The role for the p85 subunit of PI3kinase in the regulation of rab proteins

Upon activation by the platelet-derived growth factor receptor (PDGFR), phosphatidylinositol 3'-kinase (PI3K) converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate to activate the PI3K/Akt cellular survival signalling pathway within cells. The p85 subunit of PI3K has also been shown to have GTPase activating protein (GAP) activity towards Rab proteins involved in receptor endocytosis and trafficking, specifically Rab5 and Rab4. Rab5 is responsible for regulating the fusion of vesicles containing activated receptors to traffic them to intracellular early/sorting endosomes. Rab4 is responsible for regulating the exit of receptors to a recycling pathway back to the plasma membrane. The p85 RabGAP activity is responsible for deactivating Rab5 and Rab4 function by accelerating their GTPase activity, resulting in the inactive conformation of Rab5 and Rab4, and decreased vesicle fusion events during receptor trafficking. The work in this thesis was performed to understand how p85 interacts with, and regulates, Rab5 and Rab4. Glutathione S-transferase pulldown experiments showed the p85 protein was able to interact with Rab5 through its BH domain and another unidentified domain. Cells expressing a p85-R274A mutant defective for RabGAP activity displayed increased PDGFR activation and decreased degradation. To understand the mechanism of decreased PDGFR degradation, PDGFR immunoprecipitation experiments showed the PDGFR was ubiquitinated, a signal needed for multi-vesicular body sorting. Biotinylation experiments showed the PDGFR was being more rapidly endocytosed and then sequestered within the cell. Immunofluorescence experiments showed cells expressing the p85-R274A mutant clearly altered PDGFR trafficking during receptor endocytosis. These results suggest the PDGFR was not spending longer periods of time on the cell surface to continue signalling and was not lacking the modification needed to be sorted to a degradative pathway. The defective trafficking observed in p85-R274A expressing cells, over time, may block PDGFR trafficking, which prevents normal PDGFR dephosphorylation and degradation, and could be attributed to a lack of sufficient cytosolic Rab5-GDP and Rab4-GDP required to associate with new membranes and facilitate additional vesicle fusion events. The lack of lysosomal targeting allows the receptor to be sequestered in cells, but still have the ability to signal as the receptor would not be targeted to multi-vesicular bodies where signalling is abolished

Thin Sea Ice, Thick Snow, and Widespread Negative Freeboard Observed During N-ICE2015 North of Svalbard

In recent years, sea-ice conditions in the Arctic Ocean changed substantially toward a younger and thinner sea-ice cover. To capture the scope of these changes and identify the differences between individual regions, in situ observations from expeditions are a valuable data source. We present a continuous time series of in situ measurements from the N-ICE2015 expedition from January to June 2015 in the Arctic Basin north of Svalbard, comprising snow buoy and ice mass balance buoy data and local and regional data gained from electromagnetic induction (EM) surveys and snow probe measurements from four distinct drifts. The observed mean snow depth of 0.53 m for April to early June is 73% above the average value of 0.30 m from historical and recent observations in this region, covering the years 1955–2017. The modal total ice and snow thicknesses, of 1.6 and 1.7 m measured with ground-based EM and airborne EM measurements in April, May, and June 2015, respectively, lie below the values ranging from 1.8 to 2.7 m, reported in historical observations from the same region and time of year. The thick snow cover slows thermodynamic growth of the underlying sea ice. In combination with a thin sea-ice cover this leads to an imbalance between snow and ice thickness, which causes widespread negative freeboard with subsequent flooding and a potential for snow-ice formation. With certainty, 29% of randomly located drill holes on level ice had negative freeboard

A framework for automated anomaly detection in high frequency water-quality data from in situ sensors

River water-quality monitoring is increasingly conducted using automated in situ sensors, enabling timelier identification of unexpected values. However, anomalies caused by technical issues confound these data, while the volume and velocity of data prevent manual detection. We present a framework for automated anomaly detection in high-frequency water-quality data from in situ sensors, using turbidity, conductivity and river level data. After identifying end-user needs and defining anomalies, we ranked their importance and selected suitable detection methods. High priority anomalies included sudden isolated spikes and level shifts, most of which were classified correctly by regression-based methods such as autoregressive integrated moving average models. However, using other water-quality variables as covariates reduced performance due to complex relationships among variables. Classification of drift and periods of anomalously low or high variability improved when we applied replaced anomalous measurements with forecasts, but this inflated false positive rates. Feature-based methods also performed well on high priority anomalies, but were also less proficient at detecting lower priority anomalies, resulting in high false negative rates. Unlike regression-based methods, all feature-based methods produced low false positive rates, but did not and require training or optimization. Rule-based methods successfully detected impossible values and missing observations. Thus, we recommend using a combination of methods to improve anomaly detection performance, whilst minimizing false detection rates. Furthermore, our framework emphasizes the importance of communication between end-users and analysts for optimal outcomes with respect to both detection performance and end-user needs. Our framework is applicable to other types of high frequency time-series data and anomaly detection applications

Kinetochore alignment within the metaphase plate is regulated by centromere stiffness and microtubule depolymerases

During mitosis in most eukaryotic cells, chromosomes align and form a metaphase plate halfway between the spindle poles, about which they exhibit oscillatory movement. These movements are accompanied by changes in the distance between sister kinetochores, commonly referred to as breathing. We developed a live cell imaging assay combined with computational image analysis to quantify the properties and dynamics of sister kinetochores in three dimensions. We show that baseline oscillation and breathing speeds in late prometaphase and metaphase are set by microtubule depolymerases, whereas oscillation and breathing periods depend on the stiffness of the mechanical linkage between sisters. Metaphase plates become thinner as cells progress toward anaphase as a result of reduced oscillation speed at a relatively constant oscillation period. The progressive slowdown of oscillation speed and its coupling to plate thickness depend nonlinearly on the stiffness of the mechanical linkage between sisters. We propose that metaphase plate formation and thinning require tight control of the state of the mechanical linkage between sisters mediated by centromeric chromatin and cohesion

A Test of Pre-Main-Sequence Lithium Depletion Models

Despite the extensive study of lithium depletion during pre-main-sequence contraction, studies of individual stars show discrepancies between ages determined from the HR diagram and ages determined from lithium depletion (Song et al. 2002, White & Hillenbrand 2005) indicating open questions in the pre-main-sequence evolutionary models. To further test these models, we present high resolution spectra for members of the Beta Pictoris Moving Group (BPMG), which is young and nearby. We measure equivalent widths of the 6707.8 Angstrom Li I line in these stars and use them to determine lithium abundances. We combine the lithium abundance with the predictions of pre-main-sequence evolutionary models in order to calculate a lithium depletion age for each star. We compare this age to the age predicted by the HR diagram of the same model. We find that the evolutionary models under-predict the amount of lithium depletion for the BPMG given its nominal HR diagram age of ~12 Myr (Zuckerman et al. 2001), particularly for the mid-M stars, which have no observable Li I line. This results in systematically older ages calculated from lithium depletion isochrones than from the HR diagram. We suggest that this discrepancy may be related to the discrepancy between measured M-dwarf radii and the smaller radii predicted by evolutionary models.Comment: Accepted by ApJ; 21 pages, 5 figure

Ice Crystal Icing Physics Study using a NACA 0012 Airfoil at the National Research Council of Canada's Research Altitude Test Facility

This paper presents results from a study of the fundamental physics of ice-crystal ice accretion using a NACA 0012 airfoil at the National Research Council of Canada (NRC) Research Altitude Test Facility in August 2017. These tests were a continuation of work which began in 2010 as part of a joint collaboration between NASA and NRC. The research seeks to generate icing conditions representative of those that occur inside a jet engine when ingesting ice crystals. In this test, an airfoil was exposed to mixed-phase icing conditions and the resulting ice accretions were recorded and analyzed. This paper details the specific objectives, procedures, and measurements which included the aero-thermal and cloud measurements. The objectives were built upon observations and hypothesis generated from several previous test campaigns regarding mixed-phase ice-crystal icing. The specific objectives included (A) ice accretions under different wet-bulb temperatures, (B) investigations of steady-state ice shapes previously reported in the literature, (C) total water content variations in search of a threshold for accretion, and (D) probe characterization related to measuring melt fraction which is important to characterize the mixed-phase condition. The resulting ice accretions and conditions leading to such accretions are intended to help extend NASAs predictive ice-accretion codes to include conditions occurring in engine ice-crystal icing

The non-coding landscape of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive disease marked by frequent recurrence and metastasis and stagnant survival rates. To enhance molecular knowledge of HNSCC and define a non-coding RNA (ncRNA) landscape of the disease, we profiled the transcriptome-wide dysregulation of long non-coding RNA (lncRNA), microRNA (miRNA), and PIWI-interacting RNA (piRNA) using RNA-sequencing data from 422 HNSCC patients in The Cancer Genome Atlas (TCGA). 307 non-coding transcripts differentially expressed in HNSCC were significantly correlated with patient survival, and associated with mutations in TP53, CDKN2A, CASP8, PRDM9, and FBXW7 and copy number variations in chromosomes 3, 5, 7, and 18. We also observed widespread ncRNA correlation to concurrent TP53 and chromosome 3p loss, a compelling predictor of poor prognosis in HNSCCs. Three selected ncRNAs were additionally associated with tumor stage, HPV status, and other clinical characteristics, and modulation of their expression in vitro reveals differential regulation of genes involved in epithelial-mesenchymal transition and apoptotic response. This comprehensive characterization of the HNSCC non-coding transcriptome introduces new layers of understanding for the disease, and nominates a novel panel of transcripts with potential utility as prognostic markers or therapeutic targets

Whole-genome and long-read sequencing identify a novel mechanism in RFC1 resulting in CANVAS syndrome

OBJECTIVES: Cerebellar ataxia with neuropathy and bilateral vestibular areflexia syndrome (CANVAS) results from biallelic intronic pentanucleotide repeats in METHODS: We performed whole-genome sequencing (WGS) on peripheral blood DNA samples from the proband and his unaffected mother. We performed DNA long-read sequencing and synthesized complementary DNA from RNA using peripheral blood from the proband. RESULTS: WGS confirmed the maternally inherited DISCUSSION: We report an adult with CANVAS due to compound heterozygous pathogeni

Determining who responds better to a computer vs. human-delivered physical activity intervention: Results from the community health advice by telephone (CHAT) trial

Background Little research has explored who responds better to an automated vs. human advisor for health behaviors in general, and for physical activity (PA) promotion in particular. The purpose of this study was to explore baseline factors (i.e., demographics, motivation, interpersonal style, and external resources) that moderate intervention efficacy delivered by either a human or automated advisor. Methods Data were from the CHAT Trial, a 12-month randomized controlled trial to increase PA among underactive older adults (full trial N = 218) via a human advisor or automated interactive voice response advisor. Trial results indicated significant increases in PA in both interventions by 12 months that were maintained at 18-months. Regression was used to explore moderation of the two interventions. Results Results indicated amotivation (i.e., lack of intent in PA) moderated 12-month PA (d = 0.55, p \u3c 0.01) and private self-consciousness (i.e., tendency to attune to one’s own inner thoughts and emotions) moderated 18-month PA (d = 0.34, p \u3c 0.05) but a variety of other factors (e.g., demographics) did not (p \u3e 0.12). Conclusions Results provide preliminary evidence for generating hypotheses about pathways for supporting later clinical decision-making with regard to the use of either human- vs. computer-delivered interventions for PA promotion