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Investigation of the chromatin composition and structure of foreign DNA in a mammalian cellFitz-James, Maximilian Hamilton January 2018 (has links)
In order to contain many millions, or even billions of base pairs within every nucleus of a eukaryotic cell, DNA must be extensively packaged. This is achieved by association of DNA with packaging proteins, resulting in the formation of chromatin, which can lead to various degrees of compaction. The most extreme form of compaction is the highly condensed mitotic chromosome, formation of which is necessary for proper resolution and segregation of the genetic material during cell division. However, the exact nature of the structure of chromatin within the mitotic chromosome and the factors which regulate it remain subjects of debate and continued investigation. The hybrid cell line F1.1 presents a unique tool for the study of mitotic chromosome structure. This mouse cell line has been observed to present a distinct chromatin structure in mitosis assembled over a large region of DNA inserted into one of its chromosomes and originating from the fission yeast Schizosaccharomyces pombe. Direct comparison of the structure of this distinct region of chromatin with that of the adjacent endogenous chromatin could provide insight into the nature of mitotic chromosome structure as well as the properties of the chromatin which are influencing this structure. Microscopy and Hi-C analyses showed that the mitotic chromatin organising or "scaffold" proteins are not altered over the region of S. pombe chromatin, but that the amount of chromatin organised around these proteins is diminished. In accordance with the "radial-loop" model of mitotic chromosome structure, we put forward a model whereby the S. pombe chromatin is organised into smaller chromatin loops around a constant organising scaffold. Examination of the histone post-translational modifications over the region of S. pombe chromatin revealed it to be highly heterochromatic, with high levels of H3K9me3 and associated factors such as HP1α and 5meC, and low levels of activating marks. Generation of further mammalian - S. pombe fusion cell lines recapitulated both the distinct mitotic structure and the heterochromatic profile of the inserted S. pombe chromatin. However, insertion of S. pombe DNA into a mouse cell by transfection rather than fusion resulted in a large region of S. pombe DNA that lacked both a distinct structure and heterochromatin. These results suggest that H3K9me3- mediated heterochromatin may influence the structure of chromatin in mitosis, leading to an organisation into smaller chromatin loops than non-heterochromatic regions.
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'SynCheck' : new tools for dissecting Bub1 checkpoint functionsLeontiou, Ioanna January 2018 (has links)
The accurate segregation of DNA during cell division is essential for the viability of future cellular generations. Genetic material is packaged in the form of chromosomes during cell division, and chromosomes are segregated equally into two daughter cells. Chromosome mis-distribution leads to genetic disorders (e.g. Down's syndrome), aneuploidy and cancer. The spindle checkpoint ensures proper chromosome segregation by monitoring kinetochore-microtubule interactions. Upon checkpoint activation, unattached kinetochores recruit checkpoint proteins that combine to form a diffusible inhibitor (the Mitotic Checkpoint Complex-MCC). The MCC delays anaphase, thus giving cells time to fix attachment errors. Although the major checkpoint proteins were identified several years ago, we have only just begun to understand how they assemble at unattached kinetochores to generate the checkpoint signal. Yeast genetics and proteomics have revealed that kinetochores are highly complex molecular machines with almost 50 kinetochore components and ~10 components of the spindle checkpoint machinery. Such complexity makes the separation of error correction, kinetochore bi-orientation and microtubule attachment functions very challenging. To circumvent this complexity, a synthetic version of the spindle checkpoint (SynCheck), based on tetO array was engineered at an ectopic location on a chromosome arm away from kinetochores in S. pombe. This work describes that combined targeting, initially of KNL1Spc7 with Mps1Mph1 and later of Bub1 (but not Mad1) with Mps1Mph1 fragments, was able to activate the spindle checkpoint and generate a robust arrest. The system is based on, soluble complexes, which were formed between KNL1Spc7 or Bub1 with Mps1Mph1. The synthetic checkpoint or 'Syncheck' is independent of localisation of the checkpoint components to the kinetochores, to spindle pole bodies (SPBs) and to nuclear pores. By using the synthetic tethering system a Mad1-Bub1 complex was identified for the first time in S.pombe. Bub1- Mad1 complex formation is crucial for checkpoint activation. Bub1-Mad1 gets phosphorylated itself and is thought to act as an assembly platform for MCC production and thereby generation of the "wait anaphase" signal. The ectopic tetO array is an important tool, not only for generating MCC formation and activating the spindle checkpoint, but also for providing a nice system for analysing in vivo protein-protein interactions. The ectopic array is capable of not only recruiting checkpoint components, but also recruiting them in a physiological manner (similar to the unattached kinetochores). For this reason it was decided to adopt this system to examine the role of the conserved Bub1TPR domain in the recruitment of other spindle checkpoint proteins. This work represents two novel functions for the S. pombe Bub1TPR domain. For the first time in S. pombe, both in vivo tethering and in vitro experiments with purified, recombinant proteins showed that the Bub1 has the ability to homodimerise and to form a complex with Mad3BubR1 through its TPR domain. These results revealed that complex formation of Bub1 with Mad3BubR1 is important for checkpoint signalling and that the highly conserved TPR domains in BubR1Mad3 and Bub1 have key roles to play in their interactions.
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Centrosome and Mitotic Spindle Organization in Human CellsLawo, Steffen 10 January 2014 (has links)
Robust bipolar spindle formation and faithful transmission of genetic material are vital to the maintenance of genome integrity and cellular homeostasis. Chromosome segregation errors can result in aneuploidy, a hallmark of human solid tumors. The assembly of a microtubule-based mitotic spindle relies on the concerted action of centrosomes, spindle microtubules, molecular motors and nonmotor spindle proteins. Before mitosis, centrosomes need to duplicate and increase in size in order to gain sufficient microtubule nucleation activity during bipolar spindle formation. This process is called centrosome maturation and coincides with a dramatic change of centrosome structure. However, the architecture of centrosomes and the organization of centrosome components in both interphase and mitosis have long remained elusive.
In this thesis, I describe the identification and characterization of novel regulators that are essential for centrosome and mitotic spindle organization in human cells. One such regulator is human Augmin, an evolutionarily conserved eight-subunit protein complex that has essential functions for centrosome and spindle integrity. I present evidence that human Augmin promotes microtubule-dependent nucleation of microtubules by targeting microtubule-nucleating complexes to the mitotic spindle. This function of Augmin is important for generation and/or stabilization of kinetochore microtubules within the mitotic spindle, and its loss results in destabilization of kinetochore microtubules and spindle assembly errors. These errors culminate in cells displaying multipolar spindles with fragmented centrosomes and mitotic arrest. A second regulator of centrosome and spindle organization described in this thesis is CEP192. I show that CEP192 is critical for recruitment of microtubule-nucleating complexes to centrosomes and, consequently, for centrosome maturation, mitotic spindle formation, and centriole duplication. Finally, I describe novel organizational features of the centrosome using a subdiffraction microscopy approach. Because of a lack of higher-order structural information, centrosomes have traditionally been described as amorphous clouds. My results now reveal that centrosome components instead occupy separable spatial domains throughout the cell cycle and highlight the role of higher-order protein organization in the regulation of centrosome assembly and function. Collectively, this work has significantly expanded our current knowledge of centrosome architecture and biogenesis and of the mechanisms that underlie robust bipolar spindle assembly.
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Centrosome and Mitotic Spindle Organization in Human CellsLawo, Steffen 10 January 2014 (has links)
Robust bipolar spindle formation and faithful transmission of genetic material are vital to the maintenance of genome integrity and cellular homeostasis. Chromosome segregation errors can result in aneuploidy, a hallmark of human solid tumors. The assembly of a microtubule-based mitotic spindle relies on the concerted action of centrosomes, spindle microtubules, molecular motors and nonmotor spindle proteins. Before mitosis, centrosomes need to duplicate and increase in size in order to gain sufficient microtubule nucleation activity during bipolar spindle formation. This process is called centrosome maturation and coincides with a dramatic change of centrosome structure. However, the architecture of centrosomes and the organization of centrosome components in both interphase and mitosis have long remained elusive.
In this thesis, I describe the identification and characterization of novel regulators that are essential for centrosome and mitotic spindle organization in human cells. One such regulator is human Augmin, an evolutionarily conserved eight-subunit protein complex that has essential functions for centrosome and spindle integrity. I present evidence that human Augmin promotes microtubule-dependent nucleation of microtubules by targeting microtubule-nucleating complexes to the mitotic spindle. This function of Augmin is important for generation and/or stabilization of kinetochore microtubules within the mitotic spindle, and its loss results in destabilization of kinetochore microtubules and spindle assembly errors. These errors culminate in cells displaying multipolar spindles with fragmented centrosomes and mitotic arrest. A second regulator of centrosome and spindle organization described in this thesis is CEP192. I show that CEP192 is critical for recruitment of microtubule-nucleating complexes to centrosomes and, consequently, for centrosome maturation, mitotic spindle formation, and centriole duplication. Finally, I describe novel organizational features of the centrosome using a subdiffraction microscopy approach. Because of a lack of higher-order structural information, centrosomes have traditionally been described as amorphous clouds. My results now reveal that centrosome components instead occupy separable spatial domains throughout the cell cycle and highlight the role of higher-order protein organization in the regulation of centrosome assembly and function. Collectively, this work has significantly expanded our current knowledge of centrosome architecture and biogenesis and of the mechanisms that underlie robust bipolar spindle assembly.
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The relationship between proliferation and differentiation during oligodendrocyte developmentApperly, James A. January 2001 (has links)
How do precursor cells know when it is time to stop dividing and differentiate? The phenomenon of lineage-specific progenitor cells undergoing a limited period of proliferation prior to terminal differentiation is a common theme in multicellular development. Despite this, little is understood about how these two events are co-ordinated during the normal schedule of development. I have studied the question of how proliferation and differentiation are co-regulated in the oligodendrocyte lineage in the rodent optic nerve. Oligodendrocytes are post-mitotic cells that myelinate axons in the vertebrate central nervous system. They develop from precursor cells whose maturation is controlled by a timer, which is an intrinsic property of the cells, that limits proliferation. The timer seems not to control the number of divisions the cell can undergo but rather the length of time during which divisions can occur. Significant effort has been devoted to understanding how the intracellular timer regulates oligodendrocyte development. The timer consists of two components that are modulated by distinct kinds of extracellular signals. Mitogens drive a timing component whose value increases as precursor cells continue to divide. Once this value exceeds a critical threshold, it signals that the proliferative period has elapsed, and hydrophobic signalling molecules trigger an effector component that elicits cell-cycle arrest and differentiation. The value of the timing component is determined by several intracellular molecules whose activities change as the timer runs. One of these molecules is the cell-cycle inhibitor p27: it accumulates in oligodendrocyte precursor cells as they proliferate in culture. When p27 expression is high the precursor cells are more likely to stop dividing and differentiate than when it is low. In oligodendrocyte precursor cells derived from mice that lack p27, the timer runs aberrantly and cell-cycle arrest and terminal differentiation are delayed. It is not understood how the molecular mechanics of the timer control oligodendrocyte development. Does the timer serve to arrest the cell-cycle, with differentiation following by default, or is cell-cycle arrest subordinate to the programme of terminal differentiation? These questions remain unanswered, largely because of a persistent inability to experimentally manipulate the genome of oligodendrocyte precursor cells. The present study was an attempt to overcome these problems and had two aims - first, to devise a reliable system for transfecting oligodendrocyte precursor cells and second, to determine whether the timer primarily controls the timing of cell-cycle arrest. I developed a new retroviral vector that co-expresses p27 and green fluorescent protein (GFP) in precursor cells. The use of GFP allows the identification of living precursor cells that over-express p27, which can then be followed over many days in culture. My findings support previous work showing that p27 plays a role in governing the timing of oligodendrocyte differentiation. They show that over-expression of p27 promotes oligodendrocyte differentiation by advancing the value of the timing component, although it does not promote differentiation if the effector component is inoperative. The cell-cycle time of precursor cells that over-express p27 is dramatically extended, but not stopped. It appears that a firm cell-cycle arrest and entry into a quiescent state may be required to elicit terminal differentiation.
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High-Resolution Mapping of Mitotic Recombination in Saccharomyces CerevisiaeSt. Charles, Jordan Anne January 2012 (has links)
<p>Double-stranded DNA breaks are potentially lethal lesions that can be repaired in mitotic cells by either homologous recombination (HR) or non-homologous end- joining (NHEJ) pathways. In the HR pathway, the broken DNA molecule is repaired using either the sister chromatid or the homolog as a template. Mitotic recombination events involving the homolog often result in loss of heterozygosity (LOH) of markers located distal to the crossover. In humans that are heterozygous for a mutation in a tumor suppressor gene, mitotic recombination leading to LOH can be an early step in cancer development.</p><p> In my thesis research, I analyzed mitotic recombination in the yeast Saccharomyces cerevisiae using oligonucleotide-containing microarrays to detect LOH of single-nucleotide polymorphisms (SNPs). In analyzing cells treated with ionizing radiation, I performed the first whole-genome analysis of LOH events done in any organism (Chapter 2). I showed that irradiated cells had between two and three unselected LOH events. I also showed that crossovers were often associated with non- reciprocal exchanges of genetic information (gene conversion events) and that these conversion events were more complex than predicted by standard models of homologous recombination.</p><p> In Chapter 3, I describe my mapping of spontaneous crossovers in a 1.1 Mb region of yeast chromosome IV. This analysis is the first high-resolution mitotic recombination map of a substantial fraction (about 10%) of a eukaryotic genome. I demonstrated the existence of recombination "hotspots" and showed that some of these hotspots were homolog-specific. Two of the strongest hotspots were formed by closely- spaced inverted repeats of retrotransposons. I demonstrated that the hotspot activity was a consequence of a secondary DNA structure formed by these repeats. Additionally, the majority of spontaneous LOH events reflect DNA lesions induced in unreplicated chromosomes during G1 of the cell cycle, indicating that G1-initiated lesions threaten genome stability more than G2-initiated lesions.</p><p> In Chapter 4, I describe mitotic crossovers associated with DNA replication stress induced by hydroxyurea (HU) treatment. Surprisingly, most HU-induced crossovers had conversion tracts indicative of DNA lesions initiated in G1. Additionally, HU- induced recombination events were very significantly associated with solo delta elements, a 330 bp sequence that is repeated several hundred times in the yeast genome.</p> / Dissertation
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Conduits of Intratumor Heterogeneity: Centrosome Amplification, Centrosome Clustering and Mitotic FrequencyPannu, Vaishali 18 December 2014 (has links)
Tumor initiation and progression is dependent on the acquisition and accumulation of multiple driver mutations that activate and fuel oncogenic pathways and deactivate tumor suppressor networks. This complex continuum of non-stochastic genetic changes in accompaniment with error-prone mitoses largely explains why tumors are a mosaic of different cells. Contrary to the long-held notion that tumors are dominated by genetically-identical cells, tumors often contain many different subsets of cells that are remarkably diverse and distinct. The extent of this intratumor heterogeneity has bewildered cancer biologists’ and clinicians alike, as this partly illuminates why most cancer treatments fail. Unsurprisingly, there is no “wonder” drug yet available which can target all the different sub-populations including rare clones, and conquer the war on cancer. Breast tumors harbor ginormous extent of intratumoral heterogeneity, both within primary and metastatic lesions. This revelation essentially calls into question mega clinical endeavors such as the Human Genome Project that have sequenced a single biopsy from a large tumor mass thus precluding realization of the fact that a single tumor mass comprises of cells that present a variety of flavors in genotypic compositions. It is also becoming recognized that intratumor clonal heterogeneity underlies therapeutic resistance. Thus to comprehend the clinical behavior and therapeutic management of tumors, it is imperative to recognize and understand how intratumor heterogeneity arises.
To this end, my research proposes to study two main features/cellular traits of tumors that can be quantitatively evaluated as “surrogates” to represent tumor heterogeneity at various stages of the disease: (a) centrosome amplification and clustering, and (b) mitotic frequency. This study aims at interrogating how a collaborative interplay of these “vehicles” support the tumor’s evolutionary agenda, and how we can glean prognostic and predictive information from an accurate determination of these cellular traits.
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Action of the Cubitus Interruptus‐wallace Mutant in Drosophila Melanogaster: A Study of Leg Morphology on Mosaic and Haplo‐4 FliesBenner, D. B. 01 January 1987 (has links)
The fourth chromosome mutant cubitus interruptus‐Wallace(ciW) produces leg, wing, and body bristle aberrations. The effect on the wing is similar to that produced by cubitus interruptus‐dominant (ciD) which also has an influence on larval segmentation indicating that it has a regulatory function. Leg morphology of haplo‐4, ciW, and mosaic haplo‐4:diplo‐4, ci/ci+ flies was examined in an attempt to distinguish between a structural and a regulatory function by ciW. Aberrations recovered include failure of segment elongation, intersegmental gaps, duplication of bristles, and segments that are shorter than normal and of greater than normal diameter. Many of these effects are localized, suggesting that ciW may act to maintain cell positional reference. Increased local cell proliferation appears to be one manifestation of loss of the normal function.
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Mitotic cell detection in H&E stained meningioma histopathology slidesCheng, Huiwen 12 1900 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Meningioma represent more than one-third of all primary central nervous system (CNS) tumors, and it can be classified into three grades according to WHO (World Health Organization) in terms of clinical aggressiveness and risk of recurrence. A key component of meningioma grades is the mitotic count, which is defined as quantifying the number of cells in the process of dividing (i.e., undergoing mitosis) at a specific point in time. Currently, mitosis counting is done manually by a pathologist looking at 10 consecutive high-power fields (HPF) on a glass slide under a microscope, which is an extremely laborious and time-consuming process. The goal of this thesis is to investigate the use of computerized methods to automate the detection of mitotic nuclei with limited labeled data. We built computational methods to detect and quantify the histological features of mitotic cells on a whole slides image which mimic the exact process of pathologist workflow. Since we do not have enough training data from meningioma slide, we learned the mitotic cell features through public available breast cancer datasets, and predicted on meingioma slide for accuracy. We use either handcrafted features that capture certain morphological, statistical, or textural attributes of mitoses or features learned with convolutional neural networks (CNN). Hand crafted features are inspired by the domain knowledge, while the data-driven VGG16 models tend to be domain agnostic and attempt to learn additional feature bases that cannot be represented through any of the handcrafted features. Our work on detection of mitotic cells shows 100% recall , 9% precision and 0.17 F1 score. The detection using VGG16 performs with 71% recall, 73% precision, and 0.77 F1 score. Finally, this research of automated image analysis could drastically increase diagnostic efficiency and reduce inter-observer variability and errors in pathology diagnosis, which would allow fewer pathologists to serve more patients while maintaining diagnostic accuracy and precision. And all these methodologies will increasingly transform practice of pathology, allowing it to mature toward a quantitative science.
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An Investigation of Radiation-Induced Mitotic Inhibition in L-Strain Mouse CellsJohns, Robert Martin 10 1900 (has links)
<p> The variation in sensitivity of L60T cells to gamma rays has been studied as a function of position in the cell division cycle. For a dose range of 0-12,000 rads, no significant variation was found for mitotic delay. Such was not the case for sensitivity to cell killing, which was found to increase as the cells passed from G1 through S to G2 of the division cycle. The results of mitotic delay are in disagreement with results published by other workers although the survival data agree with previous reports for a similar cell line. Results reported in connection with cell cycle determinations and mitotic delay suggest that the existence of a repair cycle operating concurrently with the normal cell cycle may be postulated. The theoretical treatment of mitotic delay given by Lea is examined and is not found to describe adequately the present results. Finally, the evidence reported here suggests that mitotic delay and radiation lethality are not separate manifestations of the same phenomenon. Experimental materials for further investigation into the repair processes involved are suggested.</p> / Thesis / Master of Science (MSc)
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