Investigating the 3D chromatin architecture with fluorescence microscopy

Chromatin is an assembly of DNA and nuclear proteins, which on the one hand has the function to properly store the 2 meters of DNA of a diploid human nucleus in a small volume and on the other hand regulates the accessibility of specific DNA segments for proteins. Many cellular processes like gene expression and DNA repair are affected by the three-dimensional architecture of chromatin. Cohesin is an important and well-studied protein that affects three-dimensional chromatin organization. One of the functions of this motor protein is the active generation of specific domain structures (topologically associating domains (TADs)) by the process of loop extrusion. Studies of cohesin depleted cells showed that TAD structures were lost on a population average. Due to this finding, the question arose, to what extent the functional nuclear architecture, that can be detected by confocal and structured illumination microscopy, is impaired when cells were cohesin depleted. The work presented in this thesis could show that the structuring of the nucleus in areas with different chromatin densities including the localization of important nuclear proteins as well as replication patterns was retained. Interestingly, cohesin depleted cells proceeded through an endomitosis leading to the formation of multilobulated nuclei. Obviously, important structural features of chromatin can form even in the absence of cohesin. In the here presented work, fluorescence microscopic methods were used throughout, and an innovative technique was developed, that allows flexible labeling of proteins with different fluorophores in fixed cells. With this technique DNA as well as peptide nucleic acid (PNA) oligonucleotides can be site-specifically coupled to antibodies via the Tub-tag technology and visualized by complementary fluorescently labeled oligonucleotides. The advantages and disadvantages of PNAs as docking strands are discussed in this thesis as well as the use of PNAs in fluorescence in situ hybridization (FISH). In the next study, which is part of this work, a combination of FISH and super-resolution microscopy was used. There it could be shown that DNA segments of 5 kb can form both compact and elongated configurations in regulatory active as well as inactive chromatin. Coarse-grained modeling of these microscopic data, in agreement with published data from other groups, has suggested that elongated configurations occur more frequently in DNA segments in which the occupancy of nucleosomes is reduced. The microscopically measured distance distributions could only be simulated with models that assume different densities of nucleosomes in the population. Another result of this study was that inactive chromatin - as expected - shows a high level of compaction, which can hardly be explained with common coarse-grained models. It is possible that environmental effects that are difficult to simulate play a role here. Chromatin is a highly dynamic structure, and its architecture is constantly changing, be it through active processes such as the effect of cohesin investigated here or through thermodynamic interactions of nucleosomes as they are simulated in coarse-grained models. It will take a long time until we adequately understand these dynamic processes and their interplay

Cybathlon experiences of the Graz BCI racing team Mirage91 in the brain-computer interface discipline

Abstract Background In this work, we share our experiences made at the world-wide first CYBATHLON, an event organized by the Eidgenössische Technische Hochschule Zürich (ETH Zürich), which took place in Zurich in October 2016. It is a championship for severely motor impaired people using assistive prototype devices to compete against each other. Our team, the Graz BCI Racing Team MIRAGE91 from Graz University of Technology, participated in the discipline “Brain-Computer Interface Race”. A brain-computer interface (BCI) is a device facilitating control of applications via the user’s thoughts. Prominent applications include assistive technology such as wheelchairs, neuroprostheses or communication devices. In the CYBATHLON BCI Race, pilots compete in a BCI-controlled computer game. Methods We report on setting up our team, the BCI customization to our pilot including long term training and the final BCI system. Furthermore, we describe CYBATHLON participation and analyze our CYBATHLON result. Results We found that our pilot was compliant over the whole time and that we could significantly reduce the average runtime between start and finish from initially 178 s to 143 s. After the release of the final championship specifications with shorter track length, the average runtime converged to 120 s. We successfully participated in the qualification race at CYBATHLON 2016, but performed notably worse than during training, with a runtime of 196 s. Discussion We speculate that shifts in the features, due to the nonstationarities in the electroencephalogram (EEG), but also arousal are possible reasons for the unexpected result. Potential counteracting measures are discussed. Conclusions The CYBATHLON 2016 was a great opportunity for our student team. We consolidated our theoretical knowledge and turned it into practice, allowing our pilot to play a computer game. However, further research is required to make BCI technology invariant to non-task related changes of the EEG

Cohesin depleted cells pass through mitosis and reconstitute a functional nuclear architecture

The human genome forms thousands of “contact domains”, which are intervals of enhanced contact frequency. Some, called “loop domains” are thought to form by cohesin-mediated loop extrusion. Others, called “compartmental domains”, form due to the segregation of active and inactive chromatin into A and B compartments. Recently, Hi-C studies revealed that the depletion of cohesin leads to the disappearance of all loop domains within a few hours, but strengthens compartment structure. Here, we combine live cell microscopy, super-resolution microscopy, Hi-C, and studies of replication timing to examine the longer-term consequences of cohesin degradation in HCT-116 human colorectal carcinoma cells, tracking cells for up to 30 hours. Surprisingly, cohesin depleted cells proceed through an aberrant mitosis, yielding a single postmitotic cell with a multilobulated nucleus. Hi-C reveals the continued disappearance of loop domains, whereas A and B compartments are maintained. In line with Hi-C, microscopic observations demonstrate the reconstitution of chromosome territories and chromatin domains. An interchromatin channel system (IC) expands between chromatin domain clusters and carries splicing speckles. The IC is lined by active chromatin enriched for RNA Pol II and depleted in H3K27me3. Moreover, the cells exhibit typical early-, mid-, and late- DNA replication timing patterns. Our observations indicate that the functional nuclear compartmentalization can be maintained in cohesin depleted pre- and postmitotic cells. However, we find that replication foci – sites of active DNA synthesis – become physically larger consistent with a model where cohesin dependent loop extrusion tends to compact intervals of replicating chromatin, whereas their genomic boundaries are associated with compartmentalization, and do not change.3D FISH3D fluorescence in situ hybridization3D SIM3D structured illumination microscopyAIDauxin inducible degronANC / INCactive / inactive nuclear compartmentCTchromosome territoryCD(C)chromatin domain (cluster)CTCFCCCTC binding factorDAPI4’,6-diamidino-2-phenylindoleEdU5-Ethynyl-2’-deoxyuridineHi-Cchromosome conformation capturing combined with deep sequencingICinterchromatin compartmentMLNmultilobulated nucleusNCnucleosome clusterPBSphosphate buffered salinePBSTphosphate buffered saline with 0.02% TweenPRperichromatin regionRDreplication domainRLreplication labelingTADtopologically associating domai

Cohesin depleted cells rebuild functional nuclear compartments after endomitosis

Cohesin plays an essential role in chromatin loop extrusion, but its impact on a compartmentalized nuclear architecture, linked to nuclear functions, is less well understood. Using live-cell and super-resolved 3D microscopy, here we find that cohesin depletion in a human colon cancer derived cell line results in endomitosis and a single multilobulated nucleus with chromosome territories pervaded by interchromatin channels. Chromosome territories contain chromatin domain clusters with a zonal organization of repressed chromatin domains in the interior and transcriptionally competent domains located at the periphery. These clusters form microscopically defined, active and inactive compartments, which likely correspond to A/B compartments, which are detected with ensemble Hi-C. Splicing speckles are observed nearby within the lining channel system. We further observe that the multilobulated nuclei, despite continuous absence of cohesin, pass through S-phase with typical spatio-temporal patterns of replication domains. Evidence for structural changes of these domains compared to controls suggests that cohesin is required for their full integrity

Vaginal Delivery Is Associated with Neurochemical Evidence of Increased Neuroaxonal Remodelling in Infants from the KUNO-Kids Health Study: Cross-Sectional Analysis

Aim: Little is known about neonatal brain plasticity or the impact of birth mode on neurointegrity. As a reflection of neuroaxonal damage, the neuronal structural protein neurofilament light chain (NfL) has emerged as a highly specific biomarker. Our purpose was to test the hypothesis that vaginal delivery is associated with increased NfL in neonates. Methods: NfL concentrations were measured using single-molecule array immunoassay in umbilical cord serum from healthy term neonates enrolled in the prospective KUNO-Kids Health Study. NfL values were investigated for independent influencing factors using linear and logistic models, followed by post hoc propensity score-matching. Results: Of 665 neonates, n = 470 (70.7%) were delivered vaginally and n = 195 (29.3%) by cesarean section. Median serum NfL was significantly higher after vaginal delivery 14.4 pg/mL (11.6–18.5) compared to primary 7.5 pg/mL (6.1–8.9) and secondary cesarean delivery 9.3 pg/mL (7.5–12.0). Multivariable logistic regression models showed delivery mode and gestational age to be independently associated with NfL. Propensity score-matching analysis confirmed that assisted vaginal delivery generated higher NfL compared to vaginal (non-assisted), while lowest levels were associated with cesarean section. Interpretation: Our data confirm the significant impact of birth mode on neonatal NfL levels. The persistence of these differences and their potential long-term impact have yet to be investigated

Dietary vitamin K intake in relation to cancer incidence and mortality: results from the Heidelberg cohort of the European Prospective Investigation into Cancer and Nutrition (EPIC-Heidelberg)

BACKGROUND: Anticarcinogenic activities of vitamin K have been observed in animal and cell studies. OBJECTIVE: On the basis of the growth inhibitory effects of vitamin K as observed in a variety of cancer cell lines, we hypothesized that dietary intake of phylloquinone (vitamin K(1)) and menaquinones (vitamin K(2)) may be associated with overall cancer incidence and mortality. DESIGN: In the prospective EPIC-Heidelberg (European Prospective Investigation into Cancer and Nutrition-Heidelberg) cohort study, 24,340 participants aged 35-64 y and free of cancer at enrollment (1994-1998) were actively followed up for cancer incidence and mortality through 2008. Dietary vitamin K intake was estimated from food-frequency questionnaires completed at baseline by using HPLC-based food-composition data. Multivariate-adjusted hazard ratios (HRs) and 95% CIs were estimated by using Cox proportional hazards models. RESULTS: During a median follow-up time of >10 y, 1755 incident cancer cases occurred, of which 458 were fatal. Dietary intake of menaquinones was nonsignificantly inversely associated with overall cancer incidence (HR for the highest compared with the lowest quartile: 0.86; 95% CI: 0.73, 1.01; P for trend = 0.08), and the association was stronger for cancer mortality (HR: 0.72; 95% CI: 0.53, 0.98; P for trend = 0.03). Cancer risk reduction with increasing intake of menaquinones was more pronounced in men than in women, mainly driven by significant inverse associations with prostate (P for trend = 0.03) and lung (P for trend = 0.002) cancer. We found no association with phylloquinone intake. CONCLUSION: These findings suggest that dietary intake of menaquinones, which is highly determined by the consumption of cheese, is associated with a reduced risk of incident and fatal cancer

Optimasi Portofolio Resiko Menggunakan Model Markowitz MVO Dikaitkan dengan Keterbatasan Manusia dalam Memprediksi Masa Depan dalam Perspektif Al-Qur`an

Risk portfolio on modern finance has become increasingly technical, requiring the use of sophisticated mathematical tools in both research and practice. Since companies cannot insure themselves completely against risk, as human incompetence in predicting the future precisely that written in Al-Quran surah Luqman verse 34, they have to manage it to yield an optimal portfolio. The objective here is to minimize the variance among all portfolios, or alternatively, to maximize expected return among all portfolios that has at least a certain expected return. Furthermore, this study focuses on optimizing risk portfolio so called Markowitz MVO (Mean-Variance Optimization). Some theoretical frameworks for analysis are arithmetic mean, geometric mean, variance, covariance, linear programming, and quadratic programming. Moreover, finding a minimum variance portfolio produces a convex quadratic programming, that is minimizing the objective function ðð¥with constraintsð ð 𥠥 ðandð´ð¥ = ð. The outcome of this research is the solution of optimal risk portofolio in some investments that could be finished smoothly using MATLAB R2007b software together with its graphic analysis

Search for heavy resonances decaying to two Higgs bosons in final states containing four b quarks

A search is presented for narrow heavy resonances X decaying into pairs of Higgs bosons (H) in proton-proton collisions collected by the CMS experiment at the LHC at root s = 8 TeV. The data correspond to an integrated luminosity of 19.7 fb(-1). The search considers HH resonances with masses between 1 and 3 TeV, having final states of two b quark pairs. Each Higgs boson is produced with large momentum, and the hadronization products of the pair of b quarks can usually be reconstructed as single large jets. The background from multijet and t (t) over bar events is significantly reduced by applying requirements related to the flavor of the jet, its mass, and its substructure. The signal would be identified as a peak on top of the dijet invariant mass spectrum of the remaining background events. No evidence is observed for such a signal. Upper limits obtained at 95 confidence level for the product of the production cross section and branching fraction sigma(gg -> X) B(X -> HH -> b (b) over barb (b) over bar) range from 10 to 1.5 fb for the mass of X from 1.15 to 2.0 TeV, significantly extending previous searches. For a warped extra dimension theory with amass scale Lambda(R) = 1 TeV, the data exclude radion scalar masses between 1.15 and 1.55 TeV

Search for supersymmetry in events with one lepton and multiple jets in proton-proton collisions at root s=13 TeV

Peer reviewe

Measurement of the top quark mass using charged particles in pp collisions at root s=8 TeV

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