Genetic characterisation of six novel African swine fever viruses isolated from a pig, warthog, wild boar, and ticks

African swine fever (ASF) is a disease that affects domestic pigs and wild boars, resulting in up to 100% case fatality rate, and there is currently no effective treatment or vaccine. To date, there are 67 ASFV complete genome sequences available, but most of the sequences represent only genotypes I-V and VII-X of the 24 genotypes identified based on p72 sequencing, limiting inter and intra-genotype comparative studies. ASFVs encode several multigene families (MGFs) involved in virulence and host range which are found at the genomic termini and the majority of genomic differences between isolates are due to the composition of these MGFs. The comparison of the MGFs across ASFV isolates is of utmost importance in understanding genome variability and their contribution to virulence. The p72 gene has historically been used in phylogenetic analysis of ASFV. However, it lacks the capacity for higher resolution between isolates belonging to the same genotype. This study aimed to analyse and characterise six novel ASFV isolates of African origin from a domestic pig, warthog, wild boar and ticks in terms of genomic makeup, MGF composition and phylogenetic relationships, including identification of additional phylogenetic markers, specifically for use in discrimination between closely related isolates. Genomes of six novel isolates were sequenced and annotated by identifying open reading frames (ORFs) with a methionine START codon and performing BLASTx searches of each ORF against the NCBI data base. Differences between the genomes were analysed by generating dotplots and using Base-By-Base which showed them to be mostly collinear, but regions of difference were observed at the termini and the CCR. MGF analysis using sorting and clustering in Morpheus software, based on genotype, serogroup, country, host, virulence, and year, showed that genotype and serogroup play a role in the MGF arrangement patterns. Loci corresponding to regions of difference in the CCR were used for phylogenetic comparison to the previously identified marker p72. The tree topology of all of the alternative phylogenies differed from the current p72 classification. B117L and B169L provided slightly better resolution of genotypes I and II, respectively, and viruses from East Africa that are classified as belonging to genotype IX based on p72 were separated when using EP364R. This data adds to the pool of diverse ASFV isolates available for comparative genomics studies, and to the knowledge of ASFV in Africa. The sequencing of more diverse ASFV isolates of each genotype will help characterise the MGFs arrangement patterns among isolates. The novel alternative phylogenetic markers should further be investigated using more ASFV isolates representing the 24 genotypes described to date

Missing Heritability And Novel Germline Risk Loci In Hereditary Ovarian Cancer: Insights From Whole Exome Sequencing And Functional Analyses

While 25% of ovarian cancer (OVCA) cases are due to inherited factors, most of the genetic risk remains unexplained. This study addressed this gap by identifying previously undescribed OVCA risk loci through the whole exome sequencing (WES) of 48 BRCA1/BRCA2 wild type women diagnosed with OVCA, selected for high risk of genetic inheritance. Five clearly pathogenic variants were identified in this sample, four of which are in two genes featured on current multi-gene panels; (RAD51D, ATM). In addition, a high impact variant in FANCM (R1931*) was identified. FANCM has been recently implicated in familial breast cancer risk but is not yet featured on testing panels. Numerous rare and predicted to be damaging variants of unknown significance were detected in genes on current commercial testing panels. Also, the BRCA2 variant p.K3326*, considered benign but resulting in a 93 amino acid truncation, was overrepresented in our sample (OR= 4.95, p=0.01) and coexisted in the germline of these women with other deleterious variants, suggesting a possible role as a modifier of genetic penetrance. A candidate gene analysis detected loss of function (LOF) variants in genes involved in OVCA relevant pathways; DNA repair and cell cycle control, including FANCM, CHK1, TP53I3, REC8, HMMR, RAD1, and MCM4. Wet lab functional assessment implicated FANCM, CHK1, RAD1 and TP53I3 as having the BRCA-like phenotype typically observed in tumor suppressor genes commonly mutated the germline of women with inherited risk of breast and/or ovarian cancer. Importantly, plotting various panel genes based on cell viability and sensitivity to DNA damage after siRNA knock down correctly differentiated between moderate and high penetrant genes. This technique identified candidate genes CHK1 and RAD1 as high and TP53I3 as moderate in penetrance. The results of this project indicate that WES on study samples filtered for family history and negative for known causal variants is the most appropriate study design for identifying rare and novel high-risk variants. This study implicates novel risk loci as well as highlights the necessity of wet lab functional assessment. Importantly, this study also suggests that wet lab assays may be employed to differentiate moderate from high risk genetic loci

Virtual 3D Reconstruction of Archaeological Pottery Using Coarse Registration

The 3D reconstruction of objects has not only improved visualisation of digitised objects, it has helped researchers to actively carry out archaeological pottery. Reconstructing pottery is significant in archaeology but is challenging task among practitioners. For one, excavated potteries are hardly complete to provide exhaustive and useful information, hence archaeologists attempt to reconstruct them with available tools and methods. It is also challenging to apply existing reconstruction approaches in archaeological documentation. This limitation makes it difficult to carry out studies within a reasonable time. Hence, interest has shifted to developing new ways of reconstructing archaeological artefacts with new techniques and algorithms. Therefore, this study focuses on providing interventions that will ease the challenges encountered in reconstructing archaeological pottery. It applies a data acquisition approach that uses a 3D laser scanner to acquire point cloud data that clearly show the geometric and radiometric properties of the object’s surface. The acquired data is processed to remove noise and outliers before undergoing a coarse-to-fine registration strategy which involves detecting and extracting keypoints from the point clouds and estimating descriptions with them. Additionally, correspondences are estimated between point pairs, leading to a pairwise and global registration of the acquired point clouds. The peculiarity of the approach of this thesis is in its flexibility due to the peculiar nature of the data acquired. This improves the efficiency, robustness and accuracy of the approach. The approach and findings show that the use of real 3D dataset can attain good results when used with right tools. High resolution lenses and accurate calibration help to give accurate results. While the registration accuracy attained in the study lies between 0.08 and 0.14 mean squared error for the data used, further studies will validate this result. The results obtained are nonetheless useful for further studies in 3D pottery reassembly

Analysis of telomere maintenance in artemis defective human cell lines

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.Telomeres are physical ends of chromosomes consisting of (TTAGGG)n DNA sequence and a specialized set of proteins that protect chromosomal ends from degradation and from eliciting DNA damage response. These specialized set of proteins, known as shelterin, directly bind to telomeric DNA. In addition, some DNA double-strand break (DSB) repair proteins such as, DNA-PKcs and KU70/80, play active roles in telomere maintenance. Mouse knock-out experiments have revealed that deletion of either DNA-PKcs or Ku70/80 resulted in elevated levels of telomeric fusion, indicative of dysfunctional telomeres. Artemis protein is involved in DNA DSB repair through non-homologous end joining (NHEJ) and it is phosphorylated by DNAPKcs. Human cells defective in Artemis have been identified and shown to be radiosensitive and patients with an Artemis defective gene suffer from radiosensitive severe-combined immune deficiency syndrome (RS-SCID). Mouse cells defective in Artemis have elevated levels of telomeric fusion. We have demonstrated in this thesis that Artemis defective human cell lines show a mild telomeric dysfunction phenotype detectable at the cytological level. The nature of telomere dysfunction phenotype appears to be similar to that observed in DNAPKcs defective cells as exemplified by the presence of IR induced chromatid telomeric fusions. We have also shown that (a) DNA damage occurring within the telomeric DNA is difficult to repair or irreparable in older cells and that (b) Artemis defective older cells show higher proportion of DNA damage at telomeres than their normal counterparts. Finally, we have demonstrated that inhibition of DNA-PKcs causes (a) an increase in telomeric fusions in Artemis defective cell lines relative to both normal cell lines after inhibition and Artemis cell lines before inhibition and (b)elevated levels of DNA damage at telomeres following exposure of cells to radiation relative to both irradiated normal cells exposed to a DNA-PKcs inhibitor and irradiated Artemis defective cells but not exposed to the DNA-PKcs inhibitor. These results suggest that the effects of Artemis and DNA-PKcs on telomeres are cumulative. We have also performed (a) experiments to examine telomere function in Artemis defective cell lines after knocking down DNA-PKcs levels by RNAi and b) preliminary experiments to knock-down Artemis in DNA-PKcs defective cells. Taken together, our results suggest that the Artemis defect causes mild telomere dysfunction phenotype in human cells

Molecular and Genetic Studies of robo2 Transcriptional Regulation in the Central Nervous System of Drosophila melanogaster

Drosophila Robo2 axon guidance receptor is a member of the evolutionarily conserved Roundabout (Robo) protein family that is involved in directing axons that cross the midline to the other side of the animal body. Robo2 roles mainly depend on two factors: The functional domains of the Robo2 protein, which is extensively studied, and the dynamic transcription of robo2 in various subsets of cells throughout embryogenesis which is not fully understood. Thus, knowing robo2 enhancers that transcriptionally regulate robo2 during embryogenesis is significant. To investigate robo2 potential enhancers, we screened 17 transgenic lines of Drosophila that were generated by Janelia Research Center. These lines contain 17 fragments distributed within and around the robo2 gene. We identified six fragments that regulated robo2 expression by the GAL4-UAS-GFP system suggesting that they were promising enhancers. Using these identified regulatory fragments in addition to three fragments generated in our lab, we built the HA-Robo2 transgenic constructs. These constructs were introduced into Drosophila which allowed us to test robo2 expression and its dependent axon guidance phenotypes in the embryonic CNS. GMR28G05 and GMR28F02 fragments showed the strongest robo2 expression in the lateral pathway. To further study these fragments, we introduced them separately or together into robo2 null mutant background. We found that Robo2’s dynamic expression pattern is specified by multiple regulatory regions. We utilized these fragments to generate and characterize an equivalent set of robo2 transgenes expressing the axonal marker TauMyc instead of the HA-Robo2 and hsp70 promoter instead of robo2 promoter. The results show that GMR28F02 fragment drove strong expression of TauMyc in a subset of the lateral neurons, cell bodies, and commissural axon from which Robo2 protein is expressed. CRISPR/Cas9 system was used to further investigate the importance of our findings. Cas9 protein and specific gRNAs were used to target and delete robo2 potential enhancers (GMR28G05 and GMR28F02) separately or together. Applying bioinformatics tools and literature I predicted three transcription factors (Hb9, Nkx6.1, and Lhx2) that have a high probability to bind robo2 potential enhancers. In summary, robo2 has potential enhancers located in the first intron and upstream of the gene, and multiple enhancers more efficiently regulated robo2 expression in Drosophila