Localising and quantifying damage by means of a multi-chromosome genetic algorithm

This paper presents a structural damage detection methodology based on genetic algorithms and dynamic parameters. Three chromosomes are used to codify an individual in the population. The first and second chromosomes locate and quantify damage, respectively. The third permits the self-adaptation of the genetic parameters. The natural frequencies and mode shapes are used to formulate the objective function. A numerical analysis was performed for several truss structures under different damage scenarios. The results have shown that the methodology can reliably identify damage scenarios using noisy measurements and that it results in only a few misidentified elements. (C) 2012 Civil-Comp Ltd and Elsevier Ltd. All rights reserved.CNPq (Brazilian National Council for Technological and Scientific Development)Brazilian National Council for Scientific and Technological Development (CNPq

Functional identification and investigation of genes initiating chromosomal instability using CRISPR activation and high-throughput automated image analysis

Chromosomal instability (CIN), the dynamic state where cells experience increased structural and/or numerical chromosome segregation errors, is prevalent in cancer, where it contributes to aneuploidy and tumour evolution. Despite its profound consequences on human health, initiation of CIN in the early stages of tumourigenesis is not well understood. In this work multiple strategies were employed to investigate the effects of gene upregulation, as well as upregulation of the PIK3CA signalling pathway, a frequently altered pathway in many cancer types (Jamal-Hanjani et al. 2017; Teixeira et al. 2019). Combining CRISPR gene upregulation to model overexpression, high content imaging (HCS), and automated high-throughput image analysis, a pipeline was developed to screen for CIN and aneuploidy in a normal human cell line, RPE1. This provides a readout of micronuclei and centromere counts. Using this pipeline, upregulation of KIF11 was found to increase the proportion of cells exhibiting micronuclei, and cause significant deviation from the modal centromere count, indicating CIN and aneuploidy. Further investigation of this phenotype revealed that KIF11 upregulation causes spindle pole fragmentation, mitotic catastrophe, and chromosome congression defects. Centric and acentric lagging chromosomes were observed in cells that exhibited both normal and fragmented spindle poles. Mechanistically, KIF11 was shown to generate a force imbalance in the early stages, which could be partially rescued upon upregulation of HSET. MCF10A cell lines expressing PIK3CAH1047R at the endogenous genetic loci as a result of CRISPR genome editing were used to investigate the impact of increased signalling through the PIK3CA pathway on CIN, aneuploidy, and centrosome biology. This showed that PIK3CAH1047R increased the incidence of supernumerary centrosomes, and may play a role in structural CIN, but failed to identify any effect on numerical CIN. Finally, by chemically modulating PIK3CA activity, microtubule dynamics in response to PIK3CA pathway activation and inhibition were investigated

Unravelling the genetic base of the meiotic recombination landscapes in two varieties of the button mushroom, Agaricus bisporus

The button mushroom, Agaricus bisporus var. bisporus, is one of the most cultivated mushrooms worldwide. Even though wild isolates of this variety have a broad genetic variation, the traditional and present-day hybrids only have a very narrow genetic base. The button mushroom has a typical meiotic recombination landscape (MRL) in which crossover (CO) events are predominantly restricted to the extreme ends of the chromosomes. This has been one of the main obstacles for mushroom breeders in improving or generating new mushroom hybrids due to a considerable linkage drag. A wild variety of A. bisporus, i.e., burnettii appeared to have CO spread more evenly across the genome. The existence of two extremely different MRLs in two compatible A. bisporus varieties offers an excellent opportunity to study the genetic basis for positioning CO in meiosis. The main objective of the research presented in this thesis initially was to examine meiosis of the var. burnettii in more detail and subsequently to identify genomic regions revealing the difference in MRL of the two A. bisporus varieties. The availability of genome sequences in the bisporus variety has produced many more informative markers such as SNP. We aimed to de novo sequence one of the haplotypes of a heterokaryotic strain of the burnettii variety using the PacBio sequencing technique and resequencing the other haplotype using Illumina HiSeq. In parallel to this, we used Genotyping by Sequencing (GBS) to construct the first linkage map of the burnettii variety, showing a more or less even distribution of COs across the genome. The constructed linkage map has also proved to be a useful tool for de novo assembly of the burnettii variety genome sequence. In addition, we performed comparative genome sequence studies between the burnettii variety and the previously sequenced genomes of two of the bisporus variety homokaryons, indicating high levels of collinearity between all three genomes. The only chromosomal rearrangement to be found was on chromosome 10, where an inversion of ~ 800 kb in the burnettii variety was detected compared to the var. bisporus genomes. As a starting point for unravelling the genetic basis underlying MRL in the A. bisporus, we performed quantitative trait loci (QTL) analysis using bisporus and burnettii varieties. An inter-varietal population was developed from a cross between a constituent nucleus of the bisporus and the burnettii variety. This population contains 178 haploid progenies which were genotyped by 210 SNP markers to construct a genetic linkage map, which proves to be a solid foundation for exploring the genetic control of MRL of A. bisporus. In addition, we performed a comparative genetic mapping study using the genetic maps of the bisporus variety Horst U1, the burnettii variety Bisp119/9 and the inter-varietal hybrid by selecting markers having similar positions in these three maps. In contrast to the bisporus variety where CO events are mainly restricted to chromosome ends, the burnettii variety shows a more or less equal distribution of CO events across the entire genome. The recombination landscape of the inter-varietal hybrid shows an intermediate pattern to that of both varieties. The MRL trait is expressed as a CO event in the offspring of each individual of the inter-varietal mapping population. For this reason, the individuals of the inter-varietal mapping population were intercrossed and outcrossed to generate three types of second generation hybrids. Two compatible tester homokaryons derived from the bisporus and burnettii varieties were used for outcrossing. Subsequently, the haploid progenies from each type of second generation hybrids were isolated to generate three types of segregating populations. The haploid progenies from segregating populations were genotyped with SNP markers covering the whole length of all the chromosomes. Recombination frequencies were determined at distal ends and elsewhere on the chromosomes and used to compare recombination frequencies between chromosomes within each population as well as between segregating populations across all chromosomes. A prerequisite for successful QTL mapping the MRL is to select segregating populations in which the segregation of MRL is clear. We observed that segregating populations outcrossed with the bisporus tester homokaryon were the most useful populations to generate haploid offspring in which COs are assessed for further QTL study of MRL at the time when this research was carried out. To map genomic regions involved in the different MRLs of A. bisporus, 71 homokaryotic offspring of the inter-varietal hybrid were outcrossed with an unrelated tester homokaryon of the bisporus variety. Subsequently, the haploid progenies were isolated from each hybrid and genotyped with SNP markers. Marker pairs were generated for the end regions of chromosomes to assess CO there or anywhere else on the chromosomes for each segregating population. QTL mapping analysis revealed two QTLs located on chromosome l and three others located on chromosomes IV, VI and VII. The QTLs identified span large parts of their respective chromosomes; therefore further strategies are needed for a more precise assessment and localisation of MRL.</p

Single-molecule protein dynamics during DNA replication

Observing the processes of life that occur in all cellular organisms at the level of single molecules has allowed a deeper understanding of the dynamic processes taking place in complex biological systems. There has been a strong growth in the application of molecular biophysics to visualize in real time the behaviour of single molecules within a reaction, transforming our perception of the molecular processes that occur within a cell. A multitude of proteins participates across the genome to support the processes of replication, transcription, translation, repair and recombination. The continuous interplay of these proteins on the DNA produces unavoidable physical conflicts that have their own impact on genomic stability. Beyond the complexities of the cellular processes that involve DNA as a reaction partner, the duplex is also constantly exposed to DNA-damaging agents as a result of environmental factors such as UV radiation and oxidative stress. It comes as no surprise that replisomes frequently stall and dissociate because of encounters with DNA damage or tightly-bound protein-DNA complexes. In bacteria, such genomic instability can result in the genetic changes that drive antibiotic resistance evolution. Genomic stability is maintained through pathways that ensure continued replication by minimising the frequency or impact of collisions and identifying and repairing stalled forks. The methodologically diverse toolkit of single-molecule biophysics has been used to address a wide range of questions related to complex protein machineries. Specifically, this thesis highlights the application of single-molecule fluorescence methods to visualize and characterize DNA and the proteins that interact with it. In addition, it describes methodological advances that have been made to utilize linear DNA substrates to uncover protein dynamics. The overall goal of the projects described in this thesis was to design protocols and workflows for the production of linear DNA substrates which are (1) easily customizable to adjust for different experimental parameters and (2) which could be utilized to address a diverse range of biological questions, with a key focus on the controlled introduction of specific chemical lesions. This protocol was employed in support of answering a specific question: How do polymerase exchange dynamics affect lesion bypass mechanisms? This thesis focuses on the protein dynamics that occur at the replication fork in the context of roadblocks and lesions. For the first time, we observe replisome collisions with site-specific cyclobutane pyrimidine dimer lesions on linear substrates at the single-molecule level. This assay presents an exciting avenue to unveil further details of replication stalling and restart. Furthermore, this assay can be adapted to introduce a diverse range of roadblocks, to study dynamics of repair proteins at replication forks and observe the behavior of other replisome complexes. Classical biochemical and single-molecule techniques have provided insight into the proteins and macromolecular complexes responsible for rescue of stalled DNA replication forks. While the majority of studies have employed a reductionist approach in focusing on functions of isolated enzymes, recent work has started to explore the reconstitution of multiple-protein complexes of replication and repair pathways on single molecules of DNA. As we gain more knowledge of the dynamics and mechanisms observed at the single-molecule level, we will see emerging a more detailed picture of the molecular steps associated with the rescue stalled forks. This thesis represents an important step towards that more refined understanding

Engineering morphogenesis of Marchantia polymorpha gemmae

Morphogenesis is an apparent yet complex process: emergence of a plant shape is the result of an intricate interplay between genetic regulation, cell physiology and mechanical processes at the tissue scale. Morphogenesis spans three levels of biological organisation, forming a nested complex system. Genes, which form the lowest level of the system, are arranged into networks that control properties of cells. Cells, which form the middle level of the system, are arranged into geometric networks, and their mechanical and chemical interactions give rise to the morphology of a whole organism. Therefore, the study of plant morphogenesis relies on understanding how genetically-driven cellular interactions influence the formation of a plant shape. Clonal propagules of Marchantia polymorpha (Marchantia), also known as gemmae, are an attractive system for studying these interactions. Gemmae are small, have a simple disk-like shape and are resilient to environmental conditions. As such, they are well-suited for fluorescent microscopy and the collection of gene expression data. These features create an opportunity to study the processes of gemma morphogenesis at tissue, cellular and genetic levels through fluorescent microscopy in a single assay. In order to enable the engineering of morphogenesis in Marchantia gemmae, tools and frameworks for obtaining, storing and analysing the observations from the three levels of biological organisation should be put into place. The work presented in this thesis focuses on the development of such tools and their application in studying the role of phytohormone auxin in Marchantia development. I introduced novel sample preparation and time-lapse imaging assays for Marchantia gemmae along with image- processing methods for the estimation of tissue expansion rates with sub-cellular resolution. These methods allowed me to hypothesise a mechanism behind the regulation of cell proliferation in Marchantia and the role auxin plays in controlling this process. Together with Bernardo Pollak, I developed MarpoDB, a gene-centric representation of the Marchantia genome that enables the search and preparation of Marchantia genetic parts for assembly into synthetic DNA constructs. I used MarpoDB to extract parts and build fluorescent reporters, providing proxies for auxin biosynthesis and signalling. The reporters were then introduced into the Marchantia genome, and the gemmae of the transgenic lines were imaged to estimate the average patterns of the reporters’ expression. The collected patterns were then overlaid on the patterns of relative tissue expansion to validate the proposed role of auxin as an inhibitor of cell proliferation and the mechanism behind its transport in Marchantia.BBSR