Measuring primate gene expression evolution using high throughput transcriptomics and massively parallel reporter assays

A key question in biology is how one genome sequence can lead to the great cellular diversity present in multicellular organisms. Enabled by he sequencing revolution, RNA sequencing (RNA-seq) has emerged as a central tool to measure transcriptome-wide gene expression levels. More recently, single cell RNA-seq was introduced and is becoming a feasible alternative to the more established bulk sequencing. While many different methods have been proposed, a thorough optimisation of established protocols can lead to improvements in robustness, sensitivity, scalability and cost effectiveness. Towards this goal, I have contributed to optimizing the single cell RNA-seq method "Single Cell RNA Barcoding and sequencing" (SCRB-seq) and publishing an improved version that uses optimized reaction conditions and molecular crowding (mcSCRB-seq). mcSCRB-seq achieves higher sensitivity at lower cost per cell and shows the highest RNA capture rate when compared with other published methods. We next sought the direct comparison to other scRNA-seq protocols within the Human Cell Atlas (HCA) benchmarking effort. Here we used mcSCRB-seq to profile a common reference sample that included heterogeneous cell populations from different sources. Transfer of the acquired knowledge on single cell RNA sequencing methods to bulk RNA-seq, led to the development of the prime-seq protocol. A sensitive, robust and cost-efficient bulk RNA-seq protocol that can be performed in any molecular biology laboratory. We compared the data generated, using the prime-seq protocol to the gold standard method TruSeq, using power simulations and found that the statistical power to detect differentially expressed genes is comparable, at 40-fold lower cost. While gene expression is an informative phenotype, the regulation that leads to the different phenotypes is still poorly understood. A state-of-the-art method to measure the activity of cis-regulatory elements (CRE) in a high throughput fashion are Massively Parallel Reporter Assays (MPRA). These assays can be used to measure the activity of thousands of cis-Regulatory Elements (CRE) in parallel. A good way to decode the genotype to phenotype conundrum is using evolutionary information. Cross-species comparisons of closely related species can help understand how particular diverging phenotypes emerged and how conserved gene regulatory programs are encoded in the genome. A very useful tool to perform comparative studies are cell lines, particularly induced Pluripotent Stem Cells (iPSCs). iPSCs can be reprogrammed from different primary somatic cells and are per definition pluripotent, meaning they can be differentiated into cells of all three germlayers. A main challenge for primate research is to obtain primary cells. To this end I contributed to establishing a protocol to generate iPSCs from a non-invasive source of primary cells, namely urine. By using prime-seq we characterized the primary Urine Derived Stem Cells (UDSCs) and the reprogrammed iPSCs. Finally, I used an MPRA to measure activity of putative regulatory elements of the gene TRNP1 across the mammalian phylogeny. We found co-evolution of one particular CRE with brain folding in old world monkeys. To validate the finding we looked for transcription factor binding sites within the identified CRE and intersected the list with transcription factors confirmed to be expressed in the cellular system using prime-seq. In addition we found that changes in the protein coding sequence of TRNP1 and neural stem cell proliferation induced by TRNP1 orthologs correlate with brain size. In summary, within my doctorate I developed methods that enable measuring gene expression and gene regulation in a comparative genomics setting. I further applied these methods in a cross mammalian study of the regulatory sequences of the gene TRNP1 and its association with brain phenotypes

Effect of composition changes on the structure and properties of phosphate glasses in the pyrophosphate region

The structures of phosphate glasses in the ultra- and polyphosphate region are well-investigated. Discontinuous changes of the properties in the composition ränge around 50 mol% P₂O₅ (transition from network to chain-like structures) are known. The present work is concerned with the structural transition of chain-like phosphate glasses to pyrophosphate glasses. ³¹P MAS N M R in vestigations were used for the quantitative structural analyses of pyrophosphate and mixed pyrophosphate/orthophosphate glasses. Several chemical and physical properties of these glasses were determined in dependence on composition and structure. The in vestigated phosphate invert glasses are very sensitive to additives. Therefore, some important properties of phosphate invert glasses and glass-ceramics can be controlled by addition of relatively small and highly charged cations

Phosphate glasses and glass-ceramics for medical applications

Modified calcium phosphate glass Systems meet the requirements for the development of different biomaterials. Bioactive, longterm stable phosphate glass-ceramics and resorbable phosphate glasses were prepared in the Systems P₂O₅ - CaO - Al₂O₃ - Na₂O and P₂O₅ - CaO - MgO - Na₂O . Due to their high thermal expansion coefficients the bioactive phosphate glass-ceramics are excellently suited for coating metals. Using a salt-sintering process, porous resorbable glasses can be obtained. The properties of glassceramic coatings on cobalt/chromium alloys and the production of porous resorbable phosphate invert glasses are described

Effect of TiO2 , ZrO2 and ZnO addition on the hydrolytic durability, viscosity and crystallization behaviour of fibre glasses

Glasses of the System Na2O-K2O-MgO-CaO-BaO-AI2O3-SiO2-B2O3 with additions of TiO2, ZrO2 and ZnO were melted from the raw materials. The glasses were characterized with respect to their hydrolytic durabilities, the working ranges (difference of temperatures attributed to viscosities of 10^2 and 10^4 dPa s), the temperatures attributed to the working point (η = 10^3.5 dPa s), and the crystal growth velocities. In the most cases, the additions led to an increase in viscosity and to a slight decrease in the workability range. The crystal growth velocities decreased with the addition of Ti02 and/or Zr02, while they increased if adding ZnO. The addition of ZnO or of TiO2 and ZrO2 resulted in better chemical durability

Incentive spirometry versus active cycle of breathing technique: effect on chest expansion and flow rates in post abdominal surgery patients

Background: The patients undergoing abdominal surgery has characteristic post-operative mechanical abnormality in respiration like restrictive pattern of ventilation. Spontaneous deep breaths to restore functional residual capacity are abolished by pain. Incentive spirometry (IS) promotes frequent maximum inspiratory effort and is used for the prophylaxis and treatment of respiratory complications in post-surgery wards. Aim of the study was to compare the effect of incentive spirometry (IS) versus active cycle of breathing technique (ACBT) on flow rates and chest expansion in patients following abdominal surgery.Methods: It was prospective comparative interventional study. 90 patients posted for abdominal surgery and satisfying the inclusion criteria were included in the study. They were randomly divided through simple random sampling into two groups. Group A-IS group and Group B-ACBT group. Outcome measures were recorded as Peak inspiratory flow rate [PIFR], Peak expiratory flow rate [PEFR], Forced expiratory volume in 1 sec [FEV1] and chest expansion on pre and post-operative day five. Comparison of pre and post-operative day 1 and pre and post-operative day 5 was done using Wilcoxon signed Ranks Test for both group A and group B, further post hoc analysis was done by Tukey’s test at significance level of p<0.05. Inter as well as Intra group comparison was done. The comparison between group A and B for all the parameters was done by Mann- Whitney U test. At Statistical level of significance for Mann- Whitney U p<0.001.Results: Both IS and ACBT improve the peak flow rates and chest expansion in post abdominal surgery patients. Active cycle of breathing techniques is better technique compared to incentive spirometry in post abdominal surgery patients.Conclusions: ACBT (active cycle of breathing technique) has better results than incentive spirometry in post abdominal surgery cases

Chemical solubility of phosphate glasses in the system Na2O-CaO-MgO-P2O5-Al2O3-TiO2 in aqueous solutions of different pH values

Phosphate glasses of different composition ranges show very different solubility behaviour in aqueous solutions of pH 4 and 5.8. With inereasing P2O5 concentrations between 38 and 52 mol% , in the system Na2O-CaO-MgO-P2O5-Al2O3-TiO2 , the dissolution rate generally increases, however, the most notable increase is observed at P2O5 concentrations larger than 48 mol%. Additives of TiO2 and AI2O3 increase the stability to corrosion. Glasses with compositions in the metaphosphate range show larger dissolution rates in slightly acidic medium than glasses near the invert glass range. Glasses in the invert glass range show a stronger increase in the dissolution rates with decreasing pH value and also deceleration of the dissolution with time

Crystallization and vicosity of phosphate melts in the system Na2O-CaO-MgO-P2O5-Al2O3-TiO2

Glasses in the system Na2O-CaO-MgO-P2O5-Al2O3-TiO2 were studied with respeet to their crystallization and viscosity. Glasses in the metaphosphate range are composed of phosphate chains, and possess a comparably small tendeney to crystallization. Here, AlPO4 in a cristobalite-like modification and Ca2P2O7 are formed. Glasses in the intermediate range between metaphosphate and invert glass structures are composed of smaller phosphate units and show higher erystal growth velocities as well as higher viscosities. Additions of MgO, AI2O3 and TiO2 result in a strengthening of the glass network, and hence, inereasing viscosities and a notably smaller tendeney to crystallization

ETA og forsøket på å radikalisere baskiske identifiseringsprosesser på 1970-tallet

ETA har vært en av Baskerlands mest kjente representanter utad, og sørget for at området ofte har blitt forbundet med radikal nasjonalisme. Denne teksten tar for seg perioden der organisasjonen var på sitt mest innflytelsesrike, og med dette utgangspunkt vil den utforske hvordan ETA forsøkte å påvirke baskisk kollektiv identitet.Master i HistorieMAHF-HISHIS35

Bioactive coatings of glass-ceramics on metals

To combine the mechanical properties of high-strength base metals with the biological properties of bioactive materials, coatings of BIOVERIT® glasses and glass-ceramics on CoCr alloys and titanium have been produced. Different kinds of coating processes have been used: dipping, sputtering, plasma spraying, sintering and pasting. Dipping and pasting seem not to be suitable to produce layers because of cracks and low adhesion strength of the coatings (dipping) and the limitations in relation to the implant shape and the thickness of the layers (pasting). Using sputter techniques it is possible to produce dense layers ( < 10µim) with a high adhesion strength. Plasma spraying resuhed in layers with a thickness of 50 to 300µim, but during the plasma spraying process there is a partial evaporation of the highly volatile glass components. Sintering processes are very suitable to produce layers with a high adhesion strength; these layers are long term stable in simulated body fluid