Identification of genes involved in leukaemia and differentiation induced by activated mutants of the GM-CSF receptor β subunit.

Interleukin (IL)-3, IL-5 and granulocyte-macrophage-colony-stimulating factor (GM-CSF) are cytokines that affect the growth, survival and differentiation of many cells within the haematopoietic system. The functions of these factors are mediated by membrane bound receptor complexes that are composed of specific ligand binding subunits (α)and a common signal transducing subunit(hβc). Constitutively activated mutants of hβc have been previously identified that are able to confer factor-independent signalling in a number of haematopoietic cell lines (including FDC-P1 and FDB-1). These activated mutants fall into two classes defined by the location of the mutation and their biochemical and leukaemogenic properties. In particular, the transmembrane mutant, V449E, causes an acute myeloid leukaemia in vivo, whereas the extracellular mutants (FI∆ or I374N) cause chronic myeloproliferative disorders. The work described in this thesis used the activated hβc mutants to uncover novel transcriptional events induced by the GM-CSF/IL-3/IL-5 receptor complex and to define pathways associated with proliferation and differentiation. Large-scale gene expression profiling techniques were used to investigate the genes involved in these biological processes in the murine myelomonocytic cell line FDC-P1, and the bi-potent FDB-1 myeloid cell line, which are responsive to IL-3 and GM-CSF. Membrane arrays were used to identify differences in gene expression between I374N and V449E expressing FDC-P1 cells. This technique revealed that the gene Ptpmt1 was differentially expressed between V449E and I374N, which was subsequently confirmed by Northern blotting. This finding suggested that the phosphatase encoded by Ptpmt1 may be involved in the different outcomes induced by these two hβc mutants. Northern analysis also revealed Ptpmt1, Nab1 and Ddx26b to be regulated in response to human GM-CSF in FDC-P1 cells expressing human GM CSFα and hβc. A large-scale cDNA microarray experiment was also performed to identify genes that are selectively expressed during differentiation of FI∆ expressing FDB-1 cells, compared to proliferating V449E expressing FDB-1 cells over 24 hours. A comprehensive analysis approach was adopted to examine the microarray data and identify differentially expressed genes. Among the genes displaying differential expression were Btg1, S100a9, Cd24, and Ltf found to be differentiation-associated and Bnip3, Cd34, Myc, Nucleophosmin, and Nucleostemin found to be proliferation-associated. Hipk1, Klf6, Sp100, and Sfrs3 were also identified as potential transcriptional regulators during growth and differentiation. Northern analysis was used to confirm differences in expression for these 13 genes between FI∆ and V449E expressing FDB-1 cells. Eleven of the 13 genes examined were confirmed to be differentially expressed between FI∆ and V449E expressing FDB-1 cells over 24 hours. Furthermore, six genes (Btg1, Hipk1, Cd24, Cd34, Klf6 and Nucleostemin) examined over 72 hours revealed differences in gene expression at early (6-12 hours) and late (48-72 hours) time points. Cell surface expression of CD24 protein was also shown to be induced upon FI∆ expression or GM-CSF induced differentiation of FDB-1 cells, consistent with elevated levels of Cd24 mRNA in FI∆ cells over time. Based on their confirmed gene expression differences seen on the microarrays and Northern analysis, four genes (Btg1, Cd24, Klf6 and Nucleostemin) were selected for over-expression analysis in FDC-P1 or FDB-1 cells, in order to gain insights into the function of these genes. Optimisation of the retroviral infection process was performed so that the role of these genes in proliferation and differentiation could be investigated in the FDB-1 model. Such preliminary functional experiments in FDB-1 cells will enable prioritisation of the genes for further analysis of their function in primary cells. Thus, the work in this thesis describes the first use of microarrays to identify gene expression differences between hβc mutants with differential activities affecting myeloid growth and differentiation.Thesis (PhD)-- School of Medicine, 200

Effects of Gender and Position of Test Stimuli on Undergraduates \u27 Spatial Task Performance

This study predicted that sex differences in performance can occur where the sense of touch serves as vision, and that the position of the test array may significantly affect performance. Sixty-four college undergraduates (32 males and 32 females), with ages ranging from 18-27 (M = 20.06, SD = 1.82) were recruited from the psychology subject pool of Eastern Illinois University for participation. Apparatus consisted of templates with raised line drawings of tilted jars containing water drawn on them. The subjects were blindfolded and instructed to interpret four jar drawings at a time. The task consisted of identifying the jar with the correct water line. All subjects participated in 8 trials. Half were tested on an upright test array, and the rest on an array that was tilted. The data were analyzed using a 2 X 2 X 4 (Gender X Position of Test Array X Angle of Jar) ANOVA. The results indicated that gender was significantly related to performance of the task, and that males performed better than females did, F (1,180) = 8.1, p\u3c0.01, while the position of the test array was not, F (1,180) = .83, p\u3e0.37

Effects of Gender and Position of Test Stimuli on Undergraduates \u27 Spatial Task Performance

This study predicted that sex differences in performance can occur where the sense of touch serves as vision, and that the position of the test array may significantly affect performance. Sixty-four college undergraduates (32 males and 32 females), with ages ranging from 18-27 (M = 20.06, SD = 1.82) were recruited from the psychology subject pool of Eastern Illinois University for participation. Apparatus consisted of templates with raised line drawings of tilted jars containing water drawn on them. The subjects were blindfolded and instructed to interpret four jar drawings at a time. The task consisted of identifying the jar with the correct water line. All subjects participated in 8 trials. Half were tested on an upright test array, and the rest on an array that was tilted. The data were analyzed using a 2 X 2 X 4 (Gender X Position of Test Array X Angle of Jar) ANOVA. The results indicated that gender was significantly related to performance of the task, and that males performed better than females did, F (1,180) = 8.1, p\u3c0.01, while the position of the test array was not, F (1,180) = .83, p\u3e0.37

Genomic landscape of platinum resistant and sensitive testicular cancers

While most testicular germ cell tumours (TGCTs) exhibit exquisite sensitivity to platinum chemotherapy, ~10% are platinum resistant. To gain insight into the underlying mechanisms, we undertook undertake whole exome sequencing and copy number analysis in 40 tumours from 26 cases with platinum-resistant TGCT, and combined this with published genomic data on an additional 624 TGCTs. We integrated analyses for driver mutations, mutational burden, global, arm-level and focal copy number (CN) events, and SNV and CN signatures. Albeit preliminary and observational in nature, these analyses provide the first support for a possible mechanistic link between early driver mutations in RAS and KIT and the widespread copy number events by which TGCT is characterised

Microarrays and breast cancer clinical studies: forgetting what we have not yet learnt

This review takes a sceptical view of the impact of breast cancer studies that have used microarrays to identify predictors of clinical outcome. In addition to discussing general pitfalls of microarray experiments, we also critically review the key breast cancer studies to highlight methodological problems in cohort selection, statistical analysis, validation of results and reporting of raw data. We conclude that the optimum use of microarrays in clinical studies requires further optimisation and standardisation of methodology and reporting, together with improvements in clinical study design

Issues and tensions in public procurement of ‘green innovation’: a cross-country study

No description supplie

The Quality Control Algorithms Used in the Process of Creating the NASA Kennedy Space Center Lightning Protection System Towers Meteorological Database

The methodology and the results of the quality control (QC) process of the meteorological data from the Lightning Protection System (LPS) towers located at Kennedy Space Center (KSC) launch complex 39B (LC-39B) are documented in this paper. Meteorological data are used to design a launch vehicle, determine operational constraints, and to apply defined constraints on day-of-launch (DOL). In order to properly accomplish these tasks, a representative climatological database of meteorological records is needed because the database needs to represent the climate the vehicle will encounter. Numerous meteorological measurement towers exist at KSC; however, the engineering tasks need measurements at specific heights, some of which can only be provided by a few towers. Other than the LPS towers, Tower 313 is the only tower that provides observations up to 150 m. This tower is located approximately 3.5 km from LC-39B. In addition, data need to be QC'ed to remove erroneous reports that could pollute the results of an engineering analysis, mislead the development of operational constraints, or provide a false image of the atmosphere at the tower's location

The Quality Control Algorithms Used in the Creation of NASA Kennedy Space Center Lightning Protection System Towers Meteorological Database

No abstract availabl

Sequencing Structural Variants in Cancer for Precision Therapeutics.

The identification of mutations that guide therapy selection for patients with cancer is now routine in many clinical centres. The majority of assays used for solid tumour profiling use DNA sequencing to interrogate somatic point mutations because they are relatively easy to identify and interpret. Many cancers, however, including high-grade serous ovarian, oesophageal, and small-cell lung cancer, are driven by somatic structural variants that are not measured by these assays. Therefore, there is currently an unmet need for clinical assays that can cheaply and rapidly profile structural variants in solid tumours. In this review we survey the landscape of 'actionable' structural variants in cancer and identify promising detection strategies based on massively-parallel sequencing.This work was supported by Cancer Research UK [grant numbers A15973, A15601: 454 G.M, J.D.B], VUmc Cancer Center Amsterdam [VUmc-CCA: BY] and the Dutch 455 Cancer Society [VU 2015-7882: BY].This is the author accepted manuscript. The final version is available from Cell/Elsevier via http://dx.doi.org/10.1016/j.tig.2016.07.00

SPARC regulates transforming growth factor beta induced (TGFBI) extracellular matrix deposition and paclitaxel response in ovarian cancer cells

TGFBI has been shown to sensitize ovarian cancer cells to the cytotoxic effects of paclitaxel via an integrin receptor-mediated mechanism that modulates microtubule stability. Herein, we determine that TGFBI localizes within organized fibrillar structures in mesothelial-derived ECM. We determined that suppression of SPARC expression by shRNA decreased the deposition of TGFBI in mesothelial-derived ECM, without affecting its overall protein expression or secretion. Conversely, overexpression of SPARC increased TGFBI deposition. A SPARC-YFP fusion construct expressed by the Met5a cell line co-localized with TGFBI in the cell-derived ECM. Interestingly, in vitro produced SPARC was capable of precipitating TGFBI from cell lysates dependent on an intact SPARC carboxy-terminus with in vitro binding assays verifying a direct interaction. The last 37 amino acids of SPARC were shown to be required for the TGFBI interaction while expression of a SPARC-YFP construct lacking this region (aa 1–256) did not interact and co-localize with TGFBI in the ECM. Furthermore, ovarian cancer cells have a reduced motility and decreased response to the chemotherapeutic agent paclitaxel when plated on ECM derived from mesothelial cells lacking SPARC compared to control mesothelial-derived ECM. In conclusion, SPARC regulates the fibrillar ECM deposition of TGFBI through a novel interaction, subsequently influencing cancer cell behavior