Genetic analysis of Homologous Recombination at Arabidopsis thaliana telomeres

Telomere sind die Enden von linearen Chromosomen. Ihre Hauptfunktion ist zu verhindern, dass die Enden als DNS DoppelStrangBrüche (DSB) erkannt werden. In höheren Eukaryonten geschieht dies durch die T-loop Struktur. Bei dieser Struktur invasiert das einzelsträngige 3’ Chromosomenende die doppelsträngige telomerische DNS. Es wird angenommen, dass dazu die Maschinerie der Homologen Rekombination (HR) benötigt wird. Anders als bei einem DNS Reperaturereignis wird der T-loop jedoch nicht aufgelöst. Wie dieses unterschiedliche Verhalten entsteht wird noch nicht verstanden. Proteine der HR Maschinerie, wie der MRN Komplex (MRE11-RAD50-NBS1) sind auch am Telomer zu finden. RAD51 und die BCDX2 (RAD51B-RAD51C-RAD51D-XRCC2) und CX3 (RAD51C-XRCC3) Komplexe sind notwending für den Schritt der Stranginvasion während der HR. Manche Studien schlagen auch eine Rolle der BCDX2 and CX3 Komplexe in der Auflösung der Zwischenstrukturen der HR vor. Doch obwohl es biochemische Daten zu diesen zwei Komplexen gibt, sind keine genetischen Daten vorhanden. Die Rolle der beiden Komplexe im Stoffwechsel des T-loops sind unbekannt. In dieser Diplomarbeit verwendete ich Arabidopsis thaliana als Modellorganismus, um genetische Grundlagen der telomerischen Rekombination zu untersuchen. Mutaten in einem der fünf Gene, welche die BCDX2 and CX3 Komplexe bilden sind in Pflanzen vital. In Mäusen hingegen sind Mutationen in RAD51C und XRCC2 embryolethal. In dieser Diplomarbeit presentieren wir die ersten Daten über genetische Interaktionen zwischen den Genen, die für die Proteine der BCDX2 and CX3 Komplexe kodieren. Weiters untersuchten wir die Rolle von RAD51 und MRE11 im Stoffwechsel des T-loops. Um auf funktionelle Redundanzen innerhalb der BCDX2 and CX3 Komplexe zu testen, erzeugten wir Tripelmutanten mit ku70-/-. In dieser Mutante enstehen extrachromosomale, telomerische Ringe (t-circles), welche zur Betrachtung der HR an Telomeren verwendet werden können. Ich testete sechs verschiedene Mutantenkombinationen und konnte vier davon erzeugen. In diesen vier Tripelmutanten waren t-circles vorhanden. Ich scheiterte daran rad51c-1 xrcc3-1 ku70-1 und mre11-3 rad51c-1 ku70-1 Tripelmutanten zu erzeugen. Die Segregationsanalyse enthüllte Nicht-Mendelische Segregation der F2 Populationen beider Mutantenkombinationen. Weitere Analysen brachten eine Translokation zum Vorschein, welche für das Nicht-Mendelische Verhalten der rad51c-1 xrcc3-1 Mutantenkombination verantwortlich war. Diese Translokation verband die beiden Gene physisch. Im Falle der mre11-3 rad51c-1 Mutantenkombination kennen wir den Grund für das Nicht-Mendelische Verhalten noch nicht. Um dies zu erklären werden weitere Experimente benötigt.Telomeres are the end of linear chromosomes. The main function of telomeres is to protect the chromosome end from being recognized as a DNA Double Strand Break (DSB). In higher eukaryotes this is achieved by a T-loop structure. In this structure the single stranded 3’ chromosome end invades the duplex telomeric DNA. It is thought that this process requires the Homologous Recombination (HR) machinery. Unlike a DNA repair event, the T-loop structure is not resolved. How this bi-modal behavior at telomeres is achieved is not understood. Proteins of the HR machinery, like the MRN (MRE11-RAD50-NBS1) complex, are located at telomeres. RAD51 as well as the BCDX2 (RAD51B-RAD51C-RAD51D-XRCC2) and CX3 (RAD51C-XRCC3) complexes are necessary for the strand invasion step of HR. Some studies also suggest a resolving role of the BCDX2 and CX3 complexes. Even though biochemical data on these two complexes exist, genetic data is missing. The role of these complexes in T-loop metabolism is unknown. In this thesis I utilized Arabidopsis thaliana as a model system to examine genetic requirements of telomeric recombination. Mutations in one of the five genes building the BCDX2 and CX3 complexes are viable, which is in contrast to mice, where mutations in RAD51C and XRCC2 are embryo-lethal. In this thesis we present the first data on genetic interaction between genes encoding proteins of the BCDX2 and CX3 complexes. We also investigated the role of RAD51 and MRE11 in T-loop metabolism. To test for functional redundancies within the BCDX2 and CX3 complexes, we generated double and triple mutant combinations of recombination genes with ku70-/-. This mutation results in extra-chromosomal telomeric circles (t-circles) that can be used to monitor HR at telomeres. I tested six different mutant combinations and obtained triple mutants for four of them. In all four triple mutant combinations, t-circles were present. However, I failed to obtain rad51c-1 xrcc3-1 ku70-1 and mre11-3 rad51c-1 ku70-1 combinations. Segregation analysis revealed a Non-Mendelian segregation in F2 populations derived from both crossings. Further analysis revealed a translocation to be the cause for the Non-Mendelian behavior in the rad51c-1 xrcc3-1 combination. This translocation physically linked the two genes. In case of the mre11-3 rad51c-1 combination we do not know the reason for the Non-Mendelian behavior yet. Further experiments will be needed to answer this question

An augmented reality study for public participation in urban planning

Ongoing urbanisation processes invoke immense construction activities, for which citizens often participate in planning. Yet, imagining planned buildings based on visual representations is a highly demanding task. While traditional methods, such as construction spans, 2D, or 3D visualisation often fail to offer a complete picture, we propose Augmented Reality (AR) as a more adequate tool. We first present an evaluation of the suitability of AR compared to construction spans for a future building and assess which degree of abstraction of AR is most effective, as well as difficulty of interpreting them correctly. In a between-subjects field study we compare construction spans and a prototype AR application including three levels of detail (LOD) of the same building project. Participants solve two estimation tasks using the construction spans and six estimation tasks using the AR application, before answering a questionnaire on the different visualisation methods. We find participants are confident about the potential of AR, but no significant differences between the different LOD groups in subjective assessment. Results suggest that previous knowledge (e.g. in GIS) may have a positive impact on dimension estimation performance. Also, details, such as façade elements or windows, could facilitate estimation tasks because they allow inferences about a building’s size

Electronic transient spectroscopy from the deep UV to the NIR: unambiguous disentanglement of complex processes

Complex multi-stage relaxation and reaction pathways after the optical excitation of molecules makes the disentanglement of the underlying mechanisms challenging. We present four examples that a new transient spectrometer with excitation fully tunable from the deep UV to the IR and 225 to 1700 nm probing allows for an analysis with greatly reduced ambiguity. The temporal resolution of about 50 fs allows us to resolve all relevant processes. For each example there is a new twist in the sequence of relaxation steps that had previously been overlooked. In malachite green it appears that the importance of the phenyl twisting has been overemphasized and rather a charge transfer state should be considered. In TINUVIN-P the predicted twisting as the driving motion for the ultrafast IC is confirmed and leads to a resolution of the earlier puzzle that the sub-5 ps regime shows kinetics deviating from a pure cooling process despite the sub-ps proton transfer cycle. For the bond cleavage of Ph2CH-Cl and Ph2CH-Br the degree of electron transfer within the radical pair can now be determined quantitatively and leads to a profound understanding of the long term cation yield. For the first time coherent wavepacket motion in the photoproducts is reported. Last but not least the measurement of the GSB recovery in the deep UV allows for the surprising result, that even after S-2 excitation of cyclopentenones the triplet states are reached with near unity probability within a few picoseconds

Objective assessment of motor fatigue in multiple sclerosis using kinematic gait analysis: a pilot study

<p>Abstract</p> <p>Background</p> <p>Fatigue is a frequent and serious symptom in patients with Multiple Sclerosis (MS). However, to date there are only few methods for the objective assessment of fatigue. The aim of this study was to develop a method for the objective assessment of motor fatigue using kinematic gait analysis based on treadmill walking and an infrared-guided system.</p> <p>Patients and methods</p> <p>Fourteen patients with clinically definite MS participated in this study. Fatigue was defined according to the Fatigue Scale for Motor and Cognition (FSMC). Patients underwent a physical exertion test involving walking at their pre-determined patient-specific preferred walking speed until they reached complete exhaustion. Gait was recorded using a video camera, a three line-scanning camera system with 11 infrared sensors. Step length, width and height, maximum circumduction with the right and left leg, maximum knee flexion angle of the right and left leg, and trunk sway were measured and compared using paired t-tests (α = 0.005). In addition, variability in these parameters during one-minute intervals was examined. The fatigue index was defined as the number of significant mean and SD changes from the beginning to the end of the exertion test relative to the total number of gait kinematic parameters.</p> <p>Results</p> <p>Clearly, for some patients the mean gait parameters were more affected than the variability of their movements while other patients had smaller differences in mean gait parameters with greater increases in variability. Finally, for other patients gait changes with physical exertion manifested both in changes in mean gait parameters and in altered variability. The variability and fatigue indices correlated significantly with the motoric but not with the cognitive dimension of the FSMC score (R = -0.602 and R = -0.592, respectively; P < 0.026).</p> <p>Conclusions</p> <p>Changes in gait patterns following a physical exertion test in patients with MS suffering from motor fatigue can be measured objectively. These changes in gait patterns can be described using the motor fatigue index and represent an objective measure to assess motor fatigue in MS patients. The results of this study have important implications for the assessments and treatment evaluations of fatigue in MS.</p

Can clinical case discussions foster clinical reasoning skills in undergraduate medical education? A randomised controlled trial

OBJECTIVE Fostering clinical reasoning is a mainstay of medical education. Based on the clinicopathological conferences, we propose a case-based peer teaching approach called clinical case discussions (CCDs) to promote the respective skills in medical students. This study compares the effectiveness of different CCD formats with varying degrees of social interaction in fostering clinical reasoning. DESIGN, SETTING, PARTICIPANTS A single-centre randomised controlled trial with a parallel design was conducted at a German university. Study participants (N=106) were stratified and tested regarding their clinical reasoning skills right after CCD participation and 2 weeks later. INTERVENTION Participants worked within a live discussion group (Live-CCD), a group watching recordings of the live discussions (Video-CCD) or a group working with printed cases (Paper-Cases). The presentation of case information followed an admission-discussion-summary sequence. PRIMARY AND SECONDARY OUTCOME MEASURES Clinical reasoning skills were measured with a knowledge application test addressing the students' conceptual, strategic and conditional knowledge. Additionally, subjective learning outcomes were assessed. RESULTS With respect to learning outcomes, the Live-CCD group displayed the best results, followed by Video-CCD and Paper-Cases, F(2,87)=27.07, p<0.001, partial η2=0.384. No difference was found between Live-CCD and Video-CCD groups in the delayed post-test; however, both outperformed the Paper-Cases group, F(2,87)=30.91, p<0.001, partial η2=0.415. Regarding subjective learning outcomes, the Live-CCD received significantly better ratings than the other formats, F(2,85)=13.16, p<0.001, partial η2=0.236. CONCLUSIONS This study demonstrates that the CCD approach is an effective and sustainable clinical reasoning teaching resource for medical students. Subjective learning outcomes underline the importance of learner (inter)activity in the acquisition of clinical reasoning skills in the context of case-based learning. Higher efficacy of more interactive formats can be attributed to positive effects of collaborative learning. Future research should investigate how the Live-CCD format can further be improved and how video-based CCDs can be enhanced through instructional support

Challenging the Postwar Narrative: The Art and Agenda of Boris Lurie

Art history is shaped, studied, and taught based on narratives, artistic movements, and the biographies of celebrated artists. While contributing to an understanding of prevalent traditions and artists working in those traditions, these narratives are also constructions of inclusion and exclusion that establish art historical placement for certain artists while relegating others to historical obscurity. It is clear what happens to the critical fortunes of artists who are placed within these narratives. Yet what happens to the artists who do not fit within any of the categories established by these constructions? Are they then to be understood as simply minor artists or perhaps even “outsider artists?” Using the example of Boris Lurie and his critical fortune within the context of the standard art historical narrative of American art of the post World War Two period, this thesis argues for an expanded vision of modern and contemporary art that would accommodate lesser-known artists and offer a nuanced understanding of what American art has been after 1945