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Genome Wide DNA Replication Timing in Human Pluripotent and Leukemic Cell TypesUnknown Date (has links)
Accurate replication of DNA once and only once per cell cycle is an essential process for all living organisms. Despite many studies aimed at understanding this phenomenon, no mechanism describing where and when replication initiates in mammalian cells has yet been elucidated. However, it is well established that DNA is replicated as megabase-sized chromosomal segments called domains in a specific temporal order during S phase. The order in which these segments replicate is called the replication timing program. In recent years, approaches using microarray technology have been developed to study replication timing throughout the entire genome (Hiratani et al., 2008; Hiratani et al., 2010; Woodfine et al., 2004). These approaches have allowed the study of genome-wide replication timing patterns throughout various stages of mouse embryonic stem cell development, and led to the discovery that replication timing is a cell-type specific, developmentally regulated event with epigenetic significance (Hiratani et al., 2010; Ryba et al., 2010). Here I use the genome-wide replication timing assay to study replication timing patterns in human cell types. Specifically, I have investigated whether significant differences in replication timing exist in human embryonic stem cell lines cultured under various growth conditions. In collaboration with other stem cell biologists I have performed genome wide replication timing analysis on primed and naïve human pluripotent cell types. Finally, in a separate but related project, I have used the genome wide replication timing assay to compare replication timing in normal B lymphoblasts to replication timing in established leukemic cell lines and leukemia patient samples in order to probe for the existence of leukemia specific changes in replication timing that may be linked to known genetic subtypes of leukemia or prognoses. / A Thesis Submitted to the Department of Biological Science in Partial Fulfillment
of the Requirements for the Degree of Master of Science. / Spring Semester, 2011. / March 31, 2011. / Leukemia, Pluripotency, Stem Cell, DNA Replication / Includes bibliographical references. / David Gilbert, Professor Directing Thesis; Akash Gunjan, Committee Member; Karen McGinnis, Committee Member; Yanchang Wang, Committee Member.
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The Subcellular Localization of the Transcription Factor YY1 during Cellular Life and DeathUnknown Date (has links)
One of the events leading to a cell's commitment to a new cell cycle resulting in cell division is upregulation of the replication-dependent histone gene family. Our laboratory has previously identified a coding region activating sequence (CRAS) present in all replication-dependent histone genes. Furthermore, we showed that the Yin Yang-1 (YY1) protein binds to the alpha element within the conserved CRAS and that this binding activity is essential for correct regulation of this histone gene family. Thus, YY1 plays a central role in gene regulation in the cell cycle, specifically at the G1/S phase transition. Here, we report a mechanistic link between DNA status in the cell and localization of YY1 in the cell. We present several lines of evidence that support YY1 involvement in cellular life and death processes. Confocal microscopic studies show that YY1 subcellular localization in the cell is responsive to DNA synthesis checkpoint events. At the onset of DNA synthesis as cells enter S phase, YY1 pattern of localization changes from the cytoplasm to the nucleus. Later, past the midpoint of S phase, YY1 is primarily cytoplasmic again. Inhibition of DNA synthesis in CHO cells leads to loss of YY1 in the nucleus, and overriding DNA synthesis checkpoints restores YY1 nuclear localization. Moreover, use of apoptosis-inducing agents in HeLa cells leads to translocation of YY1 to the nucleus, very early in the apoptosis process, and regardless of the status of DNA replication in the cell. These results clearly suggest a role of YY1 in global survival or death processes. Also, in our efforts to understand the events leading to a cell's decision to divide again, we performed genomewide gene expression studies using cells in G1 and S phases of the cell division cycle. Utilizing synchronous cell populations, obtained by mitotic shake-off method, and human microarray gene chips, we identified genes up and down regulated in early time points of the cell cycle. We have previously identified 874 genes to be periodically expressed in the human cell cycle using HeLa cells. Using RNA samples collected every 15 minutes for a period of 2 hours post-mitotically, we present here a detailed gene expression profiling of gene activity after cells exit mitosis and begin entry into a new cycle. We also present evidence from histone array data and analysis of stress-induced and mechanical-stress induced genes to prove that our approach, which utilizes the mitotic shake-off technique, is a stress-free method to synchronize mammalian cells. Replication-dependent histone gene expression is tightly linked to the onset of DNA synthesis in the eukaryotic cell cycle. Understanding the protein-gene regulatory network that precedes and then promotes the expression of this gene family will enable us to better understand the signaling that controls cellular growth and proliferation. / A Dissertation Submitted to the Department of Biological Science in Partial
Fulfillment of the Requirements for the Degree of Doctor of Philosophy. / Spring Semester, 2007. / December 15, 2006. / Histone Genes, Gene Expression, CDNA Microarray, Cell-Cycle, YY1, Apoptosis, Mitotic Shakeoff, Subcellular Localization / Includes bibliographical references. / Myra Hurt, Professor Directing Dissertation; Cathy Levenson, Outside Committee Member; Hank Bass, Committee Member; Lloyd Epstein, Committee Member; Thomas C. S. Keller, III, Committee Member.
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The Dynamics of Replication Timing, Chromatin Compartments, and Gene Expression Changes during Lineage Specification of Stem CellsUnknown Date (has links)
The temporal order in which segments of the genome are duplicated is referred to as the replication timing (RT) program. RT is
established in each cell cycle coincident with the repositioning and anchorage of chromosomes in early G1. In general, segments that
replicate in early S are organized into transcriptionally permissive chromatin, and segments that replicate in late S are assembled into
repressive chromatin. During human embryonic stem cell (hESC) differentiation, segments of the genome undergo changes in RT, which are
accompanied by changes in chromatin compartments, and transcriptional activity. Determining the order these changes occur during hESC
differentiation required defining cell cycle parameters for hESCs. First, we demonstrate that the fluorescence ubiquitination cell
cycle indicator (Fucci) system is incapable of demarcating G1/S cell cycle transitions. Instead, we employed a combination of fluorescent
PCNA to monitor S phase progression, cytokinesis to demarcate mitosis, and fluorescent nucleotides to label early and late replicating DNA
and track 3D organization. We find that re-localization and anchorage of chromosomes were completed prior to the onset of S phase, even in
the context of an abbreviated G1 phase. Furthermore, we find that single hESCs preferentially differentiate from G1. We show changes in RT
are remarkably coincident with transcription; although, neither is sufficient for the other to occur. We also show changes in RT accompany
cell commitment during the first cell cycle and precede changes in chromatin compartments. Finally, we find that in hESCs, domains that
switch from early to late replication interact more frequently with late replicating chromatin, suggesting hESCs may be poised to quickly
repress early to late switching domains upon stimulation. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the
requirements for the degree of Doctor of Philosophy. / Summer Semester 2016. / July 18, 2016. / Includes bibliographical references. / David Gilbert, Professor Directing Dissertation; Timothy Megraw, University Representative; Hank
Bass, Committee Member; Brian Chadwick, Committee Member; Jonathan Dennis, Committee Member.
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An investigation of the phenomenon of colcemid induced endoreduplication in Chinese hamster ovary cellsUnknown Date (has links)
by Joan T. Hare. / Thesis (M.S.)--Florida State University. / Bibliography: leaves 55-58. / Florida State University faculty publication.
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Vasoactive amine mediation of endothelial cell movement and barrier function in vitroBottaro, Donald Paul January 1986 (has links)
Investigations were conducted to characterize endothelial cell
(EC) movement and barrier function in vitro and to investigate their
potential interrelationship. Specifically, the mediation of EC
movement and barrier function by vasoactive amines, and the relevance
of this mediation to a potential mechanism by which blood platelets
may help maintain microvascular integrity, was examined. The effects
of the platelet constituents serotonin (5-HT) and norepinephrine (NE)
and the effects of histamine on bovine aortic endothelial cell (BAEC)
and vascular smooth muscle cell (VSMC) movement were quantitated using
a phagokinetic tracking assay. BAEC movement was significantly reduced
by 5-HT, NE, and histamine, while VSMC motility was significantly
enhanced by 5-HT and histamine, but reduced by NE. The use of specific
receptor antagonists revealed that the 5-HT- and NE-associated
inhibition of BAEC movement may be mediated by beta-adrenergic
receptors, and the histamine-associated inhibition may be partially
mediated by H-1 receptors.
An assay to measure the passage of a trypan blue dye-bovine serum albumin conjugate (TB-BSA) across cells grown on microcarriers was
used to compare the barriers provided by EC and other cell types. VSMC
or 3T3 fibroblasts impeded TB-BSA diffusion significantly less than
BAEC, suggesting that barrier formation may be an EC-specific
phenomenon. Treatment of BAEC with 5-HT or NE significantly impeded
TB-BSA diffusion relative to untreated controls. In contrast,
histamine treatment significantly increased TB-BSA diffusion. The
amine-associated effects were dose-dependent and cell-specific, and in
some cases appeared to be receptor-mediated. BAEC and pulmonary
microvessel EC (PMEC) barriers were quantitatively comparable, but
significantly more permeable than that observed for cerebral
microvessel EC (CMEC). Glutaraldehyde fixation and low temperature
reduced TB-BSA passage across BAEC by <30%, indicating that the bulk
of tracer movement occurred via intercellular diffusion. Treatment
with cytochalasin resulted in significant BAEC and CMEC barrier loss,
suggesting that microfilament bundles are involved EC junctional
maintenance. Collectively, the results suggest a dynamic model of
vascular permeability in which intercellular macromolecular diffusion
may be regulated by EC junctional apposition, and responsive to
physiologic -agents that affect EC movement.
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Nitric oxide mediated effects on bone cellsO'Shaughnessy, Margaret Clare January 2001 (has links)
No description available.
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Thermoresponsive magnetic colloidal gels for in vitro cell expansionBraim, Shwana January 2016 (has links)
Recent studies and clinical trials have shown the potential of cell-based therapies for the treatment of a number of diseases and organ/ tissue damages. However, limited availability of some therapeutically important cells (i.e. adult stem cells) still remain as main challenges in the development of tissue engineering through to the clinic. Healthy cells are required in large numbers to form a tissue-engineered construct and primary cells must therefore be expanded in vitro for both scientific and clinical applications. Various strategies have been developed to expand cells in vitro with increasing emphasis on 3D matrices because it can provide microenvironments which more closely mimic in vivo systems. In this way the inherent difficulties associated with 2D culture such as loss of phenotype could be overcome. Moreover, 3D matrices provide higher surface areas to support expansion of larger cell numbers compared to monolayer culture. Although each 3D method has certain advantages, there is no single technique that can be used to produce material assemblies that address all the fundamental problems linked to 3D cell seeding (penetration into the scaffold), passaging (use of enzymes), and harvesting (cell yield). Recently, thermally reversibly-associating particles have been studied for the growth and support of multiple cell types and for delivery of therapeutic cells. But coupling of thermoresponsive properties to magnetic microspheres would enhance the 3D culture and expansion of multiple cell types, and facilitate rapid recovery of the expanded cell population by simple magnetic separation. In this study, it was proposed that the thermoresponsive properties would allow simple cell seeding at temperatures below the LCST of polymer stabiliser when the suspension is flowing and upon heating to above the LCST cells would be encapsulated and cultured within the particle gels (every cells surrounded by a number of particles, as the size of the particles are much smaller than the cells). The magnetic responsive property would allow efficient and scaffold free cell recovery after expansion without the need for using trypsin or enzymatic treatment. The ‘switchable’ component of reversibly associating colloidal microparticles were prepared via two different strategies. In the first strategy, thermoresponsive PDEGMA was physically adsorbed onto the surface of PS microspheres, whereas, in the second strategy, PDEGMA was chemically grafted from functionalised PCMS microspheres via SI-ATRP. The most simple method i.e. physical adsorption is rapid and can be adapted to many microparticle surfaces but has the drawback of possible desorption of polymer chains during extended use. The chemical grafting method i.e. the formation of covalent bonds between the polymer corona and the microparticle core provides robust and well defined materials but is more complex and time-consuming. In both cases, particle aggregation in their suspensions occurred on increasing the temperature to above the LCST of PDEGMA, but could be reversed by cooling the suspensions back to below the LCST. This confirmed the presence of the thermoresponsive polymer on the surface of the microspheres using both methods (adsorption and grafting). Rheological measurements demonstrated that the viscoelasticity of the prepared particle gels can be tuned, enabling these gels to have the mechanical properties that should facilitate their applications as 3D cell scaffolds for in vitro expansion of cells. Cell culture studies showed that these microparticle based scaffolds can support expansion of clinically relevant cell types (human MSC) and allowed efficient cell recovery after proliferation without the need for using trypsin or enzymatic treatment. Overall, those results suggest that the designed scaffolds had great potential for 3D in vitro cell expansion. The new developed materials have excellent biocompatibility, allow simple and rapid cell seeding and cell recovery after expansion, and possess mechanical strength and stability to support cell growth and proliferation. The materials developed and studied in this thesis may represent a significant contribution to the fields of biomaterials, tissue engineering, 3D cell culture and even bio-separation.
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Mechanisms of Actomyosin Contractility in CellsStachowiak, Matthew R. January 2011 (has links)
Many fundamental cellular processes hinge on the ability of cells to exert contractile force. Contractility is used by cells to divide, to migrate, to heal wounds, and to pump the heart and move limbs. Contractility is mediated by the actin and myosin cytoskeleton, a dynamic and responsive meshwork that assembles into various well-defined structures used by the cell to accomplish specific tasks. While muscle contraction is well-characterized, the contraction mechanisms of actomyosin structures in nonmuscle cells are relatively obscure. Here we elucidate the contraction mechanisms of two prominent and related actomyosin structures: the contractile ring, which constricts to divide the cell during cytokinesis, and the stress fiber, which is anchored to the extracellular matrix and allows the cell to exert contractile forces on its surroundings. In the first part of the thesis, we develop a mathematical model to characterize the constriction mechanism of contractile rings in the Schizosaccharomyces pombe model organism. Our collaborators observed that after digesting the cell wall to create protoplasts, contractile rings constricted by sliding along the plasma membrane without cleaving the cell. This novel approach allowed direct comparison of our model predictions for the ring constriction rate and ring shape to the experimental data, and demonstrated that the contractile ring's rate of constriction is determined by a balance between ring tension and external resistance forces. Our results describe a casual relationship between ring organization, actin turnover kinetics, tension, and constriction. Ring tension depends on ring organization through the actin and myosin concentrations and their statistical correlations. These correlations are established and renewed by actin turnover on a timescale much less than the constriction time so that rapid actin turnover sets the tension and provides the mechanism for continuous remodeling during constriction. Thus, we show that the contractile ring is a tension-producing machine regulated by actin turnover whose constriction rate depends on the response of a coupled system to the ring tension. In the second part of the thesis we examine the contraction mechanisms of stress fibers, which have a sarcomeric structure reminiscent of muscle. We developed mathematical models of stress fibers to describe their rapid shortening after severing and to describe how the kinetics of sarcomere contraction and expansion depend on actin turnover. To test these models, we performed quantitative image analysis of stress fibers that spontaneously severed and recoiled. We observed that after spontaneous severing, stress fibers shorten by ~80% over ~15-30 s, during which ~50% of the actin initially present was disassembled. Actin disassembly was delayed by ~50 s relative to fiber recoil, causing a characteristic increase, peak, and decay in the actin density after severing. Model predictions were in excellent agreement with the observations. The model predicts that following breakage, fiber shortening due to myosin contractile force increases actin filament overlap in the center of the sarcomeres, which in turn causes compressive actin-actin elastic stresses. These stresses promote actin disassembly, thereby shortening the actin filaments and allowing further contraction. Thus, the model identifies a mechanism whereby coupling between actin turnover and mechanical stresses allows stress fibers to dynamically adjust actin filament lengths to accommodate contraction.
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Control of Neuronal Circuit Assembly by Gtpase RegulatorsSommer, Julia January 2011 (has links)
One of the most remarkable features of the central nervous system is the exquisite specificity of its synaptic connections, which is crucial for the functioning of neuronal circuits. Thus, understanding the cellular and molecular mechanisms leading to the precise assembly of neuronal circuits is a major focus of developmental neurobiology. The structural organization and specific connectivity of neuronal circuits arises from a series of morphological transformations: neuronal differentiation, migration, axonal guidance, axonal and dendritic arbor growth and, eventually, synapse formation. Changes in neuronal morphology are driven by cell intrinsic programs and by instructive signals from the environment, which are transduced by transmembrane receptors on the neuronal cell surface. Intracellularly, cytoskeletal rearrangements orchestrate the dynamic modification of neuronal morphology. A central question is how the activation of a neuronal cell surface receptor triggers the intracellular cytoskeletal rearrangements that mediate morphological transformations. A group of proteins linked to the regulation of cytoskeletal dynamics are the small GTPases of the Rho family. Small RhoGTPases are regulated by GTPase exchange factors (GEF) and GTPase activating proteins (GAP), which can switch GTPases into "on or off" states, respectively. It is thought, that GEFs and GAPs function as intracellular mediators between transmembrane receptors and RhoGTPases, to regulate cytoskeletal rearrangements. During my dissertation I identified the GAP α2-chimaerin as an essential downstream effector of the axon guidance receptor EphA4, in the assembly of neuronal locomotor circuits in the mouse. Furthermore, I identified two novel neuronal GAPs, mSYD-1A and mSYD-1B, which interact with components of the presynaptic active zone and which may contribute to presynaptic assembly downstream of synaptic cell surface receptors.
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Cell Size Control in Fission YeastPan, Kally Zhang January 2013 (has links)
Among all living organisms, there is almost much variety in cell size as there is for cell function and cell type. However, within each cell type, cells stay remarkably faithful to a defined size over generations. Many factors have been found to influence this ability to specify and maintain cell size, yet clear mechanisms have yet to be elucidated. The fission yeast Schizosaccharomyces pombe is an ideal model organism whose simple but conserved cell biology has led to the identification of many important cell size regulators common to all eukaryotes. In this thesis, I have quantitatively analyzed the dynamics and localization of several key players of cell size regulation, which lead to a new physical model on cell size regulation based on the localization and accumulation of a size sensing kinase cdr2p. In this model, cdr2p molecules accumulate in proportion to cell size into complexes called midsomes, which localize to the cortex at the central section of the cell. Upon reaching the desired cell size, cdr2p accumulation surpasses a concentration threshold and the cell will divide. This accumulation is partly facilitated by the key negative regulator pom1p, which prevents midsome formation at the cell tip. Evidence also suggests that the ER serves a role in confining midsome localization to the medial plasma membrane, perhaps by providing a physical link to the nucleus. Together, this work elucidates a mechanistic understanding of how cell size can be determined and controlled.
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