Modeling of forms and technologies of achievement of tops of professionalism of pedagogical activity

In this article we discuss the experience of developing the concept of modeling forms and technologies of achievement of tops of professionalism of pedagogical activity on the basis of competence approachВ представленной работе обсуждается опыт разработки концепции моделирования форм и технологий достижения вершин профессионализма педагогической деятельности на основе компетентностного подход

Modern methods of organization of educational process in the conditions of real inclusion

In this article we discuss the experience in a differentiated way, the group forms of organization of educational process at school on the basis of the discipline “Technology”Обсуждается опыт реализации дифференцированно-групповой формы организации образовательного процесса в общеобразовательной школе на примере учебной дисциплины «Технология

Statistical Methods and Models for Modern Genetic Analysis.

The Genome-Wide Association Study (GWAS) is the predominant tool to search for genetic risk variants that contribute to complex human disease. Despite the large number of GWAS findings, variants implicated by GWAS are themselves unlikely to fully explain the heritability of many diseases. In this dissertation, we propose statistical methods to augment GWAS and further our understanding of the genetic causes of complex disease. In the first project, we consider the challenges of a gene-environment analysis performed as a follow-up to a significant initial GWAS result. It is known that effect estimates based on the same data that showed the significant GWAS result suffer from an upward bias called the “Winner's Curse." We show that the initial GWAS testing strategy can induce bias in both follow-up hypothesis testing and estimation for gene-environment interaction. We propose a novel bias-correction method based on a partial likelihood Markov Chain Monte Carlo algorithm. In the second project, we shift attention to rare genetic variants that have low power of being detected by GWAS. We propose the Cumulative Minor Allele Test (CMAT) to pool together multiple rare variants from the same gene and test for an excessive burden of rare variants in either cases or controls. We show the CMAT performs favorably across a range of study designs. Notably, the CMAT accommodates probabilistic genotypes, extending applicability to low-coverage and imputed sequence data. We use a simulation analysis to validate study designs that combine sequenced and imputed samples as a means to improve power to detect rare risk variants. Determining conditions that optimize imputation accuracy is important for successful application. In the final project, we propose a coalescent model of genotype imputation that allows fast, analytical estimates of imputation accuracy across complex population genetic models. We use our model to compare the performance of custom-made reference panels drawn from the same source population as imputation targets to publicly available reference panels (i.e. 1000 Genomes Project) that may differ in ancestry from the targets.Ph.D.BiostatisticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/89761/1/mattz_1.pd

Assessing adaptation of the cancer kinome in response to targeted therapies

Cancer cells are dependent on protein kinase signalling networks to drive proliferation and to promote survival, and, accordingly, kinases continue to represent a major target class for development of anti-cancer therapeutics. Kinase inhibitors nevertheless have yielded only limited success with many different malignancies due to the inability of single agents to sustain a durable clinical response. Cancer cell kinomes are highly resilient and able to bypass targeted kinase inhibition, leading to tumour resistance. A novel platform has been developed to analyse the activity of the expressed kinome using MIBs (multiplexed inhibitor beads), which consist of Sepharose beads with covalently immobilized inhibitors that preferentially bind activated kinases. Coupling MIB capture with MS (MIB-MS) allows simultaneous determination of the activity of over 75% of the expressed kinome, facilitating high-throughput assessment of adaptive kinase responses resulting from deregulated feedback and feedforward regulatory mechanisms. The adaptive response frequently involves transcriptional up-regulation of specific kinases that allow bypass of the targeted kinase. Understanding how the kinome reprogrammes to targeted kinase inhibition will allow novel therapeutic strategies to be developed for durable clinical responses

Patterning pallet arrays for cell selection based on high-resolution measurements of fluorescent biosensors

Pallet arrays enable cells to be separated while they remain adherent to a surface and provide a much greater range of cell selection criteria relative to that of current technologies. However there remains a need to further broaden cell selection criteria to include dynamic intracellular signaling events. To demonstrate the feasibility of measuring cellular protein behavior on the arrays using high resolution microscopy, the surfaces of individual pallets were modified to minimize the impact of scattered light at the pallet edges. The surfaces of the three-dimensional pallets on an array were patterned with a coating such as fibronectin using a customized stamping tool. Micropatterns of varying shape and size were printed in designated regions on the pallets in single or multiple steps to demonstrate the reliability and precision of patterning molecules on the pallet surface. Use of a fibronectin matrix stamped at the center of each pallet permitted the localization of H1299 and mouse embryonic fibroblast (MEF) cells to the pallet centers and away from the edges. Compared to pallet arrays with fibronection coating the entire top surface, arrays with a central fibronectin pattern increased the percentage of cells localized to the pallet center by 3-4 fold. Localization of cells to the pallet center also enabled the physical separation of cells from optical artifacts created by the rough pallet side walls. To demonstrate the measurement of dynamic intracellular signaling on the arrays, fluorescence measurements of high spatial resolution were performed using a RhoA GTPase biosensor. This biosensor utilized fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) to measure localized RhoA activity in cellular ruffles at the cell periphery. These results demonstrated the ability to perform spatially resolved measurements of fluorescence-based sensors on the pallet arrays. Thus, the patterned pallet arrays should enable novel cell separations in which cell selection is based on complex cellular signaling properties

A RhoC Biosensor Reveals Differences in the Activation Kinetics of RhoA and RhoC in Migrating Cells

RhoA and RhoC GTPases share 92% amino acid sequence identity, yet play different roles in regulating cell motility and morphology. To understand these differences, we developed and validated a biosensor of RhoC activation (RhoC FLARE). This was used together with a RhoA biosensor to compare the spatio-temporal dynamics of RhoA and RhoC activity during cell protrusion/retraction and macropinocytosis. Both GTPases were activated similarly at the cell edge, but in regions more distal from the edge RhoC showed higher activation during protrusion. The two isoforms differed markedly in the kinetics of activation. RhoC was activated concomitantly with RhoA at the cell edge, but distally, RhoC activation preceded RhoA activation, occurring before edge protrusion. During macropinocytosis, differences were observed during vesicle closure and in the area surrounding vesicle formation

Kinome and Transcriptome Profiling Reveal Broad and Distinct Activities of Erlotinib, Sunitinib, and Sorafenib in the Mouse Heart and Suggest Cardiotoxicity From Combined Signal Transducer and Activator of Transcription and Epidermal Growth Factor Receptor Inhibition

BACKGROUND: Most novel cancer therapeutics target kinases that are essential to tumor survival. Some of these kinase inhibitors are associated with cardiotoxicity, whereas others appear to be cardiosafe. The basis for this distinction is unclear, as are the molecular effects of kinase inhibitors in the heart. METHODS AND RESULTS: We administered clinically relevant doses of sorafenib, sunitinib (cardiotoxic multitargeted kinase inhibitors), or erlotinib (a cardiosafe epidermal growth factor receptor inhibitor) to mice daily for 2 weeks. We then compared the effects of these 3 kinase inhibitors on the cardiac transcriptome using RNAseq and the cardiac kinome using multiplexed inhibitor beads coupled with mass spectrometry. We found unexpectedly broad molecular effects of all 3 kinase inhibitors, suggesting that target kinase selectivity does not define either the molecular response or the potential for cardiotoxicity. Using in vivo drug administration and primary cardiomyocyte culture, we also show that the cardiosafety of erlotinib treatment may result from upregulation of the cardioprotective signal transducer and activator of transcription 3 pathway, as co-treatment with erlotinib and a signal transducer and activator of transcription inhibitor decreases cardiac contractile function and cardiomyocyte fatty acid oxidation. CONCLUSIONS: Collectively our findings indicate that preclinical kinome and transcriptome profiling may predict the cardiotoxicity of novel kinase inhibitors, and suggest caution for the proposed therapeutic strategy of combined signal transducer and activator of transcription/epidermal growth factor receptor inhibition for cancer treatment

MicroRNA 9-3p Targets 1 Integrin To Sensitize Claudin-Low Breast Cancer Cells to MEK Inhibition

MEK1/2 inhibitors such as AZD6244 are in clinical trials for the treatment of multiple cancers, including breast cancer. Targeted kinase inhibition can induce compensatory kinome changes, rendering single therapeutic agents ineffective. To identify target proteins to be used in a combinatorial approach to inhibit tumor cell growth, we used a novel strategy that identified microRNAs (miRNAs) that synergized with AZD6244 to inhibit the viability of the claudin-low breast cancer cell line MDA-MB-231. Screening of a miRNA mimic library revealed the ability of miR-9-3p to significantly enhance AZD6244-induced extracellular signal-regulated kinase inhibition and growth arrest, while miR-9-3p had little effect on growth alone. Promoter methylation of mir-9 genes correlated with low expression of miR-9-3p in different breast cancer cell lines. Consistent with miR-9-3p having synthetic enhancer tumor suppressor characteristics, miR-9-3p expression in combination with MEK inhibitor caused a sustained loss of c-MYC expression and growth inhibition. The β1 integrin gene (ITGB1) was identified as a new miR-9-3p target, and the growth inhibition seen with small interfering RNA knockdown or antibody blocking of ITGB1 in combination with MEK inhibitor phenocopied the growth inhibition seen with miR-9-3p plus AZD6244. The miRNA screen led to identification of a druggable protein, ITGB1, whose functional inhibition synergizes with MEK inhibitor

MAP3K4 Controls the Chromatin Modifier HDAC6 during Trophoblast Stem Cell Epithelial-to-Mesenchymal Transition

The first epithelial-to-mesenchymal transition (EMT) occurs in trophoblast stem (TS) cells during implantation. Inactivation of the serine/threonine kinase MAP3K4 in TS cells (TSKI4 cells) induces an intermediate state of EMT, where cells retain stemness, lose epithelial markers, and gain mesenchymal characteristics. Investigation of relationships among MAP3K4 activity, stemness, and EMT in TS cells may reveal key regulators of EMT. Here, we show that MAP3K4 activity controls EMT through the ubiquitination and degradation of HDAC6. Loss of MAP3K4 activity in TSKI4 cells results in elevated HDAC6 expression and the deacetylation of cytoplasmic and nuclear targets. In the nucleus, HDAC6 deacetylates the promoters of tight junction genes, promoting the dissolution of tight junctions. Importantly, HDAC6 knockdown in TSKI4 cells restores epithelial features, including cell-cell adhesion and barrier formation. These data define a role for HDAC6 in regulating gene expression during transitions between epithelial and mesenchymal phenotypes