmental process of the organ. Thus, it is required to capture subcellular events in a larger spatio-temporal scale context, which has been up to now a technical challenge. In developmental biology, light microscopy has always been an important tool. The dilemma of light microscopy, in particular fluorescence microscopy, is that molecules receive high light intensities that might change the conformation of molecules, which can have signaling or toxic effects. In Light Sheet-based Fluorescence Microscopy (LSFM), the energy required for a single recording is reduced by several orders of magnitude compared to other fluorescence microscopy techniques. During the last ten years, LSFM has emerged as a preferred tool to capture all cells during embryogenesis of the zebrafish Danio rerio, the fruit fly Drosophila melanogaster or recently the red flour beetle Tribolium castaneum for a period of several days. The motivation of this work was to gain new insights in developmental related processes of plant organs. The aim of this work was to establish a protocol for imaging plant growth over a long period of time using LSFM and perform comprehensive analyses at the cellular level. Plants have to cope with a variety of environmental conditions, therefore the conditions inside the microscope chamber had to be brought under control. The sample preparation methods and the standardized conditions at a physiological level allowed the study of gravity response, day-night rhythms, organ shape development as well as the intracellular dynamic events of the cytoskeleton and endosomal compartments in an unprecedented manner. Several of these projects were successfully published in collaborations with Prof. Jozef Šamaj (Palacký University Olomouc, Czech Republic), Prof. Niko Geldner (University of Lausanne, Switzerland), Prof. Malcom Bennett (University of Nottingham, UK) and Dr. Jürgen Kleine-Vehn (University of Natural Resources and Life Sciences, Austria). The main part of my work focused on the formation of lateral roots in Arabidopsis thaliana and was conducted in close collaboration with Dr. Alexis Maizel (University of Heidelberg, Germany). Previously, most experiments that describe lateral root formation have been performed on a small number of cells and for short periods of time. Capturing the complete process of lateral roots is an ambitious goal, because first, the primordium of a lateral root is located deep inside the primary root and imaging quality is impaired due to scattering of the overlaying tissue. Second, the process takes about 48 h, i.e. the plant has to be kept healthy for the whole period. Third, the amount of excitation light required for the spatio-temporal might have phototoxic effects that lead to a stop of growth at least in conventional microscopic techniques. In Arabidopsis embryogenesis, the sequence of cell divisions is relatively invariant. However, whether lateral root organogenesis follows particular cell division patterns has been unknown. The complete process of lateral root formation was captured from the first cell division until after the emergence from the main root. Images of a nuclei marker and a plasmamembrane marker were recorded every 5 min for a time period of up to 64 h. The positions and cell divisions of all cells were tracked manually. In collaboration with Alexander Schmitz (Goethe University Frankfurt am Main, Germany) and Dr. Jens Fangerau (University of Heidelberg, Germany), comprehensive analyses of the data were performed. A lateral root forms from initially 8-15 founder cells, arranged in a patch of 5-8 parallel files. The occurrence of new cell layers by periclinal divisions, as well as the sequence of layer generation was conserved and resembles the sequence suggested by Malamy and Benfey in 1997. Besides this stereotyped occurrence of periclinal divisions, radial divisions were found to appear stochastically, following no particular pattern. A large variability was also found in the contribution of founder cells and cell files to the final lateral root. In summary, the results suggest that a stereotyped pattern of cell divisions at particular developmental stages and a dynamically adapted control of cell divisions exist in parallel. Both properties allow a controlled but flexible development of the organ according to variations in cell topology and mechanical properties of the surrounding tissue. This work shows that LSFM, the sample preparation methods and controlled environmental conditions allow to capture and analyse the development of plants over several days at high resolution in an unprecedented manner.
During this study clumped isotope analysis of carbonates was established at the Goethe University of Frankfurt, Germany. Therefore, preparation protocols and analytical parameters were elaborated to obtain precise and accurate Δ47 data. Briefly, analyte CO2 was cleaned cryogenically using glass extraction lines to remove traces of water that enable re-equilibration of C–O bonds in the gases. Furthermore, analyte CO2 was passed through a gas chromatograph (GC) to clean it from contaminants that produce isobaric interferences with m/z 47. Initially, phosphoric acid digestions of carbonates was conducted at 25 °C in McCrea-type reaction vessels. Afterwards samples were reacted at 90 °C using a common acid bath. Mass spectrometric analyses were performed using a MAT 253 equipped with a dual inlet system. Δ47 values were directly projected to the absolute scale using CO2 gases equilibrated at distinct temperatures.
In cooperation with Stefano Bernasconi and his research group at ETH Zurich we studied the non-linearity that occurs for the measurement of m/z 47. This effect results from secondary electrons created by the m/z 44 beam. These electrons cause a negative background on the m/z 47 collector. A correction procedure was proposed that relies on the determination of the negative background on the m/z 47 Faraday cup. This approach might reduce time-consuming analyses of heated gases which were used so far to account for the observed non-linearity. However, the suggested correction of the negative background on the m/z 47 cup is only applicable if the slit width of the m/z 44 beam is significantly wider than that of the m/z 47 beam.
This thesis, furthermore, presents a comparison of the different phosphoric acid digestion techniques which are commonly used for carbonate clumped isotope analysis. For calcitic and aragonitic material digested at 25 °C in McCrea-type vessels we observed that the sample size has an effect on Δ47 data: higher mean Δ47 values and a larger scatter of data were received for samples <7 mg than for larger aliquots. For carbonate samples digested at 90 °C in a common acid bath no sample size effect was determined. We assume that secondary re-equilibration of CO2 with water preferentially occurs at 25 °C producing the observed differences. However, a sample size effect can be avoided if reaction temperature is increased to 90 °C.
In order to make carbonate Δ47 data obtained from acid digestions at 90 °C comparable to Δ47 data received from reactions at 25 °C the difference of the acid fractionation factores (Δ47*25-90) between both temperatures has to be known. For the determination of the Δ47*25-90 value we have considered Δ47 data made at 25 °C from samples >7 mg only. For calicte and aragonite we obtained differences in fractionation factores of 0.075‰ and 0.066‰, respectively. These Δ47*25-90 values are coincident with the theoretical prediction of 0.069‰ proposed for calcite (Guo et al., 2009).
Moreover, this dissertation comprises a calibration study of the clumped isotope thermometer based on various natural calcites that grew between 9 and 38 °C. The samples include a brachiopod shell, a bivalve shell, an eggshell of an ostrich and foraminifera tests which formed from distinct biomineralizing processes. Furthermore we included an authigenic carbonate crystallized from biological-induced precipitation. The following linear relationship between 1/T2 and Δ47 was determined (with Δ47 in ‰ and T in K):
Δ47 = 0.0327 (± 0.0026) x 106 / T2 + 0.3030 (± 0.0308) (R2 = 0.9915)
This equation differs from the pioneering Ghosh et al. (2006a) calibration. However, our regression line is statistically indistinguishable from that of Henkes et al. (2013) which is based on aragonitic mollusks and calcitic brachiopod shells. Both studies have in common that calibration data were, at first, directly referenced to the absolute scale. In addition, both datasets rely on similar digestion techniques. Furthermore, the two calibrations are conform with the theoretical prediction of Guo et al. (2009).
The calcite calibration of the clumped isotope paleothermometer received in this study was applied to Δ47 data measured for Silurian brachiopods shells from Gotland/Sweden. Prior to isotopic analysis the fossils were intensively investigated for their preservation state (CL, SEM, trace elements). The lowest T(Δ47) values of ca. 28 to 33 °C were estimated from ultrastructurally well-preserved regions of some shells. For these samples also the lowest δ18Ow values of Silurian seawater were determined. These estimates of ca. −1‰ confirm the assumption that the δ18O value of the Silurian ocean was buffered to (0 ± 1)‰.
Nevertheless, most studied shells were characterized by a patchwork of pristine and altered shell portions resulting in elevated T(Δ47) values which plot mostly between 40 and 60 °C. Our results indicate that the clumped isotopic composition of the shells were altered at low water-rock ratios, not affecting the δ18O values. Δ47 and δ18O data of associated diagenetic phases (sparitic and micritic phases of the inner fillings of the fossils) provide evidence that the sparitic cements grew during several diagenetic events which occurred at different temperatures in fluid-buffered systems. We, furthermore, conclude that the micritic phases lithified at a very early diagenetic stage with the δ18O values being most probably close to a Silurian seawater composition