Estimation filtering for Deep Water Navigation

The navigation task for Unmanned Underwater Vehicles is made difficult in a deep water scenario because of the lack of bottom lock for Doppler Velocity Log (DVL). This is due to the operating altitude that, for this kind of applications, is typically greater than the sensor maximum range. The effect is that the velocity measurements are biased by sea currents resulting in a rapidly increasing estimation error drift. The solution proposed in this work is based on a distributed, cooperative strategy strongly relying on an acoustic underwater network. According to the distributed philosophy, an instance of a specifically designed navigation filter (named DWNF - Deep Water Navigation Filter) is executed by each vehicle. Each DWNF relies on different Extended Kalman Filters (EKFs) running in parallel on-board: one for own navigation state estimation (AUV-EKF), the other ones for the navigation state of the remaining assets (Asset-EKF). The AUV-EKF is designed to simultaneously estimate the vehicle position and the sea current for more reliable predictions. The DWNF builds in real-time a database of past measurements and estimations; in this way it can correctly deal with delayed information. An outlier detection and rejection policy based on the Mahalanobis distance associated to each measurement is implemented. The experimental validation of the proposed approach took place in a deep water scenario during the Dynamic Mongoose’17 exercise off the South coast of Iceland (June-July 2017); preliminary analysis of the results is presented

Real-time underwater positioning and navigation of an AUV in deep waters

Due to the absence of GPS, navigation of autonomous vehicles underwater requires the integration of various measurements to provide the best location estimate. Usually in littoral waters, adequate navigational accuracy may be obtained by integrating odometry measurements provided by a Doppler Velocity Log (DVL) into an Inertial Navigation System (Aided INS). However, due to the bulk attenuation of seawater at the acoustic centre frequency at which DVLs typically operate, odometry estimates become increasingly unreliable when the vehicle flies more than 200 m above the bottom (depending on the DVL central frequency). Such a case occurs during experiments in deep waters. This work addresses a theoretical and experimental study on the feasibility of navigating the AUVs using a multi-input Extended Kalman Filter (EKF) integrating proprioceptive measurements (i.e., INS data and speed-over-water observations from DVL) with a set of exteroceptive sensor data, when available. The filter was integrated on-board the CMRE Ocean Explorer Class Version C (OEX-C) AUVs, and tested at sea for the first time in deep water during the NATO exercise Dynamic Mongoose’17 off the South coast of Iceland (June-July 2017)

Disruption of TTDA Results in Complete Nucleotide Excision Repair Deficiency and Embryonic Lethality

The ten-subunit transcription factor IIH (TFIIH) plays a crucial role in transcription and nucleotide excision repair (NER). Inactivating mutations in the smallest 8-kDa TFB5/TTDA subunit cause the neurodevelopmental progeroid repair syndrome trichothiodystrophy A (TTD-A). Previous studies have shown that TTDA is the only TFIIH subunit that appears not to be essential for NER, transcription, or viability. We studied the consequences of TTDA inactivation by generating a Ttda knock-out (Ttda2/2) mouse-model resembling TTD-A patients. Unexpectedly, Ttda2/2 mice were embryonic lethal. However, in contrast to full disruption of all other TFIIH subunits, viability of Ttda2/2 cells was not affected. Surprisingly, Ttda2/2 cells were completely NER deficient, contrary to the incomplete NER deficiency of TTD-A patient-derived cells. We further showed that TTD-A patient mutations only partially inactivate TTDA function, explaining the relatively mild repair phenotype of TTD-A cells. Moreover, Ttda2/2 cells were also highly sensitive to oxidizing agents. These findings reveal an essential role of TTDA for life, nucleotide excision repair, and oxidative DNA damage repair and identify Ttda2/2 cells as

Gene expression levels and TFIIH amount in <i>Ttda^−/−</i> ES cells.

<p>(A) Relative expression levels of mRNAs neighboring genes encoding Synaptojamin 2 (<i>Synj2</i>), Serine active site containing 1 (<i>Serac1</i>), Trichothiodystrophy group A (<i>Ttda</i>) and Tubby like protein 4 (<i>Tulp4</i>) in <i>Ttda^−/−</i> (n = 2), <i>Ttda^+/−</i> (n = 2) and wild-type (n = 2) ES cells as determined by quantitative RT-PCR. The levels were normalized to <i>Gapdh</i> and the error bars indicate SEM between experiments. (B) Representative confocal microscope pictures of <i>XpbYFP^+f/+f Ttda^−/−</i>, <i>XpbYFP^+f/+f Ttda^+/−</i> and <i>XpbYFP^+f/+f</i> MEFs isolated from 10.5-day-old embryos. (C) Confocal images of ES cells isolated from <i>XpbYFP^+f/+f</i> mouse model (left panel) and from <i>XpbYFP^+f/+f Ttda^−/−</i> mouse (right panel). The green signal is the direct fluorescence of the YFP tagged protein. The white bar measures 10 mm. (D) Confocal images of EU incorporation into ES cells isolated from <i>XpbYFP^+f/+f</i> mouse model and from <i>XpbYFP^+f/+f Ttda^−/−</i> mouse (upper panel) and MEF's isolated from the same mouse models (lower panel). Two EU incubation times have been performed: 30 minutes (left panels) and 120 minutes (right panels). The white bar measures 10 mm.</p

Mutant TTDA protein accumulation at local UV damage.

<p>(A) Schematically representation of the predicted TTDA polypeptide length (in amino acids) in human wild-type cells (TTDA^WT), TTD-A patient cells (TTDA^M1T, TTDA^L21P and TTDA^R56X) and the <i>Ttda</i> knock-out cells (<i>Ttda^−/−</i>). The red star represents the mutation found in TTDA^L21P and the red part of the <i>Ttda^−/−</i> bar represents intronic encoded non-sense amino acids. (B) Representative confocal images of <i>Ttda^−/−</i> MEFs expressing TTDA^WT-GFP before (t = 0 min) and after local UV-damage infliction (t = 5 min) in a selected area inside the nucleus (dashed circle). (C) Accumulation kinetics of TTDA^WT-GFP, TTDA^L21P-GFP and TTDA^R56X-GFP to local UV-C (laser-induced) DNA damage expressed in <i>Ttda^−/−</i> MEFs. Graphs represents the mean GFP-derived fluorescence intensity at the damaged spot at the indicated time points from approximately 12 cells. (D) Representative confocal microscope images of UV-induced UDS of <i>Ttda^−/−</i> MEFs transiently co-transfected with an empty GFP vector (as a marker for transfected cells) in combination with a vector containing TTDA^WT or TTDA^M1T. Cells were seeded on cover slips and transfected 2 days before the experiment. Cells were irradiated with 16 J/m² and subsequently labeled for 2 hours with EdU. Cells were fixed and stained for EdU incorporation (UDS and S-phase DNA synthesis, red) and GFP using antibodies against GFP (transfected cells, green). The intense red labeled cells are cells in S-phase. (E) The percentage of UDS signal in the nucleus was quantified by measuring the average fluorescence intensity from at least 25 cells positively transfected (containing GFP) and non-S-phase cells with TTDA^WT, TTDA^M1T, TTDA^R56X or TTDA^L21P. The error bars indicate the SEM.</p

Genotyping and offspring from <i>Ttda^+/−</i> mice matings.

<p>Genotyping of offspring from matings of <i>Ttda^+/−</i> mice, distributed over male and females, obtained number and percentage of offspring compared to the theoretical expected figures assuming a Mendalian inheritence pattern.</p

γH2AX signaling is abolished in <i>Ttda^−/−</i> MEFs.

<p>Cell cycle dependent analysis of XPC and γH2AX recruitment to local UV-damage in wild-type (A), <i>Xpa^−/−</i> (B) and <i>Ttda^−/−</i> (C) MEFs. Cells were seeded on cover slips and the next day irradiated with 60 J/m² through a filter containing 5 µm pores and subsequently labeled with EdU for 1 hour. After fixation cells were assayed for DNA synthesis using EdU and Alexa Fluor 647 azide (cell cycle marker, pink) and by immuno-fluorescent staining using antibodies against γH2AX (green) and XPC (red). Dashed circles indicate typical examples of cells in S-phase, closed circles indicate typical examples of G1/G2 cells.</p

Knock-down of mutant hTTDA results in complete NER deficiency.

<p>(A) Relative expression levels of TTDA mRNA as determined by quantitative RT-PCR in TTD1BR-sv cells (TTD-A) and TTD1BR-sv cells stably expressing shRNAs for respectively: #non-targeting (NT), #3398, #3399, #3400, #3401 or #3402. The levels were normalized to <i>Tubulin</i> and the error bars indicate SEM between two independent experiments. (B) Colony forming ability after different doses of UV irradiation of MRC5-sv (wild-type), XP12RO-sv (XP-A), TTD1BR-sv and TTD1BR-sv cells stably expressing shRNA: #non-targeting (NT), #3398, #3399, #3400, #3401 and #3402. The percentage of surviving cells was plotted against the applied UV-dose, measured by counting surviving colonies of two independent experiments. The error bars indicate the SEM. (C) Quantitative immuno-fluorescence to determine the relative amount of XPB (TFIIH) in MRC5-sv (wild-type), TTD1BR-sv (TTD-A) and TTD1BR-sv cells stably expressing shRNA (#3398 or #3402). Confocal microscope pictures were used to quantify the average intensity of XBP and MDC1 (internal control) in >100 cells and error bars indicate SEM.</p

Generation of <i>Ttda</i> knock-out mice.

<p>(A) Schematic presentation of the mouse <i>Ttda</i> genomic locus, <i>Ttda</i> targeting construct and <i>Ttda</i> locus after CagCre recombination. Roman numbered boxes represent exons: white boxes are non-coding exon (parts) and black boxes are coding exon (parts). In the targeting construct LoxP sites are indicated with gray arrows and the dashed box represents the Neomycin selectable marker driven by a PGK promoter. The positions of NheI restriction sites, size of the NheI restriction fragments and the position of probe A used for DNA blot screening of digested ES cell DNA are indicated. The short arrows indicate the position and direction of primers used for genotyping. (B) DNA blot analysis of NheI-digested ES cell DNA using the probe A. (C) Genotype analysis with diagnostic PCR using primers 1F, 1R, 2R and NeoR in the different combinations (A, B and C).</p

Average litter size.

<p>Average litter size of matings between two <i>Ttda^+/−</i> animals compared to matings between <i>Ttda^+/−</i> and wild-type mice.</p