Development of a new crimp-quality-monitoring system for manually operated tools

This thesis is concerned with the development of a novel technical solution that is aimed to improve the quality of cable crimp terminations in the electronics industry. The automotive, aero and military vehicle industries are of special interest due to their stringent requirements for guaranteed quality assurances in relation to their safety-critical products. The emphasis is on handheld crimping tools, where a new scheme for measuring the relevant crimp force is proposed using a small embedded computer to process information; so as to make an on-instrument statement regarding the crimp process quality. The thesis includes a detailed review of the current technical practices in the cable termination business, with a particular emphasis on the quality estimations for crimped connections using the crimp force monitoring (CFM) scheme. A comprehensive study of the state-of-the-art crimp force monitoring schemes and technologies is presented. A gap in the tool chain is identified whereby the industry has a preference for the use of hand tools in some selected processes; but the CFM scheme cannot be currently integrated directly with such hand tools. The bulk of the work in this thesis is thus focused on the development of a technical solution that will address this need for integration of hand tools with the CFM scheme. The concept is to develop a fully embedded subsystem that can be attached directly to a hand tool and this system will perform all of the signal processing and the statistical analysis so that each crimped joint can be trusted to meet the relevant stated quality parameters. In the course of development work a fully functional prototype subsystem is developed for the CFM solution, for application to hand tools. Analytical and statistical measurement and assessment algorithms were developed to a standard, which allows a final quality rating to be applied to crimped joint connections. Such algorithms are designed to be embedded directly onto the instrument’s microcomputer. The new system has been evaluated and tested in the course of this research project and it has currently been accepted by a number of industries for a planned volume qualification assessment

Anatomy and neurophysiological data for "Vocal learning-associated convergent evolution in mammalian proteins and regulatory elements"

Dataset and companion notebook in Matlab for reproducing the neuroanatomy and neurophysiological result figures in "Vocal learning-associated convergent evolution in mammalian proteins and regulatory elements" by:Morgan E. Wirthlin1,2†‡, Tobias A. Schmid3†, Julie E. Elie3,4†, Xiaomeng Zhang1§, Amanda Kowalczyk1,2, Ruby Redlich, Varvara A. Shvareva5, Ashley Rakuljic5, Maria B. Ji6, Ninad S. Bhat5, Irene M. Kaplow1,2, Daniel E. Schäffer1¶, Alyssa J. Lawler2,7§, Andrew Z. Wang1, BaDoi N. Phan1,2, Siddharth Annaldasula1, Ashley R. Brown1,2, Tianyu Lu1, Byungkook Lim8, Eiman Azim9, Nathan L. Clark10, Wynn K. Meyer11, Sergei L Kosakovsky Pond12, Maria Chikina, Michael M. Yartsev3,4#, Andreas R. Pfenning1,2*#Affiliations:1Department of Computational Biology, Carnegie Mellon University; Pittsburgh, PA 15213, USA.2Neuroscience Institute, Carnegie Mellon University; Pittsburgh, PA 15213, USA.3Helen Wills Neuroscience Institute, University of California, Berkeley; Berkeley, CA 94708, USA4Department of Bioengineering, University of California, Berkeley; Berkeley, CA 94708, USA.5Department of Molecular and Cell Biology, University of California, Berkeley; Berkeley, CA 94708, USA.6Department of Psychology, University of California, Berkeley; Berkeley, CA 94708, USA.7Department of Biological Sciences, Carnegie Mellon University; Pittsburgh, PA 15213, USA.8Neurobiology section, Division of Biological Science, University of California, San Diego; La Jolla, CA 92093, USA.9Molecular Neurobiology Laboratory, Salk Institute for Biological Studies; La Jolla, CA 92037, USA10Department of Biological Sciences, University of Pittsburgh; Pittsburgh, PA 15213, USA.11Department of Biological Sciences, Lehigh University; Bethlehem, PA 18015, USA.12Department Of Biology, Temple University, Philadelphia, PA 19122, USA.*Corresponding author. Email: [email protected].†These authors contributed equally to this work‡Present address: Allen Institute for Brain Science; Seattle, WA 98109, USA.§Present address: Broad Institute, Massachusetts Institute of Technology; Cambridge, MA 02142, USA.¶Present Address: Massachusetts Institute of Technology; Cambridge, MA 02139#These authors jointly supervised the workZoonomia Consortium Members listed at the end of the Main Text document</p

QTL mapping of <i>let-60 ras</i> modifiers.

<p><b>(A)</b> RAS/MAPK signaling induces three VPCs. P6.p receives most of the inductive EGF signal from the anchor cell and activates the EGFR/RAS/MAPK pathway inducing the 1° cell fate (green arrows). Lateral signaling via the Notch pathway induces the 2° cell fate in the neighboring VPCs P5.p and P7.p (red arrows). The remaining VPCs (blue) adopt the non-vulval 3° cell fate. <b>(B)</b> Crossing scheme to generate the <i>let-60(n1046gf)</i> miRILs. Hawaii males (red) were crossed with Bristol <i>let-60(n1046gf)</i> mutants (blue). For each example animal, the two chromosomes IV carrying the <i>n1046</i> mutation and another arbitrary chromosome pair are shown. Random segregation of the two parental genomes was allowed except for the <i>let-60(gf)</i> mutation that was kept homozygous from F2 generation onwards. After ten generations of self-fertilization to drive all regions to homozygosity, 228 independent miRILs were obtained. <b>(C)</b> Genotypes and phenotypes of the <i>let-60(gf)</i> miRILs sorted by increasing VI. Genotypes determined by FLP mapping [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005236#pgen.1005236.ref015" target="_blank">15</a>] are plotted on the y-axis versus the miRIL numbers on the x-axis. Hawaii genotypes are indicated with red, Bristol genotypes with blue and missing genotypes with gray colors. The VIs for each miRIL are shown below the genotypes. Error bars indicate the standard error of the mean. <b>(D)</b> QTL mapping identified three regions (QTL1 through QTL3) above the threshold LOD score of 3 (dotted red line). In each of the panels showing chromosomes I through X, the locations of the FLP markers used for genotyping are indicated on the x-axis with vertical lines. For the exact locations of the FLPs used, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005236#sec011" target="_blank">Materials and Methods</a> and [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005236#pgen.1005236.ref015" target="_blank">15</a>].</p

AMX-2 negatively regulates RAS/MAPK signaling.

<p><b>(A)</b> Fine-mapping of QTL1 with ILs. For each IL, the regions containing the Hawaii (black) genome in the Bristol (grey) background are indicated, and the corresponding VIs are plotted below. Black columns indicate the average VI of three independent lines carrying an introgression and gray columns the average VI of three sibling lines without introgression. Dashed boxes indicate the QTL1a and QTL1b sub-regions. <b>(B)</b> Allele-specific effects of <i>amx-2</i> RNAi compared to empty vector controls. <b>(C)</b> Two copies of Bristol but not Hawaii <i>amx-2</i> rescue the increased VI of <i>amx-2(ok1235); let-60(n1046gf)</i> double mutants. <b>(D)</b> Epistasis analysis of <i>amx-2(ok1235)</i>. The dashed line indicates the wild-type VI of 3. <b>(E-H)</b> Expression pattern of a transcriptional <i>P</i>_amx-2::<i>gfp</i> reporter in the pharynx and head neurons <b>(E)</b>, the adult vulva <b>(F)</b>, the intestine <b>(G)</b> and some rectal cells <b>(H)</b> of L4 larvae. The scale bar is 10μm. <b>(I)</b> Tissue-specific <i>amx-2</i> RNAi. Knock-down in the intestine but not the vulval cells increases the VI of <i>let-60(n1046gf)</i> mutants <b>(J)</b> Quantitative PCR of <i>amx-2</i> and <i>amx-1</i>. Expression levels were normalized to the N2 wild-type Bristol strain. Error bars in <b>(A)</b> to <b>(I)</b> indicate the standard error of the mean and in <b>(J)</b> the standard deviation measured in three independent experiments. The numbers of animals scored are shown inside the columns. *** indicates p<0.001, ** p<0.01, *<0.05 and n.s. p>0.1 in a Student’s t-test.</p

5-HIAA inhibits RAS/MAPK signaling and MPK-1 phosphorylation in multiple organs of <i>C</i>. <i>elegans</i>.

<p><b>(A)</b> Partial suppression of the germline defect in <i>let-60(ga89ts)</i> mutants treated with 5-HIAA and grown at 25°C. The images show the gonads of 5-HIAA treated (top) and untreated (bottom) young adults. Note the regularly stacked oocytes in 5-HIAA treated and the irregularly stacked and smaller oocytes in untreated animals. <b>(B)</b> Partial suppression of the duct cell duplication phenotype in <i>let-60(n1046gf)</i> mutants by 5-HIAA. The images show the single duct cell in a 5-HIAA treated <i>let-60(n1046gf)</i> L4 larva (top) and the two duct cells in an untreated larva (bottom). The arrows point at the nuclei of the duct cells expressing the <i>lin-48</i>::<i>gfp</i> marker. <b>(C)</b> MPK-1 phosphorylation in total extracts of <i>let-60(n1046gf)</i> single and <i>amx-2(ok1235); let-60(n1046gf)</i> double mutant larvae treated with 4mM 5-HT or 5-HIAA. The ratios of phosphoMPK-1 to total MPK-1 levels were determined in three independent experiments as described in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005236#pgen.1005236.ref011" target="_blank">11</a>] and Materials and Methods. Values were normalized to the ratios in untreated animals. The numbers of animals scored are indicated in brackets or inside the columns. *** indicates p<0.001 and ** p<0.01 in a Student’s t-test</p

Systemic inhibition of RAS/MAPK signaling by Serotonin and its metabolites.

<p><b>(A)</b> Function of MAOA in DA and 5-HT degradation. <b>(B)</b> 5-HT levels in total extracts of wild-type and <i>amx-2(ok1235)</i> animals. <b>(C)</b> Effect of DA, 5-HT and its metabolites on the VI of <i>let-60(n1046gf)</i> single and <i>amx-2(ok1235); let-60(n1046gf)</i> double mutants. <b>(D)</b> Examples of (top) a 5-HIAA treated and (bottom) an untreated <i>let-60(n1046gf)</i> L4 larva. The normal vulva and the ectopically induced cells are underlined. <b>(E)</b> Dose-dependent reduction of the VI by 5-HT and <b>(F)</b> 5-HIAA treatments. Note in <b>(E)</b> the different sensitivities of the two strains to 1μM 5-HT. <b>(G)</b> Effect of 5-HIAA on mutations activating the EGFR/RAS/MAPK pathway at different levels. <b>(H)</b> Resistance of some 5-HT pathway mutants to 5-HIAA treatment. Error bars indicate the standard error of the mean. The numbers of animals scored are indicated in brackets or inside the columns. *** indicates p<0.001, ** p<0.01, and n.s. p>0.1 in a Student’s t-test.</p

Cyp24a1 contains an IRES element.

<p>(A) Sequence of the human cyp24a1-5′UTR. (B) Schematic representation of the bicistronic control (phpRF) and cyp24a1-5′UTR-containing (phpR-cyp-F) luciferase constructs used for reporter assays. (C) Bicistronic reporter plasmids phpRF (white bars) and phpR-cyp-F (black bars) were transfected into MCF7 cells. 24 h after transfection <i>renilla</i> and <i>firefly</i> luciferase activities were measured and data are presented as means ± SEM relative to phpRF (n≥3, ** p<0.01). (D) RNA isolated from cells transfected with phpRF or phpR-cyp-F was DNAse treated and reverse transcribed. <i>Upper panel</i>: PCR was performed with specific primers to amplify full length RL or R-cyp-L mRNAs. PCR products were visualized <i>via</i> agarose gel electrophoresis and ethidium bromide staining. Data are representative for at least 3 independent experiments. <i>Lower panel</i>: RT-qPCR analysis of the amount of <i>firefly</i> mRNA normalized to <i>renilla</i> mRNA. Data are presented as means ± SEM (n≥3). (E) <i>In vitro-</i>transcribed mRNAs of the control (hpRF, white bars) or the cyp24a1-5′UTR-containing vector (hpR-cyp-F, black bars) were transfected into MCF7 cells. 24 h after transfection <i>renilla</i> and <i>firefly</i> luciferase activities were measured. Luciferase activities are given relative to hpRF and data are presented as means ± SEM (n≥3, ** p<0.01).</p

Polysome profile of MCF7 cells.

<p>Representative profile of MCF7 lysates at 254 nm as determined during polysomal fractionation (<i>upper panel</i>). Equal aliquots of RNA isolated from single fractions were analyzed using denaturing agarose gel electrophoresis to verify 28S and 18S rRNA content as indicators for ribosome distribution (<i>lower panel</i>).</p

CM induces cyp24a1 translation.

<p>MCF7 cells were treated with Ctr or CM for 4(A) and cyp24a1 (B) was analyzed in single fractions using RT-qPCR. The distribution of the respective mRNAs across the individual gradients was determined relative to the total RNA extracted from the gradients. Results from a representative experiment are given in A and B. (C+D) Changes of gapdh (C) and cyp24a1 (D) mRNA distribution induced by CM were normalized to Ctr. (E) cyp24a1 distribution (from D) was normalized to gapdh distribution (from C). Distribution changes are presented as means ± SEM (n≥3, * p<0.05, ** p<0.01, *** p<0.001).</p

Cyp24a1 translation is initiated in part cap-independently.

<p>MCF7 cells were treated with rapamycin [100 nM] for 4 h and subjected to polysomal fractionation. RNA from single fractions was isolated and gapdh (A) and cyp24a1 (B) mRNA distribution changes were analyzed separately as described before. Data are presented as means ± SEM (n≥3).</p