Characterisation of a MAPKK kinase from <em>Craterostigma plantagineum</em> : Interaction of VIK kinases and LEA proteins

Drought is a key factor for yield loss in agriculture. However, desiccation tolerant plant species evolved various mechanisms to protect vegetative tissues from the hazardous effects of drought. These mechanisms are not fully understood and the work in this thesis contributes to the understanding of phosphorylation of stress related proteins in the desiccation tolerant plant Craterostigma plantagineum. In desiccation sensitive plant species MAPKKKs are known to be involved in multiple drought stress response pathways. However, their role in the acquisition of desiccation tolerance of resurrection plants has not been investigated up to now. In the desiccation tolerant plant species C. plantagineum several proteins show drought stress-dependent phosphorylation. The phosphorylation of these proteins was predicted to be partially mediated by MAPKKKs (Mariaux et al. 1998; Röhrig et al. 2006; Röhrig et al. 2008). An in-gel-kinase assay using the stress-related phosphoprotein CDeT11-24 as substrate demonstrated the presence of a MAPKKK in C. plantagineum (Petersen 2012). The identified MAPKKK is a homologue to the RAF-like MAPKKK VH1-interacting kinase (VIK) from A. thaliana (At1g14000) and was consequently named CpVIK. Based on this observation it was hypothesised that CpVIK and CDeT11-24 are functionally coupled and interact with each other. In this study in silico analyses demonstrated that CpVIK belongs to the tandem-repeat ankyrin motif containing MAPKKKs (ANKMAPKKKs) that are widely distributed in various taxa. Based on their early occurrence during evolution already in bacterial genomes and the conservation in plants as well as metazoa, it is assumed that the ANKMAPKKKs have essential functions which are not yet fully understood. Co-immunoprecipitation experiments performed in this work verified a physical interaction of CDeT11-24 and CpVIK. In vitro assays confirmed phosphorylation of CDeT11-24 by the enzymatic action of CpVIK. An increased expression of CpVIK on the RNA and protein level was observed upon abiotic stresses, including dehydration. Phosphorylation of the CpVIK protein revealed to be stress-dependent. CDeT11-24 and CpVIK showed to be co-expressed and the proteins are co-localised in the cytosol. Together with previous findings, showing high expression of VIK orthologs in seeds of desiccation sensitive species, it is proposed that VIK participates in the acquisition of drought tolerance in vegetative tissues of desiccation tolerant C. plantagineum as well as in seeds of desiccation sensitive plant species. In addition to an involvement of CpVIK in drought stress, for most VIK orthologous genes, an increase of gene expression in mature leaves compared to young leaves is observed indicating importance of VIK in later stages of plant development. In A. thaliana plants no phenotypic differences were detected for AtVIK knock-out mutant plants (Δvik) in comparison to wild type plants upon dehydration or salt stress at different developmental stages. However, a lower germination rate of Δvik seeds was observed especially under salt stress conditions. Comparative seed proteome analyses verified aberrant phosphoproteins in Δvik seeds including RD29B, the closest homologue to the CDeT11-24 LEA-like C. plantagineum protein. Mass spectrometric analyses demonstrated that five phosphorylation sites were absent in the RD29B protein extracted from Δvik seeds in comparison to wild type. This represents an in vivo evidence for a functional coupling of VIK and RD29B. This thesis demonstrated a correlation of VIK proteins with LEA proteins in C. plantagineum and A. thaliana. Identification of further downstream targets in seeds of Arabidopsis and vegetative tissue of C. plantagineum will contribute to the understanding of the mechanisms of desiccation tolerance

Modulating episodic memory formation using non-invasive brain stimulation

Oscillatory activity in the beta frequency range accompanies the formation of long-term memories. Beta power decreases have frequently been shown to correlate with memory formation. However, the causal relationship between beta desynchronization and episodic memory encoding remains unclear. This thesis investigates the causal role beta oscillations play in memory formation and explores ways in which non-invasive brain stimulation can be used to test these causal mechanisms. More specifically, this thesis investigates whether increasing beta power impairs memory formation and whether decreasing beta power improves memory. We used two different non-invasive brain stimulation techniques: tACS was used to increase beta power and impair memory formation, while rTMS was used as a means of decreasing beta power and enhancing memory performance. Chapters 2 and 3 indicate that transient beta tACS does not modulate beta oscillations and does not impair memory formation, while slow rTMS effectively enhanced memory formation by modulating beta power in remote areas, in Chapter 4. This thesis emphasises that negative results are not only important, but necessary to advance our understanding of how non-invasive brain stimulation can help us unravel the causal role that beta oscillatory activity plays in the formation of episodic memories

Probing the causal involvement of dlPFC in directed forgetting using rTMS-A replication study

The forgetting of previously remembered information has, for a long time, been explained by purely passive processes. This viewpoint has been challenged by the finding that humans show worse memory for specific items that they have been instructed to forget. The dorsolateral prefrontal cortex has, through imaging, lesion and brain stimulation studies, been implied in controlling such active forgetting processes. In this study, we attempted to solidify evidence for such a causal role of the dlPFC in directed forgetting by replicating an existing rTMS study (Hanslmayr S, 2012) in a preregistered within-participant design. We stimulated participants at the dlPFC (BA9) or vertex using 45s of 1Hz rTMS after instructions to forget previously remembered words in a list-method directed forgetting paradigm and tested for effects on the amount of forgotten information. Contrary to the study we were attempting to replicate, no significant increase in forgetting under dlPFC stimulation was found in our participants. However, when combining our results with the study we were attempting to replicate, dlPFC stimulation led to significantly increased directed forgetting in both studies combined. We further explored if the rTMS parameters used here and in earlier work (Hanslmayr S, 2012) influenced inhibitory processing at their time of delivery or in a more persistent manner. Unaltered incongruency and negative priming effects in a Stroop task conducted directly after stimulation suggests that our rTMS stimulation did not continue to influence inhibitory processing after the time of stimulation. As the combined evidence for increased directed forgetting due to rTMS dlPFC stimulation is still quite weak, additional replications are necessary to show that directed forgetting is indeed causally driven by an active prefrontal process

Episodic memory retrieval functionally relies on very rapid reactivation of sensory information

Episodic memory retrieval is assumed to rely on the rapid reactivation of sensory information that was present during encoding, a process termed “ecphory.” We investigated the functional relevance of this scarcely understood process in two experiments in human participants. We presented stimuli to the left or right of fixation at encoding, followed by an episodic memory test with centrally presented retrieval cues. This allowed us to track the reactivation of lateralized sensory memory traces during retrieval. Successful episodic retrieval led to a very early (∼100–200 ms) reactivation of lateralized alpha/beta (10–25 Hz) electroencephalographic (EEG) power decreases in the visual cortex contralateral to the visual field at encoding. Applying rhythmic transcranial magnetic stimulation to interfere with early retrieval processing in the visual cortex led to decreased episodic memory performance specifically for items encoded in the visual field contralateral to the site of stimulation. These results demonstrate, for the first time, that episodic memory functionally relies on very rapid reactivation of sensory information

Probing the causal involvement of dlPFC in directed forgetting using rTMS—A replication study

The forgetting of previously remembered information has, for a long time, been explained by purely passive processes. This viewpoint has been challenged by the finding that humans show worse memory for specific items that they have been instructed to forget. The dorsolateral prefrontal cortex has, through imaging, lesion and brain stimulation studies, been implied in controlling such active forgetting processes. In this study, we attempted to solidify evidence for such a causal role of the dlPFC in directed forgetting by replicating an existing rTMS study (Hanslmayr S, 2012) in a preregistered within-participant design. We stimulated participants at the dlPFC (BA9) or vertex using 45s of 1Hz rTMS after instructions to forget previously remembered words in a list-method directed forgetting paradigm and tested for effects on the amount of forgotten information. Contrary to the study we were attempting to replicate, no significant increase in forgetting under dlPFC stimulation was found in our participants. However, when combining our results with the study we were attempting to replicate, dlPFC stimulation led to significantly increased directed forgetting in both studies combined. We further explored if the rTMS parameters used here and in earlier work (Hanslmayr S, 2012) influenced inhibitory processing at their time of delivery or in a more persistent manner. Unaltered incongruency and negative priming effects in a Stroop task conducted directly after stimulation suggests that our rTMS stimulation did not continue to influence inhibitory processing after the time of stimulation. As the combined evidence for increased directed forgetting due to rTMS dlPFC stimulation is still quite weak, additional replications are necessary to show that directed forgetting is indeed causally driven by an active prefrontal process

On the effectiveness of event-related beta tACS on episodic memory formation and motor cortex excitability

Background: Transcranial alternating current stimulation (tACS) is widely used to entrain or modulate brain oscillations in order to investigate causal relationships between oscillations and cognition. Objective: In a series of experiments we here addressed the question of whether event-related, transient tACS in the beta frequency range can be used to entrain beta oscillations in two different domains: episodic memory formation and motor cortex excitability. Methods: In experiments 1 and 2, 72 healthy human participants engaged in an incidental encoding task of verbal and non-verbal material while receiving tACS to the left and right inferior frontal gyrus (IFG) at 6.8 Hz, 10.7 Hz, 18.5 Hz, 30 Hz, 48 Hz and sham stimulation for 2s during stimulus presentation. In experiment 3, tACS was administered for 10s to M1 at the individual motor beta frequency of eight subjects. We investigated the relationship between the size of TMS induced MEPs and tACS phase. Results: Beta tACS did not affect memory performance compared to sham stimulation in experiments 1 and 2. Likewise, in experiment 3, MEP size was not modulated by the tACS phase. Conclusions: Our findings suggest that event-related, transient tACS in the beta frequency range cannot be used to modulate the formation of episodic memories or motor cortex excitability. These null-results question the effectiveness of event-related tACS to entrain beta oscillations and modulate cognition

Разработка и исследование алгоритмов решения геодезических задач с использованием Web-технологий

Объектом исследования данной работы являются базовые задачи геодезии. Цель работы – разработка и введение в эксплуатацию Web-приложения выполняющего вычисления возникающие при решении базовых задач геодезии с максимальной автоматизацией.Research and modification of algorithms for solving basic problems of geodesy. Development web-based applications based on compiled algorithms

Probing the causal role of prestimulus interregional synchrony for perceptual integration via tACS

The phase of prestimulus oscillations at 7–10 Hz has been shown to modulate perception of briefly presented visual stimuli. Specifically, a recent combined EEG-fMRI study suggested that a prestimulus oscillation at around 7 Hz represents open and closed windows for perceptual integration by modulating connectivity between lower order occipital and higher order parietal brain regions. We here utilized brief event-related transcranial alternating current stimulation (tACS) to specifically modulate this prestimulus 7 Hz oscillation, and the synchrony between parietal and occipital brain regions. To this end we tested for a causal role of this particular prestimulus oscillation for perceptual integration. The EEG was acquired at the same time allowing us to investigate frequency specific after effects phase-locked to stimulation offset. On a behavioural level our results suggest that tACS did modulate perceptual integration, however, in an unexpected manner. On an electrophysiological level our results suggest that brief tACS does induce oscillatory entrainment, as visible in frequency specific activity phase-locked to stimulation offset. Together, our results do not strongly support a causal role of prestimulus 7 Hz oscillations for perceptual integration. However, our results suggest that brief tACS is capable of modulating oscillatory activity in a temporally sensitive manner