Cell fate restriction in Caenorhabditis elegans is orchestrated by precise chromatin organization and transcription factor activity

The plasticity of cells in a multicellular organism is progressively lost during differentiation. This loss is reflected in studies involving the ectopic misexpression of fate-specifying or terminal selector transcription factors (TFs). These TFs can efficiently activate target genes in undifferentiated cells, but lose this ability as cells differentiate. While this phenomenon of cell fate restriction is widely observed, the mechanisms orchestrating it are poorly understood. In this thesis, I have used the ubiquitous overexpression of Zn-finger-TF CHE-1 as a tool to understand the mechanisms that restrict cell fate in Caenorhabditis elegans. When CHE-1 is ubiquitously expressed at embryonic stages, it activates target gene expression in many cell types, while in adults it can only act in a few neurons. To uncover factors that inhibit plasticity of all other adult cells, I first performed an RNAi screen against chromatin-associated factors. Using this approach I found that the removal of either the PRC2 complex, which deposits the H3K27me3 mark, or loss of proteins that indirectly regulate domains of H3K27me3, allows CHE-1 and two other terminal selector TFs to activate target genes in the germline. These data show that the correct distribution of H3K27me3 is crucial for the restriction of germ cell fate. I next took a candidate approach to identify genes that regulate fate restriction in other cell types. We hypothesized that terminal selector TFs themselves, in addition to specifying cellular identity by controlling large gene sets, may also act to inhibit plasticity. To test this, I first assayed the activity of CHE-1 in mutants of COE-TF unc-3, the terminal selector for a subset of cholinergic motor neurons (MNs). I found that in contrast to wildtype MNs, unc-3 mutant MNs remain plastic as CHE-1 can induce expression of target genes in these cells even at the adult stage. This phenotype is also observed in four of six additional terminal selector mutants tested. I further found that the removal of met-2, a protein required for H3K9 methylation, or mes-2, a PRC2 component, also makes differentiated cholinergic MNs amenable to the activity of CHE-1. Preliminary evidence suggests that met-2 may act in the same pathway as unc-3. These results raise the exciting possibility that selector TFs play a role in restricting cell fate by organizing the heterochromatin domains in differentiated cells. Overall, in this work I provide functional evidence to show that specific chromatin-modifying enzymes restrict the fate of germ cells and that both fate-specifying TFs and chromatin-modifying enzymes are required for the fate restriction in neurons

Investigating genetic variation in Alzheimer’s disease

Alzheimer’s disease (AD) is the most common form of dementia, now the leading cause of death in the UK, which affects more than 35 million people worldwide. Genome-wide association studies identified 20 genetic loci associated with disease susceptibility, however these only exerted small effects on risk. Next-generation sequencing is now being employed to identify more of the missing heritability. This project utilised whole-exome sequencing to explore genetic variation using the Brains for Dementia Research (BDR) resource, a well-characterised cohort of neuropathologically confirmed samples. Exome-wide and candidate gene approaches were employed to assess coding variants for association with AD, using single-variant and burden tests. Coding variants in other neurodegenerative disease genes were also analysed as potential susceptibility factors for AD. Furthermore, polygenic risk scores (PRS) were generated to explore the ability to classify case and control individuals based on their genetic profiles. A synonymous variant in PILRA (rs2405442) was nominally associated with 3-fold increased risk of AD, also contributing strongly to PILRA burden. It was previously linked to AD through risk gene ZCWPW1; however, it has not been directly associated until now. Additional variants in GWAS gene ABCA7 (rs3764645, rs3752234, rs3752237, rs4147915) and rare variants in CLU were also implicated, further supporting their roles in AD susceptibility. A variant in PD gene LRRK2 (rs35303786) inferred protection against AD, implicating potential pleiotropy across the two diseases. PRS could distinguish AD cases from controls with 85.3% accuracy and also identified controls with high PRS but no cognitive impairments. This could be useful for identifying individuals at risk of developing AD in the future. We have uncovered tentative associations both in established and newly identified loci; highlighting several interesting candidates for further investigation. Although there remains a large amount of missing heritability, we hope that as the BDR resource grows, we will achieve increased power to detect significant associations with AD

Assessing the impact of administering the 'Challenges of Living with Cystic Fibrosis' questionnaire on the self-efficacy of caregivers of children with cystic fibrosis

INTRODUCTION Cystic Fibrosis is a multi-organ, genetic disorder with the most pronounced effects observed in the lungs, pancreas and the gastrointestinal system. From a young age, patients suffer from chronic pulmonary infections, poor digestion and malabsorption of nutrients from the gut. As a result, patients have to perform numerous therapeutic tasks regularly to remain healthy. These treatments come at the expense of a considerable treatment burden and burden of care to the patient and family involved. METHODS The ‘Challenges of Living with Cystic Fibrosis Questionnaire’ (CLCF-Q) has been developed to measure the burden felt by caregivers of children with CF. The aim of this study was to investigate the effect of administering the CLCF-Q to caregivers during their child’s annual review on the carer’s self-efficacy. Self-efficacy was measured by the ‘Cystic Fibrosis Self-Efficacy Questionnaire’ (CFSE-Q). Participants were randomised into the intervention or control groups. Those in the intervention group completed the CLCF-Q during the annual review process and received feedback at a later date. All participants completed the CFSE-Q at baseline and endpoint to measure their self-efficacy. RESULTS Thirty seven participants (17 Intervention, 20 Control) completed the whole study. The CLCF-Q identified some of the burdens faced by caregivers of children with CF. Total self-efficacy scores ranged from 36-53 in the intervention group and 37-51 in the control group at baseline. End-point scores ranged from 38-54 for the intervention group and 31-53 for the control group. Statistical analysis revealed that the self-efficacy of the intervention group remained stable over time and that of the control group decreased significantly by the end of the study. Self-efficacy scores decreased for six items and increased for six items in the intervention group. Only two items had higher scores in the control group and 10 items had lower scores at the end of the study. DISCUSSION The CLCF-Q may be used as a tool to measure burden in caregivers of children with CF. It helps health care providers recognise the burdens faced by these families. The feedback process aims to involve the caregiver and encourage them in areas of treatment which they find challenging. The combination of these two processes as an intervention may be used to maintain a high level of self-efficacy in caregivers of children with CF. The CFSE-Q was able to detect changes in self-efficacy and may be used in future self-efficacy trials in caregivers of patients with CF

Flowcharts improve periodontal diagnosis by dental and dental hygiene students

BACKGROUND: In 2017, the American Academy of Periodontology and the European Federation of Periodontology updated the classification of periodontal and peri-implant diseases and conditions. The goal of the present crossover study was to develop straightforward, illustrative flowcharts and determine their impact on the accuracy and speed of diagnosing periodontal conditions by predoctoral dental students (DS) and dental hygiene students (DHS). METHODS: Two flowcharts (a decision-tree flowchart and one based on the periodontal disease/condition entity) were developed using updated diagnostic determinants proposed by the 2017 classification. A total of 26 second-, third-, and fourth-year DS (DS2, DS3, and DS4, respectively) and second-year DHS (DHS2) took a mock examination consisting of 10 periodontal clinical cases. The participants first diagnosed periodontal conditions using only their curricula-based knowledge (control) and then using the flowcharts (test). They also completed an optional post-examination questionnaire to provide feedback on the flowcharts. Statistical significance was detected at RESULTS: Combined test groups had significantly higher accuracy in diagnosing periodontal conditions compared to controls (73.5% vs 50.0%, respectively), with the most substantial improvement in DS2 (66.3% vs 30%, respectively) and DHS2 (70.0% vs 41.4%, respectively). Combined test groups also completed the examination more quickly compared to controls (14.92 vs 20.85 minutes, respectively). The participants provided positive feedback and constructive criticism on the flowcharts, and also suggested converting them into application software. CONCLUSION: The flowcharts significantly improved the accuracy of diagnosing periodontal conditions in academic settings, especially among junior, less experienced participants

Investigating splicing variants uncovered by next-generation sequencing the Alzheimer’s disease candidate genes, CLU, PICALM, CR1, ABCA7, BIN1, the MS4A locus, CD2AP, EPHA1 and CD33

Late onset Alzheimer’s disease (LOAD), the most common cause of late onset dementia, has a strong genetic component. To date, 21 disease-risk loci have been identified through genome wide association studies (GWAS). However, the causative functional variant(s) within these loci are yet to be discovered. This study aimed to identify potential functional splicing mutations in the nine original GWAS-risk genes: CLU, PICALM, CR1, ABCA7, BIN1, the MS4A locus, CD2AP, EPHA1 and CD33. Target enriched next generation sequencing (NGS) was used to resequence the entire genetic region for each of these GWAS-risk loci in 96 LOAD patients and in silico databases were used to annotate the variants for functionality. Predicted splicing variants were further functionally characterised using splicing prediction software and minigene splicing assays. Following in silico annotation, 21 variants were predicted to influence splicing and, upon further annotation, four of these were examined utilising the in vitro minigene assay. Two variants, rs881768 A>G in ABCA7 and a novel variant 11: 60179827 T>G in MS4A6A were shown, in these cell assays, to affect the splicing of these genes. The method employed in the paper successfully identified potential splicing variants in GWAS-risk genes. Further investigation will be needed to understand the full effect of these variants on LOAD risk. However, these results suggest a possible pipeline in order to identify putative functional variants as a result of NGS in disease-associated loci although improvements are needed within the current prediction programme in order to reduce the number of false positives

Spatial search using the discrete time quantum walk

We study the quantum walk search algorithm of Shenvi et al. (Phys Rev A 67:052307, 2003) on data structures of one to two spatial dimensions, on which the algorithm is thought to be less efficient than in three or more spatial dimensions. Our aim is to understand why the quantum algorithm is dimension dependent whereas the best classical algorithm is not, and to show in more detail how the efficiency of the quantum algorithm varies with spatial dimension or accessibility of the data. Our numerical results agree with the expected scaling in 2D of O(√N log N}) , and show how the prefactors display significant dependence on both the degree and symmetry of the graph. Specifically, we see, as expected, the prefactor of the time complexity dropping as the degree (connectivity) of the structure is increased

Quantum walks: a comprehensive review

Quantum walks, the quantum mechanical counterpart of classical random walks, is an advanced tool for building quantum algorithms that has been recently shown to constitute a universal model of quantum computation. Quantum walks is now a solid field of research of quantum computation full of exciting open problems for physicists, computer scientists, mathematicians and engineers. In this paper we review theoretical advances on the foundations of both discrete- and continuous-time quantum walks, together with the role that randomness plays in quantum walks, the connections between the mathematical models of coined discrete quantum walks and continuous quantum walks, the quantumness of quantum walks, a summary of papers published on discrete quantum walks and entanglement as well as a succinct review of experimental proposals and realizations of discrete-time quantum walks. Furthermore, we have reviewed several algorithms based on both discrete- and continuous-time quantum walks as well as a most important result: the computational universality of both continuous- and discrete- time quantum walks.Comment: Paper accepted for publication in Quantum Information Processing Journa

Saltatory remodeling of Hox chromatin in response to rostrocaudal patterning signals

Hox genes controlling motor neuron subtype identity are expressed in rostrocaudal patterns that are spatially and temporally collinear with their chromosomal organization. Here we demonstrate that Hox chromatin is subdivided into discrete domains that are controlled by rostrocaudal patterning signals that trigger rapid, domain-wide clearance of repressive histone H3 Lys27 trimethylation (H3K27me3) polycomb modifications. Treatment of differentiating mouse neural progenitors with retinoic acid leads to activation and binding of retinoic acid receptors (RARs) to the Hox1–Hox5 chromatin domains, which is followed by a rapid domain-wide removal of H3K27me3 and acquisition of cervical spinal identity. Wnt and fibroblast growth factor (FGF) signals induce expression of the Cdx2 transcription factor that binds and clears H3K27me3 from the Hox1–Hox9 chromatin domains, leading to specification of brachial or thoracic spinal identity. We propose that rapid clearance of repressive modifications in response to transient patterning signals encodes global rostrocaudal neural identity and that maintenance of these chromatin domains ensures the transmission of positional identity to postmitotic motor neurons later in development.Leona M. and Harry B. Helmsley Charitable TrustNational Institutes of Health (U.S.) (Grant P01 NS055923)Smith Family Foundatio

Observations of extensive gene expression differences in the cerebellum and potential relevance to Alzheimer's disease

Objectives: In order to determine how gene expression is altered in disease it is of fundamental importance that the global distribution of gene expression levels across the disease-free brain are understood and how differences between tissue types might inform tissue choice for investigation of altered expression in disease state. The aim of this pilot project was to use RNA-sequencing to investigate gene expression differences between five general areas of post-mortem human brain (frontal, temporal, occipital, parietal and cerebellum), and in particular changes in gene expression in the cerebellum compared to cortex regions for genes relevant to Alzheimer’s disease, as the cerebellum is largely preserved from disease pathology and could be an area of interest for neuroprotective pathways. Results: General gene expression profiles were found to be similar between cortical regions of the brain, however the cerebellum presented a distinct expression profile. Focused exploration of gene expression for genes associated with Alzheimer’s disease suggest that those involved in the immunity pathway show little expression in the brain. Furthermore some Alzheimer’s disease associated genes display significantly different expression in the cerebellum compared with other brain regions, which might indicate potential neuroprotective measures