1 |
A method for the parametric center problem, with a strictly monotone polynominal-time algorithm for linear programmingJanuary 1989 (has links)
by Robert M. Freund and Kok-Choon Tan. / Includes bibliographical references.
|
2 |
Issues for slot transmission in a passive optical star network based on WDMJanuary 1992 (has links)
Ghassan Semaan, Pierre Humblet. / Caption title. / Includes bibliographical references (p. 10-11). / Supported by ARO. DAAL03-86-K-0171
|
3 |
The forces that center the mitotic spindle in the C. elegans embryoGarzon-Coral, Carlos 31 March 2015 (has links) (PDF)
The precise positioning of the mitotic spindle to the cell center during mitosis is a fundamental process for chromosome segregation and the division plane definition. Despite its importance, the mechanism for spindle centering remains elusive. To study this mechanism, the dynamic of the microtubules was characterized at the bulk and at the cortex in the C. elegans embryo. Then, this dynamic was correlated to the centering forces of the spindle that were studied by applying calibrated magnetic forces via super-paramagnetic beads inserted into the cytoplasm of one- and two-cell C. elegans embryos. Finally, these results were confronted with the different centering models: cortical pushing model, cortical pulling model and the cytoplasmic pulling model.
This thesis shows that: (i) The microtubules dynamic of the spindle aster is controlled spatially in the C. elegans embryo, with not rescues and catastrophes in the cytoplasm but in the centrosome and the cortex, respectively. (ii) The centering mechanism of the spindle behaved roughly as a damped spring with a spring constant of 18 12 pN/ m and a drag coefficient of 127 65 pN s/ m (mean SD). This viscoelastic behavior is evidence of a centering force that recovers and/or maintains the position of the spindle in the cell center. (iii) It seems to be two mechanisms that recover/maintain the spindle position. A fast one that may work for transient displacements of the spindle and a slow one that work over large and long perturbations. (iv) The centering forces scale with the cell size. The centering forces are higher in the two-cell embryo. This result argues against a centering mechanism mediated by cytoplasmic factors. It seems to be a limit for the relation of centering force to size, as the forces found in the four-cell embryo are comparable to the single-cell ones. (v) The centering forces scale with the amount of microtubules in the cell. This strengthens the belief that the microtubules are the force transmission entities of the centering mechanism. (vi) The boundary conditions are important to maintain the centering forces. A transient residency time of microtubules at the cortex, which is controlled by cortical catastrophe factors, is indispensable for a proper force transmission by the microtubules. (vii) The elimination of cortical catastrophe factors provides evidence for microtubules buckling, which is taken as a proof of polymerization forces. (viii) The cortical pulling forces mediated by the gpr-1/2 pathway do not seem to be involved in centering and it is proposed they are present in the cell for off-center positioning purposes. (ix) The forces generated by vesicle transport are enough to displace the spindle and they are suggested to be auxiliary forces to centering. (x) The forces associated with the spindle change dramatically during cell division. From metaphase to anaphase the forces associated with the spindle scale up to five times. This behavior was consistent during the development of the embryo as the same pattern was observed in the one-, two- and four-cell embryo. (xi) The higher forces found during anaphase are not cortical pulling (via pgr-1/2 pathway) depended, and it is proposed the spindle is `immobilised' by tethering or by an unknown cortical pulling pathway.
To this date, this thesis presents the most complete in-vivo measurements of the centering forces in association with the microtubules dynamics. Taken together the results constrain molecular models of centering. This thesis concludes that most probably the predominant forces of the spindle centering mechanism during mitosis are generated by astral microtubules pushing against the cortex.
Additionally, this thesis presents the most complete map of forces during cell division during development, which will prove to be indispensable to understand the changes the spindle undergoes when it changes its function.
|
4 |
Live Cell Imaging of Intracellular Uptake of Contaminant Molecules (B[a]P) and its Effects on Different Cellular CompartmentsAli, Rizwan 08 August 2012 (has links) (PDF)
Exposure of hepatoma cell lines to the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P) is serving as a model for a systems biological study concerning the response of cells to contaminant molecules. Several aspects of the cellular distribution of the aryl hydrocarbon receptor (AhR) and its ligand B[a]P have been addressed by different live cell imaging techniques: The intracellular distribution of the B[a]P/AhR complex is visualized by means of confocal laser scaning microscopy (cLSM) and the intracellular transport rates of the complex is investigated by fluorescence recovery after photobleaching (FRAP) technique. Furthermore, cLSM image stacks of living cells are generated for the modeling of three dimensional (3-D) cell geometries. In order to prevent photochemical damage of the living cells induced by UV excitation of B[a]P, visualization is done by B[a]P’s auto fluorescence using near infrared two-photon-excitation. Murine Hepatoma 1c1c7 cells are exposed to graded concentrations of B[a]P (50 nM to 20 μM) for different incubation time periods (15 minutes to 48 hours). The highest amounts of B[a]P were found in lipid droplets and lysosomes, where the B[a]P molecules are collected and form large aggregates. We were able to work with concentrations down to 50 nM corresponding to that used for genomic and proteomic investigations. Also, for the first time imaging of B[a]P metabolites inside lipid droplets is presented in this work. The data and the model developed in this study will provide new insights into the systematic regulation of the B[a]P, the AhR as well as the receptor-ligand-complex pathway and the study will also serve as a prototype for elucidating other stress response pathways in the future.
|
5 |
Mechanism of spindle assembly in Schizosaccharomyces pombe-Winters, Lora 12 June 2017 (has links) (PDF)
At the onset of cell division microtubules growing from spindle pole bodies (SPB) interact with each other to form the mitotic spindle enabling proper chromosome positioning and segregation. However, the exact mechanism of microtubule dynamics and microtubule associated proteins (MAPs) underlying spindle assembly is still not well understood. We developed an in vivo method to observe spindle assembly in the fission yeast Schizosaccharomyces pombe by inducing depolymerization of already formed and grown spindles by subjecting the cells to low temperatures, followed by subsequent repolymerization at a permissive temperature. We observed that microtubules pivot, i.e., perform angular movement around the SPB in a random manner, exploring the intranuclear space. Eventually microtubules extending from opposite SPBs come into contact and establish an antiparallel connection thus reassembling the spindle. Mutant approaches revealed that deletion of ase1 and klp5 did not prevent spindle reassembly, however introduced aberrations during the spindle formation. Amazingly, cut7p showed direct colocalization with microtubule overlap during spindle reassembly. Abrogation of cut7p led to inability to form a functional spindle. Thus, cut7p is the main regulator of spindle formation in fission yeast. None of the mutant strains affected microtubule pivoting, confirming that microtubule pivoting is a random movement unrelated to MAPs.
|
6 |
Analysis of gene expression data from Massive Parallel Sequencing identifies so far uncharacterised regulators for meiosis with one candidate being fundamental for prophase I in male and female meiosisFinsterbusch, Friederike 01 June 2018 (has links) (PDF)
Meiosis is a specialized division of germ cells in sexually reproducing organisms, which is a fundamental process with key implications for evolution and biodiversity. In two consecutive rounds of cell division, meiosis I and meiosis II, a normal, diploid set of chromosome is halved. From diploid mother cells haploid gametes are generated to create genetic individual cells. This genetic uniqueness is obtained during prophase of meiosis I by essential meiotic processes in meiotic recombination, as double strand break (DSB) formation and repair, formation of crossovers (CO) and holiday junctions (HJs). Checkpoint mechanisms ensure a smooth progress of these events. Despite extensive research key mechanisms are still not understood. Based on an analysis of Massive Parallel Sequencing (MPS) data I could identify 2 genes, Mcmdc2 and Prr19, with high implication in meiotic recombination. In the absence of Mcmdc2 both sexes are infertile and meiocytes arrest at a stage equivalent to mid-‐pachytene in wt. Investigations of the synaptonemal complex (SC) formation revealed severe defects suggesting a role for MCMDC2 in homology search.
Moreover, MCMDC2 does not seem to be essential for DSB repair, as DSB markers of early and mid recombination nodules, like DMC1 and RPA, are decreased in oocytes. Nevertheless, late recombination nodules, which are positive for MutL homolog 1 (MLH1), do not form in both sexes. The absence of the asynapsis surveillance checkpoint mechanism in Hormad2 deficient ovaries with Mcmdc2 mutant background allowed survival of oocytes. This points into the direction that Mcmdc2 knockout oocytes get eliminated after prophase I due to failed homologous synapsis. Interestingly, MCMDC2 contains a conserved helicase domain, like the MCM protein family members MCM8 and MCM9. I therefore hyphothesize that Mcmdc2 promotes homolgy search.
|
7 |
Reconstitution of bacterial cytokinesis: the Z-ringArumugam, Senthil 13 November 2012 (has links) (PDF)
Prokaryotic cell division is one of the most fundamental processes in biology, but the dynamics and mechanics are far from being understood. In many bacteria, FtsZ, a tubulin homologue assembles into a ring-like structure – Z-ring at precisely the middle of the cell. This accurate site selection is dependent on the Min proteins. Min D and MinE self-organise into waves in vitro, and oscillate pole to pole in vivo. MinC is thought to couple the Min oscillations to FtsZ by direct interaction. The mechanism of inhibitory action of MinC on FtsZ assembly is not known. Critical to the understanding of regulation of FtsZ by MinC and other proteins and its probable role in force generation is the organisation, structure and the dynamics of the Z-ring. Current models of the FtsZ filament organization in the Z-ring argue between two different structures – (i) short overlapping protofilaments with lateral interactions and (ii) few long annealed protofilaments with or without lateral contacts.
Our observations of the characteristics of polymerization and turnover studies using fluorescence microscopy suggest that the FtsZ filament is a continuous and irresolute bundle. The results are consistent with a structure where the turnover happens throughout, and any specialised structure resulting in a GTP cap like structure can be ruled out. We show that the turnover rates and hydrolysis rates are similar arguing for a model in which subunit leaves as soon as it hydrolyses GTP. On the basis of crystal structures, we cloned the N-terminal of FtsZ, which acts as a C-terminal end capping fragment and is able to interact with monomers. The end-capping fragment, NZ can disassemble the FtsZ polymers, without influencing the GTPase activity, offering a comparable standard for the activity of MinC. On the basis of our observations, we propose a model on how MinC can disassemble FtsZ polymers. Furthermore, our data shows that the Min CDE system is sufficient to cause spatial regulation of FtsZ provided FtsZ is dynamic.
How the Z-ring takes the form of a functional helical or ring-like structure remains unclear. Extensive modelling approaches have tried to explain the ring formation and force generation. Previous studies have qualitatively shown bending of liposome membranes by FtsZ filaments. We hypothesised that the presumably intrinsically curved filaments should respond to pre-curved substrates, and the alignment should be quantifiable. This should ascertain whether or not FtsZ has intrinsic curvature and/or actively induces any force. Thus, we investigated how FtsZ filaments respond to a range of curvatures, which mimic different stages of the division process.
Our results show that the FtsZ filaments possess intrinsic curvatures as well as spontaneous twist. This facilitates the formation of Z-ring by utilizing geometrical cues. Our results are in agreement with consistent helical FtsZ polymers observed in vivo by Cryo-EM or super resolution microscopy. The alignment of filaments over a range of curvature suggests that the filaments have considerable flexibility, which strongly suggests reconsidering possible mechanisms of force generation. Moreover, the developed assay constitutes a valuable platform to further study proteins involved in modifying curvature of FtsZ filaments.
In summary, by reconstituting the FtsZ filament in vitro, we have elucidated the nature of FtsZ filaments. The dynamics of FtsZ filaments allows them to be inhibited by MinC, thus cooperating with the Min waves. The presence of intrinsic curvature and twist facilitates their formation into a ring necessary for the cell to carry out cytokinesis.
|
8 |
Optical Diffraction Tomography for Single CellsMüller, Paul 09 May 2016 (has links) (PDF)
Analyzing the structure of a single cell based on its refractive index (RI) distribution is a common and valued approach, because it does not require any artificial markers. The RI is an inherent structural marker that can be quantified in three dimensions with optical diffraction tomography (ODT), an inverse scattering technique. This work reviews the theory of ODT and its implementation with an emphasis on single-cell analysis, identifying the Rytov approximation as the most efficient descriptor for light propagation. The accuracy of the reconstruction method is verified with in silico data and imaging artifacts associated with the inverse scattering approach are addressed. Furthermore, an experimental ODT setup is presented that consists of a bright-field microscope, a phase-imaging camera, and an optical trap combined with a microfluidic chip. A novel image analysis pipeline is proposed that addresses image corrections and frame alignment of the recorded data prior to the RI reconstruction. In addition, for a rotational axis that is tilted with respect to the image plane, an improved reconstruction algorithm is introduced and applied to single, suspended cells in vitro, achieving sub-cellular resolution.
|
9 |
The role of yolk syncytial layer and blastoderm movements during gastrulation in zebrafishCarvalho, Lara 17 January 2008 (has links) (PDF)
During gastrulation, a set of highly coordinated morphogenetic movements creates the shape and internal organization of the embryo. In teleostean fishes, these morphogenetic movements involve not only the embryonic progenitor cells (deep cells) but also two extra-embryonic tissues: an outer sheet of epithelial cells (EVL) and a yolk syncytial layer (YSL). Epiboly is characterized by the spreading of the blastoderm (deep cells and EVL) to cover the large yolk cell, whereas convergence and extension leads, respectively, to mediolateral narrowing and anteroposterior elongation of the embryo. Recent studies have shown that the nuclei of the YSL undergo epiboly and convergence and extension movements similarly to the overlying deep cells, suggesting that these tissues interact during gastrulation. However, it is so far not clear whether and how the movements of YSL nuclei and deep cells influence each other. In the first part of this thesis, the convergence and extension movement of YSL nuclei was quantitatively compared to the movement of the overlying mesendodermal progenitor (or “hypoblast)” cells. This revealed that, besides the similarity in the overall direction of movement, YSL nuclei and hypoblast cell movements display differences in speed and directionality. Next, the interaction between YSL and hypoblast was addressed. The movement of the blastoderm was analyzed when YSL nuclei movement was impaired by interfering with the YSL microtubule cytoskeleton. We found that YSL and blastoderm epiboly were strongly reduced, while convergence and extension were only mildly affected, suggesting that YSL microtubules and YSL nuclei movement are required for epiboly, but not essential for convergence and extension of the blastoderm. We also addressed whether blastodermal cells can influence YSL nuclei movement. In maternal-zygotic one-eyed pinhead (MZoep) mutant embryos, which lack hypoblast cells, YSL nuclei do not undergo proper convergence movement. Moreover, transplantation of wild type hypoblast cells into these mutants locally rescued the YSL nuclei convergence phenotype, indicating that hypoblast cells can control the movement of YSL nuclei. Finally, we propose that the hypoblast influences YSL nuclei movement as a result of shape changes caused by the collective movement of cells, and that this process requires the adhesion molecule E-cadherin.
|
10 |
Experiments concerning the mechanism of cytokinesis in Caenorhabditis elegans embryos / Experimente zur Untersuchung der Zytokinese in Caenorhabditis elegansBringmann, Henrik Philipp 31 January 2007 (has links) (PDF)
In my thesis I aimed to contribute to the understanding of the mechanism of cytokinesis in C. elegans embryos. I wanted to analyze the relative contributions of different spindle parts – microtubule asters and the midzone - to cytokinesis furrow positioning. I developed a UV laser-based severing assay that allows the spatial separation of the region midway between the asters and the spindle midzone. The spindle is severed asymmetrically between one aster and the midzone. I found that the spindle provides two consecutive signals that can each position a cytokinesis furrow: microtubule asters provide a first signal, and the spindle midzone provides a second signal. The use of mutants that do not form a midzone suggested that the aster-positioned furrow is able to divide the cell alone without a spindle midzone. Analysis of cytokinesis in hypercontracile mutants suggests that the aster-positioned cytokinesis furrow and the midzone positioned furrow inhibit each other by competing for cortical contractile elements. I then wanted to identify the molecular pathway responsible for cytokinesis furrow positioning in response to the microtubule asters. To this end, I performed an RNAi screen, which identified a role for LET-99 in cytokinesis: LET-99 appeared to be required for aster-positioned cytokinesis but not midzone-positioned cytokinesis. LET-99 localizes as a cortical band that overlaps with the cytokinesis furrow. Mechanical displacement of the spindle demonstrated that the spindle positions cortical LET-99 at the site of furrow formation. The furrow localization of LET-99 depended on G proteins, and consistent with this finding, G proteins are also required for aster-positioned cytokinesis. (Anlage: Quick time movies, 466, 67 MB)
|
Page generated in 0.0408 seconds