Lifetime Surface Phosphor Thermometry - Technique Developments, Sources of Error, and Applications

Temperature is not only a fundamental aspect of everyday life, but it is also an essential metric for thermal conversion systems concerning efficiency, emissions, and component lifetime estimation. Therefore, a vast range of different methods to measure temperature has been developed. The technique of focus in this thesis is lifetime-based surface phosphor thermometry.In phosphor thermometry, one leverages the changes in luminescence of materials called phosphors to measure temperature. The spectral changes can result from shifts in the relative intensity of emission lines in the spectra or shifts in emission line wavelength with temperature. In addition, changes in the lifetime of the phosphorescence signal with temperature can be utilized for thermometry. Inorganic phosphors are ceramic substances that can operate with a range of luminescence activators. Most often, the luminescence originates from trivalent lanthanide ions or transition metal ions doped in a host crystal.Surface phosphor thermometry has the benefit of being a remote sensing technique, where one coats a phosphor on a surface of interest. This phosphor coating is then excited with a remote source, for example, pulsed laser light, and the phosphor luminescence is detected remotely. In this thesis, technology developments, error sources analysis, and applications of phosphor thermometry are presented. The technology developments present a high-temperature calibration system and a method for analyzing the lifetime components in the luminescence decay of phosphors in detail. An analysis of upconversion phosphors for temperature measurements beneath thermal barrier coatings and an investigation of high-temperature phosphors to 1900 K and their sensitivities to the oxygen content of the gas environment are also included in this work.The investigations into potential error sources include how PMT nonlinearity effects can influence the measured lifetime with excitation frequency from 10 Hz up to 10 kHz. In addition, the phosphor luminescence decay time impact of high repetition excitation was also investigated and analyzed to see potential error sources for temperature measurements.Phosphor thermometry was also used in two applications, including measuring the surface temperature of burning wood pellets and the surface temperature of a combustion nozzle in a down-scaled gas turbine combustor using hydrogen-enriched methane as fuel

Development of Mechanosensory Innervation in the Frog, Xenopus Laevis

ABSTRACT DEVELOPMENT OF MECHANOSENSORY INNERVATION IN THE FROG, XENOPUS LAEVIS by Peter Feuk The University of Wisconsin-Milwaukee, 2017 Under the Supervision of Dr. R. David Heathcote This study aims to investigate whether a specific target cell in the epidermis of the African clawed frog, Xenopus laevis, guides the initial outgrowth and pattern of Rohon-Beard (RB) cells and their survival. RB cells are primary mechanosensory neurons present during the early developmental stages of X. laevis. These neurons provide sensory input to the frog throughout embryonic and larval development before initiating apoptosis around the start of metamorphosis. The innervation of embryonic skin cells by RB neurons exhibits a distinct pattern that features encircling of a specific subset of epidermal cells. We hypothesize that encircled cells could be a recently discovered cell type that synthesizes and secretes serotonin. To test whether these small secretory cells (SSC) play a chemoattractive role in the innervation of primary mechanosensory neurons in X. laevis, we used a variety of approaches. By immunolabeling RB processes in the epidermis, we established the wild-type pattern and quantity of innervation. Gain of function and loss of function tests utilizing pharmacological modulators of serotonin, directly examined the role of SSCs. We showed that serotonin levels can be over or under expressed, and that there is a quantifiable response of the resulting mechanosensory innervation. RB neurons innervating larval skin lacking serotonin showed a lower frequency of encircled SSCs as well as a greater number of retraction bulbs. We also showed a correlation between behavior and serotonin levels in the epidermis. Loss of function treatments showed a higher proportion of animals failing to respond to stimuli and an increase in inappropriate escape responses. Finally, we related changes in X. laevis larval skin and mechanosensory neurons during metamorphosis to tissue remodeling and the transition to adult sensory function. This research provides insight into axonal guidance and the patterning of mechanosensory innervation in X. laevis. It stands as an example of how the innervation of the vertebrate skin is established and maintained between developmental stages

Global and unbiased detection of splice junctions from RNA-seq data

SplitSeek can be used to detect novel splicing events in SOLiD RNA-seq data without the need for a pre-defined library

Identification of novel exons and transcribed regions by chimpanzee transcriptome sequencing

Background: We profile the chimpanzee transcriptome by using deep sequencing of cDNA from brain and liver, aiming to quantify expression of known genes and to identify novel transcribed regions. Results: Using stringent criteria for transcription, we identify 12,843 expressed genes, with a majority being found in both tissues. We further identify 9,826 novel transcribed regions that are not overlapping with annotated exons, mRNAs or ESTs. Over 80 % of the novel transcribed regions map within or in the vicinity of known genes, and by combining sequencing data with de novo splice predictions we predict several of the novel transcribed regions to be new exons or 3 ' UTRs. For approximately 350 novel transcribed regions, the corresponding DNA sequence is absent in the human reference genome. The presence of novel transcribed regions in five genes and in one intergenic region is further validated with RT-PCR. Finally, we describe and experimentally validate a putative novel multi-exon gene that belongs to the ATP-cassette transporter gene family. This gene does not appear to be functional in human since one exon is absent from the human genome. In addition to novel exons and UTRs, novel transcribed regions may also stem from different types of noncoding transcripts. We note that expressed repeats and introns from unspliced mRNAs are especially common in our data. Conclusions: Our results extend the chimpanzee gene catalogue with a large number of novel exons and 3 ' UTRs an

Copy number variants and selective sweeps in natural populations of the house mouse (Mus musculus domesticus)

Copy–number variants (CNVs) may play an important role in early adaptations, potentially facilitating rapid divergence of populations. We describe an approach to study this question by investigating CNVs present in natural populations of mice in the early stages of divergence and their involvement in selective sweeps. We have analyzed individuals from two recently diverged natural populations of the house mouse (Mus musculus domesticus) from Germany and France using custom, high–density, comparative genome hybridization arrays (CGH) that covered almost 164 Mb and 2444 genes. One thousand eight hundred and sixty one of those genes we previously identified as differentially expressed between these populations, while the expression of the remaining genes was invariant. In total, we identified 1868 CNVs across all 10 samples, 200 bp to 600 kb in size and affecting 424 genic regions. Roughly two thirds of all CNVs found were deletions. We found no enrichment of CNVs among the differentially expressed genes between the populations compared to the invariant ones, nor any meaningful correlation between CNVs and gene expression changes. Among the CNV genes, we found cellular component gene ontology categories of the synapse overrepresented among all the 2444 genes tested. To investigate potential adaptive significance of the CNV regions, we selected six that showed large differences in frequency of CNVs between the two populations and analyzed variation in at least two microsatellites surrounding the loci in a sample of 46 unrelated animals from the same populations collected in field trappings. We identified two loci with large differences in microsatellite heterozygosity (Sfi1 and Glo1/Dnahc8 regions) and one locus with low variation across the populations (Cmah), thus suggesting that these genomic regions might have recently undergone selective sweeps. Interestingly, the Glo1 CNV has previously been implicated in anxiety–like behavior in mice, suggesting a differential evolution of a behavioral trai

"GenotypeColour™": colour visualisation of SNPs and CNVs

<p>Abstract</p> <p>Background</p> <p>The volume of data available on genetic variations has increased considerably with the recent development of high-density, single-nucleotide polymorphism (SNP) arrays. Several software programs have been developed to assist researchers in the analysis of this huge amount of data, but few can rely upon a whole genome variability visualisation system that could help data interpretation.</p> <p>Results</p> <p>We have developed <it>GenotypeColour™ </it>as a rapid user-friendly tool able to upload, visualise and compare the huge amounts of data produced by Affymetrix Human Mapping GeneChips without losing the overall view of the data.</p> <p>Some features of <it>GenotypeColour™ </it>include visualising the entire genome variability in a single screenshot for one or more samples, the simultaneous display of the genotype and Copy Number state for thousands of SNPs, and the comparison of large amounts of samples by producing "consensus" images displaying regions of complete or partial identity. The software is also useful for genotype analysis of trios and to show regions of potential uniparental disomy (UPD). All information can then be exported in a tabular format for analysis with dedicated software. At present, the software can handle data from 10 K, 100 K, 250 K, 5.0 and 6.0 Affymetrix chips.</p> <p>Conclusion</p> <p>We have created a software that offers a new way of displaying and comparing SNP and CNV genomic data. The software is available free at <url>http://www.med.unibs.it/~barlati/GenotypeColour</url> and is especially useful for the analysis of multiple samples.</p

Discovery of Human Inversion Polymorphisms by Comparative Analysis of Human and Chimpanzee DNA Sequence Assemblies

With a draft genome-sequence assembly for the chimpanzee available, it is now possible to perform genome-wide analyses to identify, at a submicroscopic level, structural rearrangements that have occurred between chimpanzees and humans. The goal of this study was to investigate chromosomal regions that are inverted between the chimpanzee and human genomes. Using the net alignments for the builds of the human and chimpanzee genome assemblies, we identified a total of 1,576 putative regions of inverted orientation, covering more than 154 mega-bases of DNA. The DNA segments are distributed throughout the genome and range from 23 base pairs to 62 mega-bases in length. For the 66 inversions more than 25 kilobases (kb) in length, 75% were flanked on one or both sides by (often unrelated) segmental duplications. Using PCR and fluorescence in situ hybridization we experimentally validated 23 of 27 (85%) semi-randomly chosen regions; the largest novel inversion confirmed was 4.3 mega-bases at human Chromosome 7p14. Gorilla was used as an out-group to assign ancestral status to the variants. All experimentally validated inversion regions were then assayed against a panel of human samples and three of the 23 (13%) regions were found to be polymorphic in the human genome. These polymorphic inversions include 730 kb (at 7p22), 13 kb (at 7q11), and 1 kb (at 16q24) fragments with a 5%, 30%, and 48% minor allele frequency, respectively. Our results suggest that inversions are an important source of variation in primate genome evolution. The finding of at least three novel inversion polymorphisms in humans indicates this type of structural variation may be a more common feature of our genome than previously realized

Copy number variations in East-Asian population and their evolutionary and functional implications

Recent discovery of the copy number variation (CNV) in normal individuals has widened our understanding of genomic variation. However, most of the reported CNVs have been identified in Caucasians, which may not be directly applicable to people of different ethnicities. To profile CNV in East-Asian population, we screened CNVs in 3578 healthy, unrelated Korean individuals, using the Affymetrix Genome-Wide Human SNP array 5.0. We identified 144 207 CNVs using a pooled data set of 100 randomly chosen Korean females as a reference. The average number of CNVs per genome was 40.3, which is higher than that of CNVs previously reported using lower resolution platforms. The median size of CNVs was 18.9 kb (range 0.2–5406 kb). Copy number losses were 4.7 times more frequent than copy number gains. CNV regions (CNVRs) were defined by merging overlapping CNVs identified in two or more samples. In total, 4003 CNVRs were defined encompassing 241.9 Mb accounting for ∼8% of the human genome. A total of 2077 CNVRs (51.9%) were potentially novel. Known CNVRs were larger and more frequent than novel CNVRs. Sixteen percent of the CNVRs were observed in ≥1% of study subjects and 24% overlapped with the OMIM genes. A total of 476 (11.9%) CNVRs were associated with segmental duplications. CNVS/CNVRs identified in this study will be valuable resources for studying human genome diversity and its association with disease

An enhanced method for targeted next generation sequencing copy number variant detection using ExomeDepth [version 1; peer review: 1 approved, 1 approved with reservations]

Copy number variants (CNV) are a major cause of disease, with over 30,000 reported in the DECIPHER database. To use read depth data from targeted Next Generation Sequencing (NGS) panels to identify CNVs with the highest degree of sensitivity, it is necessary to account for biases inherent in the data. GC content and ambiguous mapping due to repetitive sequence elements and pseudogenes are the principal components of technical variability. In addition, the algorithms used favour the detection of multi-exon CNVs, and rely on suitably matched normal dosage samples for comparison. We developed a calling strategy that subdivides target intervals, and uses pools of historical control samples to overcome these limitations in a clinical diagnostic laboratory. We compared our enhanced strategy with an unmodified pipeline using the R software package ExomeDepth, using a cohort of 109 heterozygous CNVs (91 deletions, 18 duplications in 26 genes), including 25 single exon CNVs. The unmodified pipeline detected 104/109 CNVs, giving a sensitivity of 89.62% to 98.49% at the 95% confidence interval. The detection of all 109 CNVs by our enhanced method demonstrates 95% confidence the sensitivity is ≥96.67%, allowing NGS read depth analysis to be used for CNV detection in a clinical diagnostic setting