Deepr: A Convolutional Net for Medical Records

Feature engineering remains a major bottleneck when creating predictive systems from electronic medical records. At present, an important missing element is detecting predictive regular clinical motifs from irregular episodic records. We present Deepr (short for Deep record), a new end-to-end deep learning system that learns to extract features from medical records and predicts future risk automatically. Deepr transforms a record into a sequence of discrete elements separated by coded time gaps and hospital transfers. On top of the sequence is a convolutional neural net that detects and combines predictive local clinical motifs to stratify the risk. Deepr permits transparent inspection and visualization of its inner working. We validate Deepr on hospital data to predict unplanned readmission after discharge. Deepr achieves superior accuracy compared to traditional techniques, detects meaningful clinical motifs, and uncovers the underlying structure of the disease and intervention space

An ATM and ATR dependent checkpoint inactivates spindle assembly by targeting CEP63

The effects of ATM and ATR signalling induced by chromosomal breakage have been described extensively in modulating cell cycle progression up to the onset of mitosis. However, DNA damage checkpoint responses in mitotic cells are not well understood. This thesis reports on the effects of double strand breaks on the progression of mitosis. We found ATM and ATR activation can occur in mitotic Xenopus laevis egg extract and the induction of ATM and ATR by chromosomal breakages inhibits spindle assembly in both Xenopus egg extract and somatic cells. The delay in mitotic progression induced by ATM and ATR was found not to involve major spindle assembly factors activities such as, Cdk1, Plx1 and RCC1/Ran-GTP. However, normal anastral spindles formation around linear DNA coated beads, which can activate ATM and ATR, linked centrosome-driven spindle assembly to ATM and ATR dependent spindle defects. cDNA expression library screening was undertaken to identify novel ATM and ATR targets in this mitotic checkpoint pathway, through which the novel centrosomal protein XCEP63 was identified as a likely candidate. Data obtained from depletion and reconstitution of XCEP63 in Xenopus egg extract established that normal centrosome-driven spindle assembly requires XCEP63. Moreover, ATM and ATR phosphorylates XCEP63 on serine 560 and promotes delocalisation from the centrosome. ATM and ATR inhibition or addition of non-phosphorylable XCEP63 recombinant protein mutated at serine 560 prevents spindle assembly abnormalities. These findings suggest that ATM and ATR regulate mitotic events by targeting XCEP63 and centrosome-dependent spindle assembly. This pathway may provide support for DNA repair processes or regulate cell survival in the presence of mitotic DNA damage

Live-cell visualisation of chromatin for the study of radiation-induced chromosome aberrations

This thesis was submitted for the degree of Master of Philosophy and awarded by Brunel University.Chromosomes can be visualised during the metaphase stage of the cell cycle however, there are various limitations associated with the visualisation of chromatin during interphase. This is due to the fact that during that stage of the cell cycle, chromatin is decondensed and difficult to resolve by using standard techniques. The employment of fluorescence microscopy has provided great insight into the examination of the structure and dynamics of fluorescently tagged chromatin regions in living cells during interphase. Thus the in vivo stable expression of fluorescent proteins such as Enhanced Green Fluorescent Protein (EGFP) has the potential to address questions that lead to understanding dynamic responses of chromatin in response to DNA damage. In this study, chromatin sites within the nuclei of Chinese Hamster Ovary (CHO) cells were fluorescently labelled using the lac operon system, which works on the principle that EGFP-lac repressor protein will bind to sites of integrated lac operator sequence. To achieve this, CHO cells were transfected with pSV2-DHFR8.32 (lac operator) and p3‟SSdimerClonEGFP (lac repressor-EGFP fusion protein) plasmids by calcium phosphate transfection. 68 stable transfectants were stored and clone CHO-R-O-25 cells were used for further experimental analysis based on these cells containing discrete and intense fluorescent spots. It was shown that the physiological and functional characteristics of CHO-R-O-25 cells were comparable to those of control CHO DG44 cells. Time-lapse fluorescence microscopy was used to examine for any alterations in the physical structure of chromatin in response to DNA damage that was induced by radiation and a range of different chemicals. It was demonstrated that in cells exposed to DNA damaging agents, chromatin diffusion and expansions revealed by alterations in the intensity and size of the fluorescent spots within the nuclei of cells are owed to relaxation of chromatin structures in response to genome insults. The potential for this tool to study the organisational responses of chromatin in response to damage and the ulitimate relevance of any physical alterations to mechanisms for chromosome exchange formation, are described

Deep Learning for Classification of Brain Tumor Histopathological Images

Histopathological image classification has been at the forefront of medical research. We evaluated several deep and non-deep learning models for brain tumor histopathological image classification. The challenges were characterized by an insufficient amount of training data and identical glioma features. We employed transfer learning to tackle these challenges. We also employed some state-of-the-art non-deep learning classifiers on histogram of gradient features extracted from our images, as well as features extracted using CNN activations. Data augmentation was utilized in our study. We obtained an 82% accuracy with DenseNet-201 as our best for the deep learning models and an 83.8% accuracy with ANN for the non-deep learning classifiers. The average of the diagonals of the confusion matrices for each model was calculated as their accuracy. The performance metrics criteria in this study are our model’s precision in classifying each class and their average classification accuracy. Our result emphasizes the significance of deep learning as an invaluable tool for histopathological image studies

Modulation of Mcm2-7 activity by Cdtl suggests novel roles for Cdtl in the control of DNA replication

Genome duplication occurs once and only once during each cell cycle. It is a highly ordered process and is separated into the formation of different multi-protein complexes. The pre-replicative complex (preRC) is formed during G1 -phase and is composed of ORC, Cdc6, Cdtl and Mcm2-7. Mcm2-7 is the replicative helicase in eukaryotic cells and is assembled on replication origins prior to S-phase. Cdtl is an essential component of the preRC. Cdtl has been shown to interact with Mcm2-7, however neither the requirements nor the effects of this interaction have explored. In this study, I show that Cdtl forms a complex with Mcm2-7 without the need for other factors. Furthermore, Cdtl modulates the helicase, ATPase and DNA binding activity of the Mcm2-7 complex. I propose a model where Cdtl modulates Mcm2-7 helicase activity by inhibiting ATP hydrolysis by Mcm2-7, thus preventing premature DNA unwinding. Furthermore, the increase in dsDNA binding affinity for the Mcm2-7/Cdtl complex (Mcm2-7*Cdtl) assists in its loading onto replication origins during preRC assembly. These results indicate a novel role for Cdtl in Mcm2-7 modulation and DNA replication control

Biochemical and biophysical characterisation of the Saccharomyces cerevisiae cell-cycle transcription factors, SBF and MBF

MBF and SBF are two large protein complexes involved in cell-cycle-dependent transcriptional regulation in Saccharomyces cerevisiae. These protein complexes bind to DNA sequences called MCBs (Mlu I cell-cycle box) and SCBs (Swi4/Swi6 cell-cycle box) upstream of promoters of many genes that regulate the G1-S phase transition. The binding of MBF to MCBs and SBF to SCBs is highly regulated and requires the association of Mbp1p with Swi6p (MBF), and Swi4p with Swi6p (SBF) through their C-terminal regions. This thesis describes biochemical and biophysical experiments that address crucial protein-protein and protein-DNA interactions involved in SBF and MBF-mediated regulation of the budding yeast cell-cycle. Plasmids expressing various C-terminal fragments of Swi6p, Swi4p, and Mbp1p, containing the heteromerisation regions, have been constructed and their respective proteins purified for structural characterisation. Initially, hydrodynamic and chemical cross-linking data suggested that a C-terminal 18kDa fragment was dimeric. This feature was later shown to be an artefact of disulphide formation in vitro. Mutation of a single conserved cysteine residue within the C-terminus of Swi6p abrogates this dimerisation, but does not show any effect on SBF-mediated transcriptional activation in vivo. The circular dichroic (CD) analyses of the C-terminal Swi6p fragments show a substantial proportion of α-helical secondary structure, verified by comparisons of CD spectra of the full-length molecule and several N- and C-terminally deleted fragments. Limited proteolysis of the 18kDa fragment suggests a bipartite helical domain within the extreme C-terminal region that is required for SBF and MBF interaction. Mutational studies of the DNA-binding region of Mbp1p (1-124) that contains a winged helix-turn-helix motif suggest that the non-conserved residues C-terminal to the core domain are essential for DNA-binding. Mutations of Lys116 (K116A) and Lys122 (K122A) show a reduction in the apparent DNA-binding affinities for the MCB oligonucleotide duplex with respect to the wild-type protein. However, in combination, these two mutations (K116A/K122A) result in a more significant reduction in the apparent binding affinity. The results support the notion of two structurally distinct DNA-binding regions within Mbp1p and related proteins

Advances in quantitative microscopy

Microscopy allows us to peer into the complex deeply shrouded world that the cells of our body grow and thrive in. With the emergence of automated digital microscopes and software for anlysing and processing the large numbers of image that they produce; quantitative microscopy approaches are now allowing us to answer ever larger and more complex biological questions. In this thesis I explore two trends. Firstly, that of using quantitative microscopy for performing unbiased screens, the advances made here include developing strategies to handle imaging data captured from physiological models, and unsupervised analysis screening data to derive unbiased biological insights. Secondly, I develop software for analysing live cell imaging data, that can now be captured at greater rates than ever before and use this to help answer key questions covering the biology of how cells make the decision to arrest or proliferate in response to DNA damage. Together this thesis represents a view of the current state of the art in high-throughput quantitative microscopy and details where the field is heading as machine learning approaches become ever more sophisticated.Open Acces

Microarray expression analysis of meiosis and microsporogenesis in hexaploid bread wheat

BACKGROUND: Our understanding of the mechanisms that govern the cellular process of meiosis is limited in higher plants with polyploid genomes. Bread wheat is an allohexaploid that behaves as a diploid during meiosis. Chromosome pairing is restricted to homologous chromosomes despite the presence of homoeologues in the nucleus. The importance of wheat as a crop and the extensive use of wild wheat relatives in breeding programs has prompted many years of cytogenetic and genetic research to develop an understanding of the control of chromosome pairing and recombination. The rapid advance of biochemical and molecular information on meiosis in model organisms such as yeast provides new opportunities to investigate the molecular basis of chromosome pairing control in wheat. However, building the link between the model and wheat requires points of data contact. RESULTS: We report here a large-scale transcriptomics study using the Affymetrix wheat GeneChip(® )aimed at providing this link between wheat and model systems and at identifying early meiotic genes. Analysis of the microarray data identified 1,350 transcripts temporally-regulated during the early stages of meiosis. Expression profiles with annotated transcript functions including chromatin condensation, synaptonemal complex formation, recombination and fertility were identified. From the 1,350 transcripts, 30 displayed at least an eight-fold expression change between and including pre-meiosis and telophase II, with more than 50% of these having no similarities to known sequences in NCBI and TIGR databases. CONCLUSION: This resource is now available to support research into the molecular basis of pairing and recombination control in the complex polyploid, wheat