Functional characterization of the Saccharomyces cerevisiae chromatin remodeler INO80

Knowing the explicit locations of nucleosomes in a genome is a pre-requisite for understanding the regulation of genes. Predominantly at regulatory active promoter sites, regular spaced arrays phased at reference points shape the chromatin landscape. In eukaryotic cells ATP-dependent chromatin remodeler align nucleosomes at reference points and are pivotal in the formation of the stereotyped promoter pattern. Chromatin remodeler of the ISWI, CHD, SWI/SNF and INO80 family convert energy derived from ATP hydrolysis to operate on their nucleosomal substrates to accomplish nucleosome spacing, eviction and editing reactions. Recent structural elucidations provided mechanistic insights into how chromatin remodelers engage their nucleosomal substrates (Eustermann et al., 2018, Aramayo et al., 2018, Willhoft et al., 2018, Ayala et al., 2018, Farnung et al., 2017, Wagner et al., 2020, Yan et al., 2019, He et al., 2020, Han et al., 2020) and brought about a unifying DNA wave mechanism underpinning ATP-dependent DNA translocation by chromatin remodeling complexes (Yan and Chen, 2020). Understanding how phased arrays of equally spaced nucleosomes are generated by chromatin remodelers represents an ultimate long-term goal in chromatin biology. What remains unclear is the underlying mechanism that directs nucleosome positioning by chromatin remodelers in absolute terms. How do ATP-dependent chromatin remodelers generate phased arrays of regularly spaced nucleosomes? How are the distances between nucleosomes and phasing sites and between adjacent nucleosomes set? Is DNA shape read-out part of nucleosome positioning driven by chromatin remodelers? Do remodelers have intrinsic ruler-like elements that set spacing and phasing distances? The aim of this thesis was to delineate whether, and if so, what type of genomic information is read by a remodeler in the stereotypic placement of nucleosomes at physiological sites, and how the remodeler activities fit into the unifying framework of ATP-dependent DNA translocation mechanism of chromatin remodelers. To gain an insight into nucleosome positioning driven by Saccharomyces cerevisiae (S.c.) ATP-dependent chromatin remodelers, a combination of a minimalistic genome-wide in vitro reconstitution system, biochemical analysis, high-resolution structures and structure-guided mutagenesis of the S.c. INO80 model system was applied. Findings of this work would have an impact on the mechanistic understanding of nucleosome positioning driven by ATP dependent chromatin remodelers based on the ruler concept that has been described earlier for the ISW1a chromatin remodeler (Yamada et al., 2011). The ISW1a, Chd1 and ISW2 remodelers demonstrated “clamping” activity and used ruler elements to set 1 Abstract distances with a defined linker length (21-26 bp at all densities, 12-13bp at all densities, 54-58 bp at low/medium densities, respectively). Mutagenesis of the INO80 model system identified and tuned the INO80 ruler element, which is comprised of the Ino80_HSA domain of the ARP module, the NHP10 module and Ino80 N-terminal residues. Regularly spaced symmetrical arrays were generated at the Reb1 reference point sites as well as at BamHI-introduced dsDNA break sites. Nucleosome positioning on the genomic sequences of S. c., Schizosaccharomyces pombe (S.p.) as well as Escherichia coli (E.coli) showed no significant differences. Mutagenesis of residues located within the Ino80_HSA domain established a causal link between nucleosome positioning by INO80 and DNA shape read-out by the INO80_HSA domain. The spacing and phasing distances generated by ATP-dependent chromatin remodelers point towards a remodeler-intrinsic ruler activity that is independent of underlying DNA sequences and can be sensitive to nucleosome density. This study measured linker lengths set by remodeler-intrinsic ruler-like functionalities in absolute terms, which will be instrumental to dissect contributions from individual remodelers in nucleosome positioning in vivo. This provides the starting point to understand how remodeler-driven nucleosome dynamics direct stable steady-state nucleosome positions relative to DNA bound factors, DNA ends and DNA sequence elements. Sequence-dependent DNA shape features have been mainly associated with binding of transcription factors as well as general regulatory factors and more static DNA binding events. This study augments the general description of nucleosome positioning sequences for chromatin remodelers by establishing nucleosome positioning motifs based on DNA shape analysis. This study provides an intriguing framework to implement DNA shape read-out in the tracking mechanism of DNA-translocating machineries

Sudden Cardiac Death Risk in Downhill Skiers and Mountain Hikers and Specific Prevention Strategies

Sudden cardiac death (SCD) still represents an unanticipated and catastrophic event eliciting from cardiac causes. SCD is the leading cause of non-traumatic deaths during downhill skiing and mountain hiking, related to the fact that these sports are very popular among elderly people. Annually, more than 40 million downhill skiers and mountain hikers/climbers visit mountainous regions of the Alps, including an increasing number of individuals with pre-existing chronic diseases. Data sets from two previously published case-control studies have been used to draw comparisons between the SCD risk of skiers and hikers. Data of interest included demographic variables, cardiovascular risk factors, medical history, physical activity, and additional symptoms and circumstances of sudden death for cases. To establish a potential connection between the SCD risk and sport-specific physical strain, data on cardiorespiratory responses to downhill skiing and mountain hiking, assessed in middle-aged men and women, have been included. It was demonstrated that previous myocardial infarction (MI) (odds ratio; 95% CI: 92.8; 22.8–379.1; p < 0.001) and systemic hypertension (9.0; 4.0–20.6; p < 0.001) were predominant risk factors for SCD in skiers, but previous MI (10.9; 3.8–30.9; p < 0.001) and metabolic disorders like hypercholesterolemia (3.4; 2.2–5.2; p < 0.001) and diabetes (7.4; 1.6–34.3; p < 0.001) in hikers. More weekly high-intensity exercise was protective in skiers (0.17; 0.04–0.74; p = 0.02), while larger amounts of mountain sports activities per year were protective in hikers (0.23; 0.1–0.4; <0.001). In conclusion, previous MI history represents the most important risk factor for SCD in recreational skiers and hikers as well, and adaptation to high-intensity exercise is especially important to prevent SCD in skiers. Moreover, the presented differences in risk factor patterns for SCDs and discussed requirements for physical fitness in skiers and hikers will help physicians to provide specifically targeted advice

Self-attraction effect and correction on three absolute gravimeters

The perturbations of the gravitational field due to the mass distribution of an absolute gravimeter have been studied. The so called Self Attraction Effect (SAE) is crucial for the measurement accuracy, especially for the International Comparisons, and for the uncertainty budget evaluation. Three instruments have been analysed: MPG-2, FG5-238 and IMPG-02. The SAE has been calculated using a numerical method based on FEM simulation. The observed effect has been treated as an additional vertical gravity gradient. The correction (SAC) to be applied to the computed g value has been associated with the specific height level, where the measurement result is typically reported. The magnitude of the obtained corrections is of order 1E-8 m/s2.Comment: 14 pages, 8 figures, submitted to Metrologi

Strukturelles und funktionelles kardiales Remodeling bei Teilnehmern eines Gewichtsabnahmeprogrammes mit und ohne Schlafapnoe

Adipositas stellt den Hauptrisikofaktor für die Entwicklung einer obstruktiven Schlafapnoe (OSA) dar. Außerdem sind Adipositas und OSA mit Veränderungen der kardialen Funktion und Morphologie assoziiert. Gewichtsabnahme und die Therapie der OSA mit CPAP sollen zu einer Verbesserung des kardialen Remodeling führen. Unbekannt hierbei ist, inwiefern die OSA bei Patienten mit starker Adipositas das linkventrikuläre Remodeling beeinflusst. Außerdem stellt sich die Frage, ob die Gewichtsreduktion und die damit verbundene Verbesserung der OSA einen Einfluss auf das kardiale Remodeling haben bzw. die AHI-Senkung, unabhängig von der Gewichtsreduktion, eine Verbesserung der kardialen Funktion mit sich bringt. In unserer Studie ist das strukturelle kardiale Remodeling bei Adipösen mit schwerer OSA mit dem hohen Körpergewicht assoziiert, nicht mit der OSA per se. Weder erfolgreiche Gewichtsreduktion, noch eine damit verbundene Besserung der OSA konnten eine Verbesserung der linksventrikulären Struktur und Funktion bewirken

Creation of a 3D Model of the Church of St. Nicholas in Kašperské Hory

Diplomová práce se zabývá kombinací terestrického 3D skenování a fotogrammetrie, pro vytvoření texturovaného 3D modelu. Práce obsahuje teoretickou část, zabývající se principy a funkcemi pozemních laserových skenerů, GNSS a fotogrammetrie. V druhé polovině popisuji průběh, zpracování a kombinaci obou metod v programech Leica Cyclone REGISTER a Reality Capture. Jako zaměřovaný objekt byl použit kostel sv. Mikuláše u Kašperských Hor vzhledem k jeho kulturní hodnotě.The Diploma thesis is about combinig terrestrial 3D scanning and photogrammetry, to create textured 3D model. The thesis contains theoretical part, about principles and functions of terrestrial laser scanners, GNSS, and photogrammetry. In second half I‘m describing meassuring in terrain, processing and combination of both methods with using Leica Cyclone REGISTER and Reality Capture. As measured object was used church of sv. Mikuláše near Kašperské Hory due to it's cultural value.544 - Katedra geodézie a důlního měřictvívýborn

Functional characterization of the Saccharomyces cerevisiae chromatin remodeler INO80

Knowing the explicit locations of nucleosomes in a genome is a pre-requisite for understanding the regulation of genes. Predominantly at regulatory active promoter sites, regular spaced arrays phased at reference points shape the chromatin landscape. In eukaryotic cells ATP-dependent chromatin remodeler align nucleosomes at reference points and are pivotal in the formation of the stereotyped promoter pattern. Chromatin remodeler of the ISWI, CHD, SWI/SNF and INO80 family convert energy derived from ATP hydrolysis to operate on their nucleosomal substrates to accomplish nucleosome spacing, eviction and editing reactions. Recent structural elucidations provided mechanistic insights into how chromatin remodelers engage their nucleosomal substrates (Eustermann et al., 2018, Aramayo et al., 2018, Willhoft et al., 2018, Ayala et al., 2018, Farnung et al., 2017, Wagner et al., 2020, Yan et al., 2019, He et al., 2020, Han et al., 2020) and brought about a unifying DNA wave mechanism underpinning ATP-dependent DNA translocation by chromatin remodeling complexes (Yan and Chen, 2020). Understanding how phased arrays of equally spaced nucleosomes are generated by chromatin remodelers represents an ultimate long-term goal in chromatin biology. What remains unclear is the underlying mechanism that directs nucleosome positioning by chromatin remodelers in absolute terms. How do ATP-dependent chromatin remodelers generate phased arrays of regularly spaced nucleosomes? How are the distances between nucleosomes and phasing sites and between adjacent nucleosomes set? Is DNA shape read-out part of nucleosome positioning driven by chromatin remodelers? Do remodelers have intrinsic ruler-like elements that set spacing and phasing distances? The aim of this thesis was to delineate whether, and if so, what type of genomic information is read by a remodeler in the stereotypic placement of nucleosomes at physiological sites, and how the remodeler activities fit into the unifying framework of ATP-dependent DNA translocation mechanism of chromatin remodelers. To gain an insight into nucleosome positioning driven by Saccharomyces cerevisiae (S.c.) ATP-dependent chromatin remodelers, a combination of a minimalistic genome-wide in vitro reconstitution system, biochemical analysis, high-resolution structures and structure-guided mutagenesis of the S.c. INO80 model system was applied. Findings of this work would have an impact on the mechanistic understanding of nucleosome positioning driven by ATP dependent chromatin remodelers based on the ruler concept that has been described earlier for the ISW1a chromatin remodeler (Yamada et al., 2011). The ISW1a, Chd1 and ISW2 remodelers demonstrated “clamping” activity and used ruler elements to set 1 Abstract distances with a defined linker length (21-26 bp at all densities, 12-13bp at all densities, 54-58 bp at low/medium densities, respectively). Mutagenesis of the INO80 model system identified and tuned the INO80 ruler element, which is comprised of the Ino80_HSA domain of the ARP module, the NHP10 module and Ino80 N-terminal residues. Regularly spaced symmetrical arrays were generated at the Reb1 reference point sites as well as at BamHI-introduced dsDNA break sites. Nucleosome positioning on the genomic sequences of S. c., Schizosaccharomyces pombe (S.p.) as well as Escherichia coli (E.coli) showed no significant differences. Mutagenesis of residues located within the Ino80_HSA domain established a causal link between nucleosome positioning by INO80 and DNA shape read-out by the INO80_HSA domain. The spacing and phasing distances generated by ATP-dependent chromatin remodelers point towards a remodeler-intrinsic ruler activity that is independent of underlying DNA sequences and can be sensitive to nucleosome density. This study measured linker lengths set by remodeler-intrinsic ruler-like functionalities in absolute terms, which will be instrumental to dissect contributions from individual remodelers in nucleosome positioning in vivo. This provides the starting point to understand how remodeler-driven nucleosome dynamics direct stable steady-state nucleosome positions relative to DNA bound factors, DNA ends and DNA sequence elements. Sequence-dependent DNA shape features have been mainly associated with binding of transcription factors as well as general regulatory factors and more static DNA binding events. This study augments the general description of nucleosome positioning sequences for chromatin remodelers by establishing nucleosome positioning motifs based on DNA shape analysis. This study provides an intriguing framework to implement DNA shape read-out in the tracking mechanism of DNA-translocating machineries

Improved Oxygen Delivery During Cardiopulmonary Resuscitation with Interposed Abdominal Compressions

The ability of a new modification of cardiopulmonary resuscitation (CPR) to deliver oxygen to tissues was evaluated. The method utilizes standard CPR techniques with the addition of manual abdominal compressions (100 mm Hg) interposed between chest compressions, and is termed interposed abdominal compression-CPR (IAC-CPR). Oxygen delivery was measured by a spirometer in a closed circuit designed to permit positive-pressure ventilation synchronized with mechanical chest compression. Ventricular fibrillation was induced electrically in 10 anesthetized dogs. In each dog, trials of IAC-CPR and standard CPR were alternated every five minutes during a 30-minute period. Arterial and central venous blood pressures, oxygen consumption, and Fick cardiac output were monitored. The addition of interposed abdominal compression significantly (P \u3c 0.01) increased each of these hemodynamic indicators. Oxygen delivery increased from 4.12 0.39 ml O2/kg/min during standard CPR to 6.37 0.35 ml O2/kg/min during IAC-CPR. Arterial systolic blood pressure increased from 67 5 mm Hg to 90 5 mm Hg, while diastolic arterial blood pressure rose from 15 2 mm Hg to 33 3 mm Hg. Cardiac output increased from 19.9 2.6 ml/min/kg to 37.5 2.7 ml/min/kg

Cardiopulmonary resuscitation with interposed abdominal compression in dogs

This study was conducted to evaluate the hemodynamic effectiveness of a new modification of cardiopulmonary resuscitation (CPR), termed interposed abdominal compression- CPR (IAC-CPR). IAC-CPR utilizes all the steps of standard CPR with the addition of abdominal compressions interposed during the release phase of chest compression. Ventricular fibrillation was induced electrically in 10 anesthetized dogs, and either IAC-CPR or standard CPR was initiated while arterial and venous blood pressures and cardiac output were monitored. The two CPR methods were alternated every three minutes over a period of thirty minutes. The addition of interposed abdominal compressions to standard CPR improved arterial pressures and perfusion in 10/10 dogs. Brachial arterial blood pressure averaged 87/32 mmHg during IAC-CPR vs. 58/16 mmHg during standard CPR. Cardiac output (±S.E.) averaged 24.2 ±5.7 ml/min/kg during IAC-CPR vs. 13.8 ±2.6 ml/min/kg during standard CPR. IAC-CPR requires no extra mechanical equipment, and, if proven effective in human trials, may improve resuscitation success in the field and in the hospital

Weltweit unterwegs: Helen Schwenken führt in die sozialwissenschaftliche Migrationsforschung ein

Helen Schwenken: Globale Migration: Zur Einführung. Hamburg: Junius Verlag 2018. 978-3-88506-805-