The DNA Replication Initiation Machinery as a Target for Cancer Diagnosis and Therapy

Cancer is a leading cause of death worldwide. Early, accurate detection of malignancy leads to better treatment decisions and there is consequently an urgent need for new cancer biomarkers. This thesis explores the use of the DNA replication initiation machinery as a target for the development of molecular biomarkers able to provide diagnostic and prognostic information in the clinical setting and to guide treatment decisions. In a large scale multicentre study, the DNA replicative helicase protein Mcm5 is shown to be a sensitive and specific biomarker for bladder cancer detection. Further clinical studies demonstrate that Mcm5 is potentially useful for the detection of prostate and pancreaticobiliary tract cancers. In conjunction with markers of mitotic progression and DNA ploidy status, replication initiation proteins are also shown to be able to provide prognostic information in the context of penile cancer. Mcm5 is a component of the DNA replicative helicase, which is phosphorylated by the cell cycle kinase Cdc7 as a crucial step during DNA replication initiation. The work described here demonstrates that depletion of Cdc7 in a normal human diploid cell line induces a novel origin activation checkpoint at the core of which there are a number of tumour suppressor and proto-oncogene proteins that are fre

Analysing engagement in an online management programme and implications for course design

We analyse engagement and performance data arising from participants’ interactions with an in-house LMS at Imperial College London while a cohort of students follow two courses on a new online postgraduate degree in Management. We identify and investigate two main questions relating to the relationships between engagement and performance, drawing recommendations for improved guidelines to inform the design of such courses

Using graph-based modelling to explore changes in students’ affective states during exploratory learning tasks

We describe a graph-based modelling approach to exploring interactions associated with a change in students' affective state when they are working with an exploratory learning environment (ELE). Student-system interactions data collected during a user study was modelled, visualized and queried as a graph. Our findings provide new insights into how students are interacting with the ELE and the effects of the system's interventions on students' affective states

Using graph-based modelling to explore changes in students’ affective states during exploratory learning tasks

This paper describes how graph-based modelling can be used to explore interactions associated with a change in students' affective state when they are working with an exploratory learning environment (ELE). We report on a user study with an ELE that is able to detect students' affective states from their interactions and speech. The data collected during the user study was modelled, visualized and queried as a graph. We were interested in exploring if there was a difference between low- and high-performing students in the kinds of interactions that occurred during a change in their affective state. Our findings provide new insights into how students are interacting with the ELE and the effects of the system's interventions on students' affective states

Differential requirement of a distal regulatory region for pre-initiation complex formation at globin gene promoters

Although distal regulatory regions are frequent throughout the genome, the molecular mechanisms by which they act in a promoter-specific manner remain to be elucidated. The human β-globin locus constitutes an extremely well-established multigenic model to investigate this issue. In erythroid cells, the β-globin locus control region (LCR) exerts distal regulatory function by influencing local chromatin organization and inducing high-level expression of individual β-like globin genes. Moreover, in transgenic mice expressing the entire human β-globin locus, deletion of LCR-hypersensitive site 2 (HS2) can alter β-like globin gene expression. Here, we show that abnormal expression of human β-like globin genes in the absence of HS2 is associated with decreased efficacy of pre-initiation complex formation at the human ɛ- and γ-promoters, but not at the β-promoter. This promoter-specific phenomenon is associated with reduced long-range interactions between the HS2-deleted LCR and human γ-promoters. We also find that HS2 is dispensable for high-level human β-gene transcription, whereas deletion of this hypersensitive site can alter locus chromatin organization; therefore the functions exerted by HS2 in transcriptional enhancement and locus chromatin organization are distinct. Overall, our data delineate one mechanism whereby a distal regulatory region provides promoter-specific transcriptional enhancement

Activation of the β globin locus by transcription factors and chromatin modifiers

Locus control regions (LCRs) alleviate chromatin-mediated transcriptional repression. Incomplete LCRs partially lose this property when integrated in transcriptionally restrictive genomic regions such as centromeres. This frequently results in position effect variegation (PEV), i.e. the suppression of expression in a proportion of the cells. Here we show that this PEV is influenced by the heterochromatic protein SUV39H1 and by the Polycomb group proteins M33 and BMI-1. A concentration variation of these proteins modulates the proportion of cells expressing human globins in a locus-dependent manner. Similarly, the transcription factors Sp1 or erythroid Krüppel-like factor (EKLF) also influence PEV, characterized by a change in the number of expressing cells and the chromatin structure of the locus. However, in contrast to results obtained in a euchromatic locus, EKLF influences the expression of the γ- more than the β-globin genes, suggesting that the relief of silencing is caused by the binding of EKLF to the LCR and that genes at an LCR proximal position are more likely to be in an open chromatin state than genes at a distal position

Molecular architecture of the DNA replication origin activation checkpoint

The tumour suppressors Foxo3a, p15INK4B and Dkk3 serve as non-redundant molecular protectors against a Cdc7-depletion-induced DNA replication checkpoint

Performance of Mcm5 and NMP22 tests for bladder carcinoma detection for all patients with test results available.

<p>Abbreviations: CI, 95% confidence interval; NPV, negative predictive value; PPV, positive predictive value; Sens, sensitivity; Spec, specificity.</p

False positive rates for the Mcm5 and NMP22 tests across benign conditions.

<p>For each subgroup, only those patients with a test results were considered.</p><p>Abbreviations: BPH, benign prostatic hyperplasia; FPR, false positive rate; IQR, interquartile range; med, median; UTI, urinary tract infection.</p>a<p>Mann-Whitney test, comparison of test value with normal.</p>b<p>False positive rate determined using 2150-cell cut-point for Mcm5 test and 10 U/mL cut-point for NMP22 test.</p>c<p>Chi-squared test, comparison of false positive rate with Normal group.</p>d<p>Excludes 53 “other cancers” of the 1354 patients without UCC having an Mcm5 test value.</p>e<p>Excludes 47 “other cancers” of the 1201 patients without UCC having an NMP22 test value.</p

Receiver operating characteristics curves for Mcm5 and NMP22 tests for the detection of bladder cancer in all studied patients with valid test results.

<p>Receiver operating characteristics curves for Mcm5 and NMP22 tests for the detection of bladder cancer in all studied patients with valid test results.</p