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Dissertations / Theses on the topic 'Plant cell cycle'
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1
Jopson, Martin Frederick. "Plant microtubules, their associated proteins and the cell cycle." Thesis, University of East Anglia, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.318090.
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Kulaveerasingam, Harikrishna. "A molecular study of dedifferentiation and cell cycle reactivation in mechanically isolated asparagus cells." Thesis, University of Leicester, 1989. http://hdl.handle.net/2381/33631.
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Mechanically isolated cell cultures were chosen as a model system to examine wound-induced dedifferentiation at the molecular level as large quantities of physiologically and morphologically similar G1-arrested mesophyll cells could be obtained. Within 5 days of culture such non-dividing, photosynthetic cells become heterotrophic, and have completed a first nuclear division and cytokinesis. There are few changes in cell morphology during the first 2-3 days in culture. However, during this period there is a massive increase in respiration rate and total RNA synthesis. Following DNA synthesis there is a rapid cell expansion, mitosis and cytokinesis. Steady state transcript populations were monitored through the first 8 days of culture by analysis of the products of in vitro translations on 2-D gels. Large changes in gene expression were evident during the first 3 days in culture with several genes highly up-regulated and others down-regulated. Dedifferentiation can be separated into 3 different phases. Firstly, reactivation of the cell cycle during which there are few cytological or physiological changes but gross changes in the expression of genes possibly associated with wounding or stress. Secondly, DNA synthesis, first mitosis event and phragmoplast formation during which there are minor changes in transcript abundance. Finally a continuation of the cell cycle with little alteration in transcript abundance. Changes in plastid morphology are only apparent after 10-14 days resulting in the formation of proplastid like structures. However, mRNA for both large subunit ribulose bisphosphate carboxylase and small subunit ribulose bisphosphate carboxylase decrease to basal levels within a day of culture and photosynthetic capacity diminishes when the first cell division is evident. Plastid dedifferentiation can therefore be considered separately and proceeds slowly being more or less complete after 2-3 cell divisions. Dedifferentiation is therefore seen to be a complex process which involves the interaction of several factors i.e wounding and hormones and results in temporal changes in transcript abundance, changes in the mode of respiration, morphology and cell proliferation.
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Harper, John D. I. "Genetical and ultrastructural analysis of the Chlamydomonas cell cycle." Thesis, Queen's University Belfast, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.236312.
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Rafiei, Golnaz. "Studies on the role of WEE1 in the plant cell cycle." Thesis, Cardiff University, 2012. http://orca.cf.ac.uk/30683/.
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WEE 1 is a key eukaryotic cell cycle regulator. In plants it has a clear role at the DNA damage/ DNA replication checkpoints. I aimed to discover the functional significance of interactions between WEE1 and other cellular proteins in Arabidopsis thaliana and Nicotiana tabacum. First I examined effects of ectopic expression of Arabidopsis WEE1 (Arath;WEE1) in transgenic tobacco and tobacco WEE1 (Nicta;WEE1) in transgenic Arabidopsis. Western blotting using a plant WEE1 antibody showed that expression of Nicta;WEE1 in Arabidopsis caused increases in total WEE1 protein. The response of primary root length, numbers of lateral roots and primordia, and meristem length to zeocin (a DNA damaging agent) and hydroxyurea, (which perturbs DNA replication), resembled the wee1-1 insertional mutant rather than Arath;WEE1 over-expression. Expression of Arath;WEE1 in tobacco resulted in reduced WEE1 protein but also induced similar phenotypic changes as Nicta;WEE1 expression in Arabidopsis under zeocin and HU stress. I concluded that interactions with cellular proteins in the alien species resulted in down-regulation of WEE1 activity. In a yeast 2-hybrid screen Arath;WEE1 interacted with the glutathione-S-transferase protein, GSTF9. To test the functionality of this interaction I analysised the root and cell cycle phase phenotypes of single mutants: wee1-1 and gstf9 and I generated the double mutant wee1-1;gstf9. I demonstrated that both Arath;WEE1 and GSTF9 have roles in the DNA replication and damage checkpoints, but largely act in different genetic pathways. Arath;WEE1 also interacts with GF14ω, a 14-3-3 protein in a yeast 2-hybrid assay. In other eukaryotes this stabilizes WEE1. I confirmed that over-expression of GFF14ω in transgenic Arabidopsis (GFF14ω OEX) results in a very similar root phenotype to over-expression of Arath;WEE1 as predicted from a stabilization of WEE1. However the GFF14ω OEX phenotype was not abolished in a wee1-1 genetics background.indicating that Arath;WEE1is not required for the action of GF14ω.
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Spadafora, N. D. "Effect of CDC25 and WEE1 on plant cell cycle and morphogenesis." Thesis, University of Worcester, 2010. http://eprints.worc.ac.uk/745/.
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The cell cycle comprises the four phases of, G1, S-phase, G2 and mitosis. Two critical transitions are G1/S and G2/M; the latter is regulated by WEE1 kinase and CDC25 phosphatases. The scope of this thesis was to investigate the regulation of the G2/M transition of the cell cycle by WEE1 and CDC25, and how these genes interface with plant growth regulators in Arabidopsis thaliana. In Arabidopsis roots, the frequency of lateral roots was found to be increased by ectopic expression of Schizosaccharomyces pombe (Sp)cdc25e and reduced by Arath;WEE1 expression. I examined the effect of Arath;WEE1 and Spcdc25 on induction of shoots and roots in Arabidopsis hypocotyls in vitro. Hypocotyl explants from two over-expressing WEE1 lines , three T-DNA insertion lines and two expressing cdc25 (Spcdc25e) lines together with wild type (WT) were cultured on two-way gradients of kinetin (Kin) and naphthyl acetic acid (NAA). Below a threshold concentration of NAA (100 ng ml-1), WEE1 repressed morphogenesis in vitro, whereas at all NAA/Kin combinations Spcdc25 promoted morphogenesis (particularly root formation) over and above that in WT. Loss of function wee1-1 cultures were very similar to WT. Quantitative data indicated a significant increase in the frequency of root formation in Spcdc25e cultures compared with WT particularly at low Kin concentrations, and WEE1oe’s repressive effect was overcome by NAA but not Kin. In conclusion, WEE1 has a repressive effect on morphogenesis in vitro that can be overcome by auxin whereas Spcd25 by-passes a cytokinin requirement for the induction of morphogenesis in vitro. The role of CDC25 and WEE1 in DNA damage responses was also analysed. Two over-expressing Arath;CDC25 lines and T-DNA mutants showed no difference to WT either in standard conditions or zeocin-supplemented treatments. However, root length was longer in Arath;CDC25oe lines treated with hydroxyurea (HU) and lateral root number was increased compared to WT. This suggests a differential response of Arath;CDC25oe in the DNA replication (HU-induced) and DNA damage (zeocin-induced) checkpoints (Chapter 5). Finally the roles of WEE1 and CDC25 in cell cycle regulation were examined using tobacco TBY-2 cell cultures expressing Arath;WEE1, Nicotiana tabacum (Nicta)WEE1 or Arath;CDC25. Whilst Nicta;WEE1 lengthened G2 of the cell cycle, Arath;WEE1 had an unusual effect of shortening G2 phase and Arath;CDC25 had no observable effect (Chapter 6).
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Webb, Penelope 1967. "Effects of yeast cell cycle gene expression in transgenic Nicotiana tabacum." Monash University, Dept. of Biological Sciences, 2001. http://arrow.monash.edu.au/hdl/1959.1/9084.
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Vieira, Paulo. "Cell cycle maneuvering : a strategy taken by plant parasitic nematodes to induce specialized feeding sites in plant roots." Nice, 2012. http://www.theses.fr/2012NICE4114.
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He, Enuo. "Stochastic modelling of the cell cycle." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:04185cde-85af-4e24-8d06-94b865771cf1.
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Precise regulation of cell cycle events by the Cdk-control network is essential for cell proliferation and the perpetuation of life. The unidirectionality of cell cycle progression is governed by several critical irreversible transitions: the G1-to-S transition, the G2-to-M transition, and the M-to-G1 transition. Recent experimental and theoretical evidence has pulled into question the consensus view that irreversible protein degradation causes the irreversibility of those transitions. A new view has started to emerge, which explains the irreversibility of cell cycle transitions as a consequence of systems-level feedback rather than of proteolysis. This thesis applies mathematical modelling approaches to test this proposal for the Mto- G1 transition, which consists of two consecutive irreversible substeps: the metaphase-to-anaphase transition, and mitotic exit. The main objectives of the present work were: (i) to develop deterministic models to identify the essential molecular feedback loops and to examine their roles in the irreversibility of the M-to-G1 transition; (ii) to present a straightforward and reliable workflow to translate deterministic models of reaction networks into stochastic models; (iii) to explore the effects of noise on the cell cycle transitions using stochastic models, and to compare the deterministic and the stochastic approaches. In the first part of this thesis, I constructed a simplified deterministic model of the metaphase-to-anaphase transition, which is mainly regulated by the spindle assembly checkpoint (the SAC). Based on the essential feedback loops causing the bistability of the transition, this deterministic model provides explanations for three open questions regarding the SAC: Why is the SAC not reactivated when the kinetochore tension decreases to zero at anaphase onset? How can a single unattached kinetochore keep the SAC active? How is the synchronized and abrupt destruction of cohesin triggered? This deterministic model was then translated into a stochastic model of the SAC by treating the kinetochore microtubule attachment at prometaphase as a noisy process. The stochastic model was analyzed and simulation results were compared to the experimental data, with the aim of explaining the mitotic timing regulation by the SAC. Our model works remarkably well in qualitatively explaining experimental key findings and also makes testable predictions for different cell lines with very different number of chromosomes. The noise generated from the chemical interactions was found to only perturb the transit timing of the mitotic events, but not their ultimate outcomes: all cells eventually undergo anaphase, however, the time required to satisfy the SAC differs between cells due to stochastic effects. In the second part of the thesis, stochastic models of mitotic exit were created for two model organisms, budding yeast and mammalian cells. I analyzed the role of noise in mitotic exit at both the single-cell and the population level. Stochastic time series simulations of the models are able to explain the phenomenon of reversible mitotic exit, which is observed under specific experimental conditions in both model organisms. In spite of the fact that the detailed molecular networks of mitotic exit are very different in budding yeast and mammalian cells, their dynamic properties are similar. Importantly, bistability of the transitions is successfully captured also in the stochastic models. This work strongly supports the hypothesis that uni-directional cell cycle progression is a consequence of systems-level feedback in the cell cycle control system. Systems-level feedback creates alternative steady states, which allows cells to accomplish irreversible transitions, such as the M-to-G1 transition studied here. We demonstrate that stochastic models can serve as powerful tools to capture and study the heterogeneity of dynamical features among individual cells. In this way, stochastic simulations not only complement the deterministic approach, but also help to obtain a better understanding of mechanistic aspects. We argue that the effects of noise and the potential needs for stochastic simulations should not be overlooked in studying dynamic features of biological systems.
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Fülöp, Katalin. "Analysis of two plant protein complexes associated with transcription and cell cycle progression." Szegedi Tudományegyetem, 2005. http://www.theses.fr/2005PA112194.
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Su, Yingtao. "Function and regulation of myc-family bHLHZip transcription factors during the animal and plant cell cycle /." Uppsala : Dept. of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, 2008. http://epsilon.slu.se/200836.pdf.
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Douglas, Mary Elizabeth. "Cost analysis and balance-of-plant of a solid oxide fuel cell/gas turbine combined cycle." Thesis, Georgia Institute of Technology, 2003. http://hdl.handle.net/1853/17960.
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Siciliano, Ilario. "Effect of plant WEE1 on the cell cycle and development in Arabidopsis thaliana and Nicotiana tabacum." Thesis, Cardiff University, 2006. http://orca.cf.ac.uk/56100/.
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In eukaryotes the regulatory cell cycle gene, WEE1, encodes a protein kinase. In late G2, it inactivates cyclin-dependent kinase (CDKs) in the CDK-cyclinA/B complexes, by phosphorylating the CDK on tyrosine 15. This can result in a delay in mitosis. Expression of Arabidopsis thaliana homologue of WEE1 (AtWEEl) in fission yeast resulted in an elongated cell length phenotype in the same way as over expression of fission yeast weel. I have tested whether At WEE1 could also induce this effect in tobacco cells and Arabidopsis plant roots. The tobacco BY-2 cells have been transformed with AtWEEl, both under constitutive and inducible promoters. Phenotypic characteristics observed compared with the control are premature entry into mitosis and a reduced cell size through a shortening of the G2 phase with a compensatory increase in the duration of Gl phase. Hence, the phenotype and cell cycle response is the exact opposite of the known effect of expression of this gene in fission yeast. NtWEEl expression data revealed that the endogenous WEE1 expression is delayed in transgenic lines, this results in a non-inhibition of CDKA and CDKB1 which are already active in early S-phase. AtWEEl was also employed to transform Arabidopsis thaliana plants, both under constitutive and inducible promoters. The effect of AtWEEl over expression was investigated on primary root growth and lateral root development. In particular, AtWEEl over expression lead to less primary root growth and a reduction in the frequency of lateral root primordia initiated when compared with wild type. Arabidopsis transgenic plants initiated fewer primordia both per unit time and per cm of primary root.
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Novak, Zsofia A. "The role and regulation of Asterless in the centrosome cycle." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:4fadaef1-8c9e-4c70-ac59-47f35af3988e.
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Centrosomes are the main microtubule organizing centres in animal cells and are formed by a pair of centrioles together with surrounding pericentriolar material (PCM). Cycling cells duplicate their centrosomes strictly once per cell cycle. This process is driven by the semi-conservative duplication of the centrioles that are found at the centrosome core. During the exit from mitosis the two centrioles within the single inherited centrosome separate, and upon the start of S-phase each of these inherited mother centrioles assembles an adjacent daughter at its side. This process results in two complete centrosomes that can form the poles of the mitotic spindle, and thus segregate evenly to the next cell generation. The formation of a daughter centriole suppresses the initiation of new duplication events from the same templating mother centriole until this daughter separates - disengages - at the end of the cell cycle. This regulation - that acts to repress centriole amplification - is summarized in the 'licensing model of centriole duplication' (Tsou and Stearns, 2006). This model states that centriole disengagement provides the license for the re-duplication of mother centrioles. Importantly, experiments show that while abolishing centriole engagement is sufficient to allow mother centrioles to re-duplicate within the same cycle, it is insufficient to allow daughter centrioles the assembly of a granddaughter before they mature into mothers towards the end of their first cell cycle. The molecular nature of this daughter-to-mother transition remains mysterious. In this thesis I show that in Drosophila embryos the essential centriole duplication protein Asl is not incorporated into daughter centrioles as they assemble during S-phase, but is only incorporated once mother and daughter separate at the end of mitosis. The initial incorporation of Asterless (Asl) is irreversible, and is dependent on centriolar DSas-4. Crucially, Asl incorporation is essential for daughter centrioles to mature into mothers that can support centriole duplication. I propose that Asl acts as a permanent primary license that allows new centrioles to duplicate for the first time. Once acquired, this primary license is not lost but rather further regulation is taken over by the reduplication licensing mechanism, disengagement. This work extends the previously proposed licensing model to also explain how new centrioles are licensed for their first duplication event.
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Farrar, Kerrie. "Exordium : a novel gene in Arabidopsis identified by promoter trapping." Thesis, Durham University, 2000. http://etheses.dur.ac.uk/4361/.
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The aim of the project was to characterize the expression of a GUS promoter trap in a transgenic line of Arabidopsis thaliana, and to investigate the function of the tagged gene. GUS expression in the transgenic line, designated line EM2, was found to be localized to embryos, and to the regions of most active cell division in the seedling, notably the apical meristems and young leaves. The tagged gene was named EXORDIUM (EXO). Line EM2, which contains a single copy of the promoter trap T-DNA, was found to have no obvious phenotype when homozygous for the T-DNA insertion, and when grown under a range of nutritional and hormonal conditions in vitro. It was found that the expression of the GUS gene and of the native EXO gene, was each up-regulated by exogenous auxin and down-regulated by exogenous cytokinin. The cloned EXO promoter was introduced as a GUS fusion into transgenic plants of A. thaliana and found to be expressed ion the same tissues as EM2, and additionally in the root vascular tissues, leaves and siliques, although to different extents in different transgenic lines. It was found that the EXO mRNA abundance accumulated in seedlings treated with hydroxyurea, which induces cell-cycle arrest at the Gl-S transition. Further analysis demonstrated that EXO mRNA is preferentially abundant during M-phase of the cell cycle. Transgenic plants were produced containing sense and antisense versions of the EXO gene under the transcriptional control of the CaMV35S promoter. One antisense line exhibited an abberant phenotype, characterized by a reduced size and abnormal shoot branching pattern. EXO encodes a predicted protein of 314 amino acids, of unknown function.
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Tan, Yi-Hsun. "Investigation of G1 Arrest Mechanisms Induced by Sanguisorba officinalis Extracts in B16F10 Cells." Kyoto University, 2019. http://hdl.handle.net/2433/245331.
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Taylor, Marcus Andrew. "The effects of light on the cell cycle in the shoot apex of Silene coeli-rosa (L.) Godron during floral evocation." Thesis, Cardiff University, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.304803.
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Skirycz, Aleksandra. "Functional analysis of selected DOF transcription factors in the model plant Arabidopsis thaliana." Phd thesis, Universität Potsdam, 2007. http://opus.kobv.de/ubp/volltexte/2008/1698/.
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Transcription factors (TFs) are global regulators of gene expression playing essential roles in almost all biological processes, and are therefore of great scientific and biotechnological interest. This project focused on functional characterisation of three DNA-binding-with-one-zinc-finger (DOF) TFs from the genetic model plant Arabidopsis thaliana, namely OBP1, OBP2 and AtDOF4;2. These genes were selected due to severe growth phenotypes conferred upon their constitutive over-expression.
To identify biological processes regulated by OBP1, OBP2 and AtDOF4;2 in detail molecular and physiological characterization of transgenic plants with modified levels of OBP1, OBP2 and AtDOF4;2 expression (constitutive and inducible over-expression, RNAi) was performed using both targeted and profiling technologies. Additionally expression patterns of studied TFs and their target genes were analyzed using promoter-GUS lines and publicly available microarray data. Finally selected target genes were confirmed by chromatin immuno-precipitation and electrophoretic-mobility shift assays. This combinatorial approach revealed distinct biological functions of OBP1, OBP2 and AtDOF4;2.
Specifically OBP2 controls indole glucosinolate / auxin homeostasis by directly regulating the enzyme at the branch of these pathways; CYP83B1 (Skirycz et al., 2006). Glucosinolates are secondary compounds important for defence against herbivores and pathogens in the plants order Caparales (e.g. Arabidopsis, canola and broccoli) whilst auxin is an essential plant hormone. Hence OBP2 is important for both response to biotic stress and plant growth.
Similarly to OBP2 also AtDOF4;2 is involved in the regulation of plant secondary metabolism and affects production of various phenylpropanoid compounds in a tissue and environmental specific manner. It was found that under certain stress conditions AtDOF4;2 negatively regulates flavonoid biosynthetic genes whilst in certain tissues it activates hydroxycinnamic acid production. It was hypothesized that this dual function is most likely related to specific interactions with other proteins; perhaps other TFs (Skirycz et al., 2007).
Finally OBP1 regulates both cell proliferation and cell expansion. It was shown that OBP1 controls cell cycle activity by directly targeting the expression of core cell cycle genes (CYCD3;3 and KRP7), other TFs and components of the replication machinery. Evidence for OBP1 mediated activation of cell cycle during embryogenesis and germination will be presented. Additionally and independently on its effects on cell proliferation OBP1 negatively affects cell expansion via reduced expression of cell wall loosening enzymes.
Summing up this work provides an important input into our knowledge on DOF TFs function. Future work will concentrate on establishing exact regulatory networks of OBP1, OBP2 and AtDOF4;2 and their possible biotechnological applications.Biologische Prozesse, wie beispielsweise das Wachstum von Organen und ganzen Organismen oder die Reaktion von Lebewesen auf ungünstige Umweltbedingungen, unterliegen zahlreichen Regulationsmechanismen. Besonders wichtige Regulatoren sind die sogenannten Transkriptionsfaktoren. Dabei handelt es sich um Proteine, die die Aktivität von Erbeinheiten, den Genen, beeinflussen. In Pflanzen gibt es etwa 2000 solcher Regulatoren. Da sie wichtige Kontrollelemente darstellen, sind sie von großem wissenschaftlichen und biotechnologischen Interesse. Im Rahmen der Doktorarbeit sollte die Funktion von drei Transkriptionsfaktoren, genannt OBP1, OBP2 und AtDOF4;2, untersucht werden. Sie wurden bei der Suche nach neuen Wachstumsregulatoren identifiziert. Als Untersuchungsobjekt diente die in der Öffentlichkeit kaum bekannte Pflanze Ackerschmalwand, lateinisch als Arabidopsis thaliana bezeichnet. Um die Funktion der Regulatoren zu entschlüsseln, wurden an der Modellpflanze genetische Veränderungen durchgeführt und die Pflanzen dann mit molekularbiologischen und physiologischen Methoden analysiert. Es zeigte sich, dass OBP1 an der Regulation der Zellteilung beteiligt ist. Alle Lebewesen sind aus Zellen aufgebaut. Gelingt es, die Zellteilung gezielt zu steuern, kann damit beispielsweise die Produktion von pflanzlicher Biomasse verbessert werden. Das OBP1-Protein übt auch einen Einfluss auf die Zellstreckung aus und beeinflusst auch auf diesem Wege das pflanzliche Wachstum. Die beiden anderen Proteine steuern Prozesse, die im Zusammenhang mit der Bildung von Pflanzeninhaltsstoffen stehen. OBP2 ist Teil eines zellulären Netzwerkes, dass die Synthese von sogenannten Glucosinolaten steuert. Glucosinolate kommen unter anderem in Broccoli und Kohl vor. Sie fungieren als Abwehrstoffe gegen Fraßinsekten. Einigen Glucosinolaten wird auch gesundheitsfördernde Wirkung zugesprochen. Das Protein AtDOF4;2 ist Komponente eines anderen Netzwerkes, dass die Bildung von Phenylpropanoiden steuert. Diese Substanzen haben strukturelle Funktion und spielen darüber hinaus eine Rolle bei der pflanzlichen Toleranz gegenüber tiefen Temperaturen. Mit der Doktorarbeit konnte das Wissen über die Transkriptionsfaktoren erheblich erweitert und die Grundlage für interessante zukünftige Arbeiten gelegt werden. Von großer Bedeutung wird es dabei sein, die Netzwerke, in die die Transkriptionsfaktoren eingebunden sind, noch besser zu verstehen. Dann wird es möglich sein, auch Teilnetzwerke gezielt zu beeinflussen, was für biotechnologische Anwendungen, beispielsweise bei der Präzisionszüchtung von nachwachsenden Rohstoffen, von zentraler Bedeutung ist.
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Kryvych, Sergiy. "Gene expression profiling in different stages of development of Arabidopsis thaliana leaftrichomes at the single cell level." Phd thesis, Universität Potsdam, 2007. http://opus.kobv.de/ubp/volltexte/2008/1747/.
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Each organ of a multicellular organism is unique at the level of its tissues and cells. Furthermore, responses to environmental stimuli or developmental signals occur differentially at the single cell or tissue level. This underlines the necessity of precise investigation of the “building block of life” -the individual cell. Although recently large amount of data concerning different aspects of single cell performance was accumulated, our knowledge about development and differentiation of individual cell within specialized tissue are still far from being complete.
To get more insight into processes that occur in certain individual cell during its development and differentiation changes in gene expression during life cycle of A. thaliana leaf hair cell (trichome) were explored in this work. After onset of trichome development this cell changes its cell cycle: it starts endoreduplication (a modified cell cycle in which DNA replication continues in the absence of mitosis and cytokinesis). This makes trichomes a suitable model for studying cell cycle regulation, regulation of cell development and differentiation.
Cells of interest were sampled by puncturing them with glass microcapillaries. Each sample contained as few as ten single cells. At first time trichomes in initial stage of trichome development were investigated. To allow their sampling they were specifically labelled by green fluorescent protein (GFP). In total three cell types were explored: pavement cells, trichome initials and mature trichomes. Comparison of gene expression profiles of these cells allowed identification of the genes differentially expressed in subsequent stages of trichome development. Bioinformatic analysis of genes preferentially expressed in trichome initials showed their involvement in hormonal, metal, sulphur response and cell-cycle regulation. Expression pattern of three selected candidate genes, involved in hormonal response and early developmental processes was confirmed by independent method. Effects of mutations in these genes on both trichome and plant development as well as on plant metabolism were analysed.
As an outcome of this work novel components in the sophisticated machinery of trichome development and cell cycle progression were identified. These factors could integrate hormone stimuli and network interactions between characterized and as yet unknown members of this machinery. I expect findings presented in this work to enhance and complement our current knowledge about cell cycle progression and trichome development, as well as about performance of the individual cell in general.Jedes Organ eines vielzelligen Organismus weißt einzigartige Merkmale auf seiner Gewebe und Zellebene auf. Darüber hinaus, werden entwicklungsabhängige sowie aus der Umwelt empfangene Signale zelltypspezifisch interpretiert. Aus dieser Spezialisierung einzelner Zellen ergibt sich somit unmittelbar die Notwendigkeit einzelne Zellen, als Bausteine komplexer Organe, individuell zu untersuchen. Obwohl in den letzten Jahrzehnten große Datenmengen über verschiedene Aspekte einzelner Zellen akkumuliert wurden, ist das Gesamtbild der Differenzierung und Entwicklung individueller Zellen in einem vielzelligen Organismus weitgehend unbekannt. Um der Frage nachzugehen, welche Prozesse sich in einer einzelnen Zelle während ihrer Differenzierung und Entwicklung abspielen, wurden Genexpressionsprofile einzelner Blatthaarzellen der Pflanze Arabidopsis thaliana in verschiedene Entwicklungsstadien erstellt. Nach dem Beginn der Entwicklung einer Protodermalzelle in ein Blatthaar (Trichom) kommt es zu einem Umschalten des Zellzyklus; Endoreduplikation setzt ein. Dies bedeutet, dass DNA repliziert wird, aber keine Zellteilung mehr stattfindet. Aus diesem Grunde eignen sich heranwachsende Trichome besonders gut Mechanismen zu erforschen, die in Verbindung mit der Zellzyklusregulation und Zellentwicklung stehen. Die Inhalte ausgewählter Einzelzellen wurden mit Glasmikrokapillaren extrahiert. Jeweils zehn derartige Einzelzellextrakte wurden daraufhin vereint. Als besonders hervorzuheben gilt, dass es uns in dieser Studie zum ersten mal überhaupt gelang die Inhalte einzelner Trichomzellen in ganz frühen Entwicklungsstadien zu extrahieren und anschließend zu analysieren. Um die Extraktion der Inhalte dieser frühen Zellstadien überhaupt zu ermöglichen, war es erforderlich diese mit dem grün fluoreszierenden Protein (GFP) zu markieren. Neben den Trichominitialzellen wurden ausgewachsene Trichomzellen sowie Epidermiszellen (Pavementzellen) mittels der Einzelzelltechnik untersucht. Ein Vergleich der erstellten Genexpressionsprofile dieser drei Zelltypen ermöglichte es Gene zu identifizieren, die in den ausgewählten Entwicklungsstadien der Trichombildung differentiell induziert wurden. Mittels bioinformatischer Analysemethoden gelang es, Gruppen von Genen zu identifieren, die exklusiv in Trichominitialzellen exprimiert sind und den Kategorien, Hormonregulation, Metallhomeostase, Schwefelstoffwechesol sowie Zellzyklusregulation zuzuordnen sind. Weiterhin wurde das Expressionsmuster dreier ausgewählter Kandidatengene mit alternativen Techniken verifiziert. Die ausgewählten Kandidatengene gehörten den Katergorien, Hormonrespons sowie frühe Entwicklungsprozesse, an. Darüber hinaus wurden Mutanten in allen drei Gene erzeugt und der Einfluss dieser Mutationen auf die Trichomentwicklung analysiert. Ein weiterer Aspekt der Mutantenanalyse lag in der Erstellung von Metabolitenprofilen ausgewählter Mutanten. Als ein wesentliches Ziel dieser Arbeit gelang es mir bisher unbekannte Komponenten in der Trichomentwicklung und damit der Zellzyklusregulation zu identifizieren. Diese neu identifizierten Komponenten führen zu einer Integration der hormonellen Kontrolle der Zellteilung und Entwicklung mit bisher unbekannten Faktoren. Ich erwarte, dass die von mir erbrachten Ergebnisse zu einem tieferen Verständnis der Prozesse, die an der Trichomentwicklung sowie an der Zellzyklusregulation beteiligt sind, beitragen. Insbesondere, zu einem erweiterten Verständnis des Verhaltens individueller Zellen in einem vielzelligen Organismus.
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Kesoglidou, Poli Xenia. "E2F7 : a member of the E2F family with a novel mechanism of transcriptional repression." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:5694f75b-0e73-493c-a6bd-da10793aded3.
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The mammalian E2F family of transcription factors plays a crucial role in the regulation of cellular proliferation, apoptosis and differentiation. E2F7 and E2F8 are the most recently identified family members and have unusual features that distinguish them from other members in the E2F family, including two distinct DNA-binding domains that bind to DNA in a DP-independent manner. E2F7 and E2F8 have been shown to be transcriptional repressors. However, the mechanism by which E2F7 represses E2F responsive gene expression remains to be elucidated. The results presented here provide the first insight into the E2F7-mediated transcriptional mechanism. E2F7 was shown to contain a CtBP binding motif and form a complex with CtBP in both HeLa and MCF7 cells. An E2F7 deletion mutant lacking the CtBP binding motif was unable to form a complex with CtBP and repress the transcription of E2F target genes in luciferase assays, suggesting that this motif is essential for E2F7-dependent repression. Furthermore, the E2F7-CtBP complex was shown to be stable under different types of damage, such as following etoposide and UV treatment, and under different cell redox states. Interestingly, however, E2F7 was unable to repress the transcriptional activity of E2F target genes following treatment with the CtBP substrate MTOB. Moreover, E2F7 endogenous immunoprecipitations showed that E2F7 forms a complex with the chromatin-modifying enzymes HDAC1, HDAC2 and LSD1 and the co-repressor CoREST. Interestingly, via chromatin immunoprecipitations, E2F7 was shown to recruit these co-repressors to a subset of E2F-responsive promoters where they affect the activity of these promoters in a target gene-specific manner. Furthermore, results presented here suggest that CtBP could play a dual role in E2F7 function, not only being involved in E2F7-mediated repression but also in the repression of E2F7 itself as siRNA mediated CtBP depletion was shown to cause an upregulation of E2F7 protein levels. These results implicate a repertoire of co-repressors in a target gene-specific E2F7 repression mechanism, and as such define a new level of complexity in cell cycle control.
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Cabral, do Nascimento Danila. "La biogenèse des galles induites par des phyto-nématodes dépend de l'hyperactivation du cycle cellulaire : études fonctionnelles des nouveaux acteurs du cycle mitotique et de l'endocycle dans les cellules géantes." Electronic Thesis or Diss., Université Côte d'Azur (ComUE), 2019. http://theses.univ-cotedazur.fr/2019AZUR6001.
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Lors des vingt dernières années, l’utilisation d’Arabidopsis thaliana en tant que plante modèle a permis d’accélérer les découvertes biologiques dans l’étude de nombreuses interactions plante-pathogènes tant au niveau moléculaire que cellulaire. Arabidopsis réagis de manière similaire aux plantes cultivées face aux attaques de nombreux pathogènes, y compris les nématodes. Meloidogyne incognita est un nématode endoparasite de la racine des plantes causant de lourdes pertes agronomiques estimées à plusieurs milliards de dollars par an. Afin d’induire la formation de sites nourriciers dans la racine de l'hôte, les nématodes à galles. Ceci a pour effet d’induire la formation de cellules géantes à l’origine des nodosités observées sur la racine de l’hôte communément appelées galles. Lors de la formation des sites nourriciers, les cellules géantes ont pour caractéristiques d’être hypertrophiées et poly-nucléés du fait de l’absence ou de l’interruption de la cytokinèse et de nombreux cycles successifs d'endoreduplication. Ainsi, une caractéristique cruciale du développement du nématode est l’hyper activation du cycle cellulaire dans les galles. Le cycle cellulaire eucaryote est conservé et caractérisé par la phase S (réplication de l'ADN), suivie par la phase mitose (M), la cytokinèse et la division cellulaire. Celles-ci sont entrecoupées par la phase G1 (Gap 1) qui relie la fin de la mitose, et la phase G2 (Gap 2) qui relie la fin de la synthèse de l'ADN. L'endocycle est une variante du cycle cellulaire dans laquelle la réplication se produit sans mitose, induisant le doublement du contenu en ADN cellulaire pour chaque endocycle. L’hyper activation du cycle cellulaire est essentielle pour la biogenèse de la galle induite par les nématodes et un équilibre précis entre les phases mitotiques et endocycliques est essentiel pour la réussite du parasitisme. Dans le cadre de ma thèse de doctorat, nous avons étudié les conditions préalables nécessaires à l’établissement d’une galle fonctionnelle induite par M. incognita. Je me suis particulièrement concentrée sur l’étude fonctionnelle de gènes du cycle cellulaire de la plante jouant un rôle dans la mitose et l’endoreduplication lors de l’interaction plante-nématode. Pour répondre à cette question j’ai réalisé des études fonctionnelles de nouveaux acteurs du cycle cellulaire chez Arabidopsis stimulant (E2F), inhibant (ABAP1 et son-interactor) ou contrôlant (WEE1) le cycle cellulaire des galles. Les résultats obtenus démontrent que les gènes étudiés ici sont essentiels pour la formation et le maintien des sites nourriciers des nématodes. Notre étude fonctionnelle et notre analyse moléculaire montrent que l’absence ou la surexpression de facteurs de transcription E2Fa et E2Fb affectent gravement le développement de la galle et la reproduction des nématodes et perturbent le cycle cellulaire des galles, validant ainsi leur importance pour le développement du site d’alimentation des nématodes. Nous avons également cherché à déterminer si le stress induit par les nématodes déclenche un checkpoint control pendant la progression du cycle cellulaire dans les galles. Par conséquent, une analyse fonctionnelle du facteur de transcription WEE1 a été réalisée et a montré que son absence induit une mitose plus précoce dans les sites nourriciers, affectant ainsi le développement des galles et la reproduction des nématodes. L’utilisation de drogues, d’un marqueur de stress SMR7, ainsi que d’une lignée exprimant un gène rapporteur sous le contrôle du promoteur du gène PARP1 (impliqué dans la réparation de l’ADN) ont suggéré l’activation du checkpoint control G1/S dans les galles. Les données présentées dans cette thèse ont apporté des connaissances fondamentales pour une meilleure compréhension du contrôle du cycle cellulaire lors de la biogenèse de la galle, ainsi que lors du développement de la planteDuring the last twenty years, Arabidopsis thaliana has been successfully used as a model plant accelerating discoveries at the molecular and cellular level in numerous plant-pathogen interactions. Arabidopsis is susceptible to a number of pathogens including nematodes, responding to pathogen attack in a similar manner to cultivated plant species. Meloidogyne incognita is a crop parasite causing extensive economic losses of billions of dollars yearly for the agriculture worldwide. Root-knot nematodes induce feeding sites within the host root inducing up to eight giant-feeding cells within the root vascular cylinder by injecting secretions. These giant cells become hypertrophied and contain multiple enlarged nuclei as a result of numerous mitotic events of which cytokinesis is absent or interrupted. Throughout expansion, giant cells become highly polyploid via successive endoreduplication cycles. Thus, a crucial feature for root-knot nematode development is the hyperactivation of the plant host cell cycle in galls. The eukaryotic cell cycle is conserved and characterized by four phases: The S phase (DNA replication) is followed by the M phase (mitosis), cytokinesis and cell division. These are intercalated by the G1 phase (first gap) which connects the end of mitosis to the start of DNA synthesis, and the G2 phase (second gap) which connects the end of DNA synthesis to the start of mitosis. The endocycle is a variant of the cell cycle in which replication occurs without mitosis, resulting in a doubling of cellular DNA content for each endocycle round. The hyperactivation of the cell cycle is essential for the nematode-induced gall biogenesis, and a precise balance between mitotic and endocycle phases is essential for the successful compatible interaction. As a broad question of my PhD, we addressed which the pre-requisites are needed to establish a functional gall induced by root-knot nematodes. More specifically, I essentially focused on the functional studies of particular plant cell cycle genes playing a role in the mitotic and the endocycle during plant-root-knot nematode interaction. To address this question, functional studies of new players in the cell cycle of the plant host, stimulating (E2F genes), inhibiting (ABAP1 and an interactor AIP10 genes) or controlling (WEE1 gene) the cell cycle, were investigated in galls induced in our model host Arabidopsis thaliana. The results obtained demonstrate that the genes here studied are critical for, as well the formation as the maintenance of nematode feeding sites. Overall, our functional study and molecular analysis show that the absence of E2Fa and E2Fb transcription factors will severely affect gall development and nematode reproduction. As well, overexpression of E2Fa and E2Fb will disrupt the cell cycle in galls, validating their importance for nematode feeding site development. We further investigated if nematode induced stress might trigger a checkpoint control during cell cycle progression in galls. Therefore, functional analysis of the WEE1 transcription factor was performed and showed that its absence induced galls prematurely to enter mitosis, thus affecting feeding site development and nematode reproduction. Performing drug treatments and the use of the stress marker SMR7 and a DNA repair promoter reporter line PARP1 suggested the induction of checkpoint activation in galls at G1/S phase of the cell cycle. The data presented in this thesis provided fundamental knowledge for a better understanding of cell cycle control during gall biogenesis as well sideways for plant development. Currently, our results are being managed for application initially in Arabidopsis, and promising strategies will be conducted and extended towards different crop species in order to better control nematode attack. Here, we specifically aimed at generating genetically modified plants by silencing E2Fa genes as an anti-nematode strategy in the field
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Savarin, Julie. "Caractérisation de la voie TCTP (TRANSLATIONALLY CONTROLLED TUMOR PROTEIN) chez Arabidopsis thaliana : identification des régulateurs de son accumulation et importance de la voie au cours du développement embryonnaire." Thesis, Lyon, 2018. http://www.theses.fr/2018LYSEN001.
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TCTP (Translationally Controlled Tumor Protein) est une protéine très conservée chez l'ensemble des eucaryotes. C’est une protéine vitale impliquée dans divers processus essentiels, et pour de nombreux organismes son absence conduit à la létalité dès les stades embryonnaires.Chez les animaux comme chez les végétaux, TCTP joue un rôle primordial dans la croissance et le développement des individus. En plus de son implication dans l’apoptose et la réparation de l’ADN, TCTP favorise la prolifération cellulaire, et se trouve donc être un élément important de la tumorigenèse. Chez les végétaux, la forte conservation de TCTP a permis la préservation de la plupart des fonctions décrites chez les animaux, mais les facteurs qui interviennent en amont ne sont pas encore connus.Par la mise en place, la conduite et la finalisation de deux cribles génétiques utilisant la plante modèle Arabidopsis thaliana, ce travail de thèse a cherché à identifier des facteurs situés en amont de TCTP. En parallèle, une seconde étude fut menée afin de mesurer l'impact de l'absence de TCTP sur les voies de l’auxine et des cytokinines au cours du développement embryonnaire, permettant de mieux comprendre l’origine de l’embryolétalité du mutant tctpTCTP (Translationally Controlled Tumor Protein) is strongly conserved among eukaryotes. It is a vital protein implicated in various major processes, and its absence leads to early embryolethality in many organisms. In plants as in animals, TCTP is a key factor of growth and development. Implicated in apoptosis and DNA repair, TCTP is also an enhancer of cell proliferation, and is a key element of tumorigenesis. Major functions of TCTP are conserved between plants and animals, but upstream factors are not known yet. Using a genetic screen on the model plant Arabidopsis thaliana, the principal goal of this thesis was to discover regulators of TCTP.In parallel, the impact of TCTP knockout on auxin and cytokinin pathways during embryo development was investigated
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Hall-Ponselè, Andrew M. "Genetic engineering of the primary/secondary metabolic interface in tobacco BY-2 cells." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:be5a3ee3-33c7-455c-b043-409987395f98.
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The supply of precursors from primary metabolism is often overlooked when engineering secondary metabolism for increased product yields. This is because precursor supply may be assumed to be non-limiting, and it is considered difficult to engineer primary metabolism, because control of carbon flow (flux) is generally distributed among most enzymes of the pathway. The aim of this thesis was to increase the production of sterols, part of the isoprenoid class of secondary metabolites, in tobacco (Nicotiana tabacum) Bright Yellow 2 (BY-2) cell cultures. This was achieved by genetically engineering increased activity of mitochondrial citrate synthase, an enzyme of the tricarboxylic acid (TCA) cycle that is involved in the provision of cytosolic acetyl coenzyme A, the primary metabolite precursor to sterols. Metabolic flux analysis revealed that citrate synthase exerts significant control over cyclic TCA cycle flux in BY-2 cells and suggested that increasing the activity of downstream enzymes within secondary metabolism could lead to a further redirection of TCA-cycle-derived precursors into sterol biosynthesis. Attempts were made to achieve this by genetically engineering increased activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), a key enzyme of secondary metabolism involved in sterol biosynthesis. Consistent with previous research, transgenic lines had increased sterol levels. However, the high sterol phenotype was unstable, and attempts to co-express HMGR and citrate synthase genes were unsuccessful. The thesis demonstrates that increasing the provision of precursors to secondary metabolites can result in increased yields of those secondary metabolites but suggests that in most cases the activity of enzymes within secondary metabolism has a greater effect on those yields. It also reveals that single enzymes can exert significant control of flux within primary metabolism, although the control exerted by specific enzymes probably changes with the demands placed on metabolism.
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Lee, Weon-Keun. "Screening for homologues of fission yeast cell cycle genes in plants." Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621893.
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Trolet, Adrien. "Cell cycle-dependent regulation and function of ARGONAUTE 1 in plants." Thesis, Strasbourg, 2018. http://www.theses.fr/2018STRAJ110.
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Chez tous les eucaryotes, la régulation de l’expression génique est primordiale pour le contrôle du cycle cellulaire. Un large éventail de gènes, incluant des régulateurs essentiels du cycle, mais aussi d’autre gènes impliqués dans la transduction du signal, la régulation hormonale et le métabolisme sont ainsi exprimé à certaines phases du cycle. Ces changements sont contrôlés à de multiples niveaux, notamment de façon transcriptionnelle et post-traductionnelle. Chez les mammifères, il est aujourd’hui évident que les micro ARNs contribuent à cette régulation en ciblant spécifiquement les transcrits d’un grand nombre de gènes régulés au cours du cycle. Cependant, nous n’avons que très peu d’informations à ce jour concernant le rôle des petits ARNs sur le contrôle de la prolifération cellulaire chez les plantes. Mes travaux de thèse ont permis de démontrer que la perte d’AGO1 affecte la prolifération cellulaire et l’activité du méristème racinaire. Nous avons également séquencé les transcrits, les petits ARNs et le dégradome à partir de cellules BY-2 synchronisées afin de déterminer le répertoire et la fonction des petit ARNs au cours du cycle cellulaireIn all eukaryotes, regulated gene expression is key to orchestrate cell cycle progression. Not only genes encoding important core cell cycle regulators, but also genes of a variety of other factors involved in signal transduction, hormonal regulation and metabolic control are expressed at specific time points of the cell cycle. These changes in gene expression are controlled at multiple levels, including transcriptional and post-translational controls. In mammals, it became evident that microRNAs contribute to this regulation by targeting the transcripts of numerous cell cycle-regulated genes. However, in plants we still know little about the regulatory roles of small RNAs in the control of cell proliferation. During my thesis, I showed that depletion of Arabidopsis AGO1 impairs cell proliferation and root meristem activity. To further determine the repertoire and role of sRNAs in cell cycle regulation, we thus sequenced total RNAs and small RNAs, AGO1-associated small RNAs and the RNA degradome of synchronized BY2 cells at S-, G2-, M- and G1-phases of the cell cycle
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Lewis, Spenser M. "Simplified core physics and fuel cycle cost model for preliminary evaluation of LSCR fueling options." Thesis, Georgia Institute of Technology, 2014. http://hdl.handle.net/1853/51772.
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The Liquid Salt Cooled Reactor (LSCR) provides several potential benefits compared to pressurized water-cooled reactor systems. These include low operating pressure of the liquid salt coolant, the high burnup tolerance of the fuel, and the high operating temperatures which leads to increases in efficiency. However, due to inherently low heavy metal loading, the fuel cycle design presents specific challenges. In order to study options for optimizing the fuel design and fuel cycle, SCALE6.1 was used to create simplified models of the reactor and look at various parameters. The primary parameters of interest included packing factor and fuel enrichment. An economic analysis was performed on these results by developing a simple fuel cycle cost (FCC) model that could be used to compare the different options from an economic standpoint. The lithium enrichment of the FLiBe coolant was also investigated. The main focus was to understand the practical limitations associated with the Li-7 enrichment and whether it could be used for beneficial purposes. The main idea was to determine whether a lower-than-equilibrium enrichment could be used at reactor start up so that the Li-6 isotope acts as a burnable absorber. The results for the lithium enrichment study showed that the enrichment converges over time, but the amount of time required to reach steady state is much too long and the FLiBe coolant could not be utilized for reactivity control as a burnable absorber. The results found through this research provide reasonable guidelines for expected costs and narrow down the types of configurations that should be considered as fuel design options for the LSCR. Additionally, knowledge was gained on methods for modeling the system not only accurately but also efficiently to reduce the required computing power and time.
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Silva, Tereza Cristina da. "Efeitos anti-neoplásicos da raiz de Pfaffia paniculata (Ginseng brasileiro) no modelo de hepatocarcinogênese murina e em cultura de células de hepatocarcinoma humano." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/10/10133/tde-02062008-095506/.
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A raiz pulverizada de Pfaffia paniculata e seu extrato butanólico apresentam propriedades antineoplásicas, quimiopreventivas e antiangiogênicas, onde muitos indícios conferem às saponinas triterpenóides presentes nestas raízes as propriedades antitumorais observadas. Sabe-se que saponinas de diversos tipos de plantas possuem capacidade de interferir diretamente no ciclo celular de células tumorais. Assim, considerando os efeitos inibitórios das raízes e extratos de P. paniculata sobre a proliferação celular, o objetivo deste trabalho foi compreender os mecanismos envolvidos na ação quimiopreventiva desta raiz, tanto na fase de iniciação da carcinogênese hepática, quanto sobre células tumorais já estabelecidas. Inicialmente, foram avaliados os efeitos de diferentes concentrações da raiz em pó de P. paniculata adicionada à ração em camundongos submetidos ao modelo de hepatocarcinogênese, pela análise da proliferação celular, indução da apoptose e a comunicação intercelular hepática. Seqüencialmente, foram avaliados os efeitos das frações purificadas do extrato butanólico destas raízes sobre linhagem de células de carcinoma hepatocelular humano. Neste experimento foram analisadas a influência do tratamento sobre a viabilidade celular, fases e proteínas do ciclo celular, proliferação e presença de morte celular. No modelo de hepatocarcinogênese o tratamento com a raiz reduziu a proliferação celular, aumentou a apoptose e desencadeou processo inflamatório crônico em intensidades dependentes da concentração testada e não afetou a comunicação intercelular. Estes resultados indicam que os efeitos quimiopreventivos da P. Paniculata são decorrentes do controle da proliferação celular e apoptose, e são diretamente influenciados pela concentração da raiz. No experimento in vitro o tratamento reduziu a concentração das células vivas sem aumentar a concentração de células mortas, diminuiu a porcentagem de células na fase G2 do ciclo celular, reduziu a expressão dos genes das proteínas ciclinas D1, E e CDK6 e aumentou a expressão de p27, e não induziu apoptose. Estes resultados mostraram que a redução na concentração de células observadas após o tratamento com a fração do extrato butanólico de P. paniculata não foi decorrente de indução de apoptose. O tratamento parou o ciclo celular das células tumorais HepG2 em G1, inibindo proteínas importantes para a progressão do ciclo e estimulando a expressão de p27 um conhecido gene inibidor de CDKs. Os efeitos antiproliferativos observados no experimento in vivo em camundongos, reproduziram-se in vitro em células tumorais humanas, o que pode indicar que as propriedades antineoplásicas anteriormente observadas não são espécie específica. Em linhas gerais, as raízes e/ou as frações do extrato butanólico obtido a partir das raízes de P. paniculata apresentam propriedades antineoplásicas por inibir a proliferação celular e induzir apoptose in vivo e por parar o ciclo celular in vitro. Estes resultados consagram as propriedades antineoplásicas de Pfaffia paniculata e motivam o desenvolvimento de mais estudos sobre as suas potencialidades.The powdered roots of Pfaffia paniculata and their butanolic extract present antineoplastic, chemopreventive and antiangiogenic properties, and many evidences suggest that the triterpenoid saponins are the responsible for these properties. It is well known that saponins from several types of plants have the capacity to directly interfere on the tumor cell cycle. Therefore, considering the inhibitory effects of the roots and extracts of P. paniculata on cell proliferation, the aim of this study was to search for the mechanisms involved in the chemopreventive effects of this root, both in the initiation phase of the hepatocarcinogenesis and on tumor cell lineage. Initially, the effects of different concentrations of the powdered root of P. paniculata added to the mouse food were evaluated in mice submitted to hepatocarcinogenesis model. Cell proliferation, induction of apoptosis, and hepatic intercellular communication were evaluated. The effects of the purified fractions of the butanolic extract of these roots were then evaluated in human hepatocarcinoma cells. In this experiment, the influence of the treatment on cell viability, phases and proteins of cell cycle, cell proliferation and cell death were evaluated. In the hepatocarcinogenesis model, the treatment with the root decreased cell proliferation, increased apoptosis and induced a chronic inflammatory process dependent on the concentration tested, and did not affect cell communication. These results indicate that the chemopreventive effects of P. Paniculata are apparently dependent on the control of cell proliferation and apoptosis and are directly influenced by the concentration of the root. In the in vitro treatment, it has been observed a reduction in the concentration of live cells without increasing the concentration of dead cells, decreased the level of G2 phase cells, reduced the expression of proteins cyclins D1, E and CDK6, increased the expression of p27, and did not induce apoptosis. These results showed that the reduction in the concentration of cells after the treatment with the butanolic extract of P. paniculata was not due to induction of apoptosis. The treatment inhibited the progression of the cell cycle of HepG2 cells in phase G1, by the inhibition of the expression of proteins that are important to the progression of the cycle, and stimulating the expression of p27, a known inhibitor of CDKs. The antiproliferative effects observed in the in vivo experiments were repeated in the in vitro study with human tumor cells. This may indicate that the antineoplastic properties previously observed are not species-specific. In conclusion, the roots and/or butanolic extract obtained from P. paniculata present antineoplastic properties due to inhibition of cell proliferation and induction of apoptosis in vivo, and due to the cell cycle arrest in vitro. These results reinforce the antineoplastic properties of Pfaffia paniculata and motivate the development of more studies focusing on its antineoplastic potentials.
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Lebogo, Kgomotso Welheminah. "The evaluation of the effects of semi-purified extracts of Commelina benghalensis on the molecular events associated with the growth, apoptosis and cell cycle progression of Jurkat-T cells." Thesis, University of Limpopo (Turfloop Campus), 2007. http://hdl.handle.net/10386/914.
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Mbazima, Vusi G. "The Effects of Crude Methanolic Extract of Commelina benghalensis Linn on the Expression of Apoptotic and Cell Division Cycle Genes in Jurkat T and Wil-2 NSCancer Cell Lines." Thesis, University of Limpopo (Turfloop Campus), 2009. http://hdl.handle.net/10386/937.
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Thesis (Ph.D. (Biochemistry)) --University of Limpopo, 2009Commelina benghalensis Linn is used in traditional medicine in several Asian and African countries for the treatment of various ailments such as stomach irritations, burns, sore throat and feet, diarrhoea and as an anti-inflammatory agent. Recently, our laboratory showed that the crude methanolic extract of Commelina benghalensis L (CMECB) exhibits growth inhibitory and proapoptotic effects in Jurkat T and Wil-2 NS cancer cell lines. In this study, the precise molecular mechanism(s) associated with CMECB-induced growth inhibitory and apoptosis inducing effects in Jurkat T and Wil-2 NS cell lines were investigated. This was achieved by investigating the effects of the extract on the cell division cycle distribution profile as well as its effects on various cell division cycle and apoptosis regulatory genes. Ground stems of C. benghalensis L were extracted with absolute methanol to obtain a crude extract. To assess the effect of CMECB on cancer cell growth, experimental cell cultures were exposed to various concentrations (0 to 600 μg/ml) of CMECB for up to 72 hours. The results demonstrated a significant reduction in cell viability and inhibition of proliferation of experimental cell cultures as determined by the trypan blue dye exclusion assay and the Coulter counter method, respectively. Analysis of nuclear morphological changes in cells stained with Hoechst 33258 confirmed apoptosis as the mode of cell death that is associated with the growth inhibitory effects of CMECB in both the Jurkat T and Wil-2 NS cell lines. This assertion was based on the observed presence of nuclear morphological changes such as chromatin condensation and fragmentation and apoptotic bodies in cells exposed to CMECB. In order to get an insight on the pro-apoptotic mechanisms of CMECB, Western blot xxi and quantitative real-time PCR (qrt-PCR) were used to investigate the expression profiles of various apoptosis and cell division cycle regulatory genes. Qrt-PCR results showed a lack of a clear up- and/or down-regulatory effects of CMECB on the mRNA expression levels of bax and bcl-2 in both Jurkat T and Wil-2 NS cells. Western blot analyses demonstrated that CMECB induced apoptosis by facilitating Bax protein translocation from the cytosol to the mitochondria in both Jurkat T and Wil-2 NS cells. In addition, CMECB down-regulated Bcl-2 protein expression which, as a result, led to the shift in the Bax/Bcl-2 protein ratio at certain time points and concentration in both Jurkat T and Wil-2 NS cells. The modulation of the Bcl-2 family members led to mitochondrial cytochrome c release into the cytosol and activation of caspases-9 and -3; this was also confirmed by caspase activity assays and eventual degradation of PARP. Furthermore, CMECB induced Jurkat T and Wil-2 NS cell division cycle arrest at the G2/M phase as determined by flow cytometric analysis. Western blot analyses of G2/M phase regulatory proteins demonstrated that the CMECB-induced cell division cycle arrest was associated with the downregulation of cyclin B1 and Cdc2 protein expression levels. Western blot analyses results further revealed that the arrest of Wil-2 NS cells at the G2/M phase was independent of p21 protein activity. However, Jurkat T cell division cycle arrest was found to be mediated, in part, by p21. Quantitative real-time PCR results did not show a clear trend in terms of the down- or up-regulatory effects of the extracts on the G2/M phase regulatory genes. The CMECBinduced apoptosis and G2/M arrest was found to occur in a p53-independent xxii manner due to the lack and down-regulation of p53 protein levels in both Jurkat T and Wil-2 NS cells, respectively. In conclusion, CMECB induces its anticancer activity by inducing G2/M phase arrest and mitochondrial-mediated apoptosis independent of p53 protein activity. Although the study did not perform in vivo experiments to ascertain the efficacy of extracts of CMECB against specific tumour types in animal models, the present findings somehow validate the traditional use of C. benghalensis L as an anticancer agent. A more definitive study needs to be done to ascertain this assertion.
National Research Foundation and the University of Limpopo research office
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Nafati, Mehdi. "Caractérisation fonctionnelle des inhibiteurs de Cyclin-Dependent Kinase (CDK) dans le fruit de tomate (Solanum lycopersicum)." Thesis, Bordeaux 2, 2010. http://www.theses.fr/2010BOR21712/document.
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Au sein de l’unité mixte de recherche 619 de l’Institut National de Recherche Agronomique, le groupe « Organogénèse du Fruit et Endoréduplication » étudie les acteurs moléculaires prenant part au contrôle du cycle cellulaire dans le fruit de tomate. L’objet de la présente thèse est l’étude de l’inhibiteur du cycle cellulaire Kip-Related Protein, et son rôle durant le développement du fruit. Identification de motifs protéiques fonctionnels chez l’Inhibiteur de Kinase Cycline-Dependent SlKRP1 chez Solanum lycopersicum : Leur rôle dans les interactions avec des partenaires du cycle cellulaire Les Kip-related proteins (KRPs) jouent un rôle majeur dans la régulation du cycle cellulaire. Il a été montré qu’ils inhibent les complexes CDK/Cyclin et ainsi bloquent la progression du cycle cellulaire. Malgré leur manque d’homologie avec leurs homologues animaux au delà de leur motif de liaison CDK/Cyclin, localisé à l’extrémité C-terminal de la protéine dans les séquences de plante, des études antérieurs ont montré la présence de motifs conservés spécifiques aux plantes chez certaines KRPs. Nous n’avons cependant que peu d’information concernant leur fonction. Nous montrons ici que les KRPs sont distribués en deux sous groupes phylogénétiques, et que chaque sous-groupe dispose de courts motifs spécifiques conservés. Les KRPs du sous-groupe 1 disposent ainsi de six motifs conservés entre eux. Utilisant SlKRP1, qui appartient au sous-groupe 1, nous avons identifié des motifs responsables de la localisation de la protéine et de ses interactions protéine-protéine. Nous montrons que le motif 2 est responsable de l’interaction avec CSN5, une sous-unité du complexe signalosome, et que le motif 5 a un effet redondant avec le motif 3 pour ce qui est de la localisation sub-cellulaire de la protéine. Nous montrons de plus que SlKRP1 est capable de guider SlCDKA1 et SlCycD3;1 vers le noyau, et ce même en l’absence du motif de liaison CDK/Cycline précédemment référencé. Ce nouveau site d’interaction est probablement localisé dans la partie centrale de la séquence de SlKRP1. Ces résultats apportent de nouveaux indices quant au rôle de la partie encore méconnue de cette protéine. La surexpression de SlKRP1 dans le mésocarpe de tomate détruit la proportionnalité entre endoréduplication et taille cellulaire Le fruit est un organe spécialisé résultant du développement de l’ovaire après pollinisation et fertilisation, et qui offre un environnement adéquat pour la maturation des graines et leur dispersion. De part leur importance en nutrition humaine et leur importance économique, les espèces à fruit charnu ont été le sujet d’étude développementales principalement orientée vers la formation de l’ovaire, la mise à fruit et la maturation du fruit. La phase de croissance du fruit a été beaucoup moins étudiée, bien que la division cellulaire et la croissance cellulaire prenant place durant cette période soient cruciales à la détermination de la taille finale du fruit, ainsi que de sa masse et sa forme. Le développement du mésocarpe du fruit de tomate se déroule par la succession d’une phase de division cellulaire suivie d’une phase d’expansion cellulaire associée à l’endoréduplication, menant à la formation de cellules géantes (jusqu’à 0,5mm) avec des niveaux de ploïdie pouvant atteindre 256C. Bien qu’une relation évidente entre endoréduplication et croissance cellulaire ait été montrée par de nombreux exemples chez les plantes, le rôle exact de l’endoréduplication n’a toujours pas été élucidé, étant donné que la plupart des expériences induisant une modification du niveau d’endoréduplication dans la plante affectaient aussi la division cellulaire. Nous avons étudié la cinétique du dévelopement du mésocarpe de tomate au niveau morphologique et cytologique et avons étudié l’effet de la diminution du niveau d’endoréduplication sur le dévelopement du fruit en sur-exprimant l’inhibiteur du cycle cellulaire Kip-Related Protein 1 (SlKRP1) spécifiquement dans les cellules en croissance du mésocarpe de tomate. Nous montrons une proportionnalité directe entre endoréduplication et taille cellulaire durant le développement normal du fruit, ce qui nous a permis de construire un modèle de développement du mésocarpe définissant l’épaisseur du péricarpe en ne prenant en compte que le nombre de divisions cellulaires et le nombre de tours d’endoréduplication. De façon surprenante, les mésocarpes de tomate affectés dans leur niveau d’endoréduplication par la sur-expression de SlKRP1 ne sont pas affectés au niveau de la taille des cellules ou du fruit, ni dans leur contenu métabolique. Nos résultats démontrent pour la première fois qu’alors que le niveau de ploïdie est étroitement lié avec la taille des cellules et du fruit, l’endoréduplication n’est pas responsable de la croissance cellulaire du mésocarpe de tomateWithin the Joint Research Unit 619 of the National Institute of Agronomic Research (INRA), the group "Organogenesis of the Fruit and endoreduplication" examines the molecular players involved in cell cycle control in tomato fruit. The purpose of this thesis is the study of the cell cycle inhibitor Kip-Related Protein and its role during fruit development. Identification of protein motifs in the functional inhibitor of Cyclin-Dependent Kinase in Solanum lycopersicum SlKRP1: Their role in interactions with partners in the cell cycle The Kip-related proteins (KRPs) play a major role in the regulation of cell cycle. It has been shown to inhibit the CDK / Cyclin and thus block cell cycle progression. Despite their lack of homology with their counterparts in animals beyond their binding motif CDK / Cyclin, located at the C-terminal protein sequences in the plant, previous studies have shown the presence of conserved motifs plant specific in some KRPs, but there is little information about their function. We show here that the KRPs are distributed into two phylogenetic groups, and that each subgroup has specific short conserved motifs. The KRPs from subgroup 1 have six conserved motifs. Using SlKRP1, which belongs to subgroup 1, we have identified the motifs responsible for the localization of the protein and protein-protein interactions. We demonstrate that the pattern 2 is responsible for the interaction with CSN5, a subunit of the signalosome complex, and that the motif 5 is redundant with motif 3 with respect to the sub-cellular localization of the protein. We also show that SlKRP1 is capable of guiding SlCDKA1 and SlCycD3; 1 to the nucleus, even in the absence of CDK / cyclin binding motif previously referenced. This new site of interaction is probably located in the central part of the sequence of SlKRP1. These results provide new clues about the role of the little-known part of this protein. Overexpression of SlKRP1 in tomato mesocarp disrupts the proportionality between endoreduplication and cell size The fruit is a specialized organ which results from the ovary after pollination and fertilization, and provides a suitable environment for seed maturation and dispersal. Because of their importance in human nutrition and economic importance, fleshy fruit species have been the subject of study mainly focused on the developmental formation of the ovary, fruit set and fruit ripening. The stage of fruit growth has been much less studied, although cell division and cell growth taking place during this period are crucial to determining the final size of the fruit, as well as its mass and shape. The development of tomato fruit mesocarp occurs by the estate of a phase of cell division followed by a phase of cell expansion associated with endoreduplication, leading to the formation of giant cells (up to 0.5 mm) with ploidy levels of up to 256C. Although a clear relationship between endoreduplication and cell growth has been shown by many examples in plants, the exact role of endoreduplication has still not been elucidated, since most of the experiments leading to a change in the level of endoreduplication in plants also affected cell division. We studied the kinetics of the development of tomato mesocarp morphologically and cytologically and studied the effect of the reduced level of endoreduplication in the development of the fruit over-expressing the cell cycle inhibitor Kip-Related Protein 1 (SlKRP1) specifically in the growing cells of the tomato mesocarp. We show a direct proportionality between endoreduplication and cell size during normal development of the fruit, which allowed us to build a model for development of mesocarp defining the thickness of the pericarp by taking into account the number of cell divisions and the number of rounds of endoreduplication. Surprisingly, the tomato mesocarps affected in their level of endoreduplication by over-expression of SlKRP1 are not affected in terms of cell size and fruit, or on their metabolic content. Our results demonstrate for the first time that while the level of ploidy is closely linked with cell size and fruit, endoreduplication is not responsible for the cell growth of tomato mesocarp
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Lubini, Greice. "Caracterização do Gene NtCDKG;2 Expresso no Pistilo de Nicotiana tabacum L." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/17/17135/tde-22042013-141248/.
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A biologia da reprodução sexual de plantas é um campo de pesquisa de grande importância, já que a maioria dos alimentos consumidos pelo homem é composta de partes reprodutivas das plantas (frutos e sementes), oriundas do desenvolvimento de partes do pistilo fertilizado. Em Nicotiana tabacum, identificou-se um gene específico de estigma/estilete, SCI1 (Stigma/style Cell-cycle Inhibitor 1), que atua na inibição da proliferação celular (DePaoli et al., 2011). Através de ensaios de pull-down, verificou-se a interação da proteína SCI1 com uma proteína quinase dependente de ciclina (CDK) (Strini, dados não publicados). Este trabalho visou à caracterização dessa nova CDK, ortóloga da CDKG;2 de Arabidopsis. A sequência correspondente de N. tabacum (NtCDKG;2) foi amplificada por PCR, a partir de cDNAs de estigmas/estiletes, clonada e sequenciada, o que permitiu a confirmação de sua identidade. A expressão de NtCDKG;2 foi analisada nos diferentes órgãos vegetativos e reprodutivos, por qRT-PCR, o que evidenciou um perfil de expressão ubíqua. Ao estudar o perfil de expressão desse gene nos estigmas/estiletes dos doze estádios de desenvolvimento floral de N. tabacum, observa-se que NtCDKG;2 é mais expresso nos estádios tardios do desenvolvimento em direção à antese, indicando uma função importante de sua proteína ao final do desenvolvimento do pistilo. Análises de expressão de NtCDKG;2 em estigmas/estiletes, de plantas de N. tabacum com produção aumentada do hormônio auxina no pistilo, sugerem que NtCDKG;2 é regulado transcricionalmente por esse hormônio. A expressão transiente da proteína de fusão NtCDKG;2-GFP, em folhas de N. tabacum, evidenciou a localização nuclear da proteína em estudo. Também foram geradas plantas transgênicas estáveis com superexpressão e com silenciamento por RNAi de NtCDKG;2. Apesar dos altos níveis de transcritos de NtCDKG;2 nas plantas de superexpressão e dos baixos níveis nas plantas silenciadas, não foram observadas alterações fenotípicas macroscópicas nessas plantas. Adicionalmente, obteve-se a expressão da proteína NtCDKG;2, fusionada a uma tag de histidina em sua porção N-terminal, em células de Escherichia coliBL21(DE3)CodonPlusRP. Através dos estudos realizados neste trabalho e análises conjuntas da literatura, é possível propor que NtCDKG;2 codifique uma proteína que está envolvida no controle do ciclo celular nos estigmas/estiletes de N. tabacum.The biology of plant sexual reproduction is a research field of great importance, since most of the food consumed by humans is composed of plant reproductive parts (fruits and seeds), originated by the development of fertilized pistil parts. In Nicotiana tabacum, it was identified a stigma/style-specific gene, SCI1 (Stigma/style Cell-cycle Inhibitor 1), which acts in the inhibition of cell proliferation (DePaoli et al., 2011). Through pull down assays, the interaction of the SCI1 protein with a cyclin-dependent protein kinase (CDK) was verified (Strini, unpublished). This work aimed the characterization of this new CDK, orthologous to the Arabidopsis CDKG;2. The N. tabacum corresponding sequence (NtCDKG;2) was PCR amplified, from stigmas/styles cDNAs, cloned and sequenced, which allowed the confirmation of its identity. The NtCDKG;2 expression was analyzed in the different vegetative and reproductive organs, by qRT-PCR, evidentiating an ubiquitous expression pattern. Studying the expression pattern of this gene in stigmas/styles of the twelve stages of N. tabacum flower development, it was observed that NtCDKG;2 is more expressed at the later developmental stages towards anthesis, indicating an important function of its protein in the end of pistil development. NtCDKG;2 expression analyses in stigmas/styles of N. tabacum plants with an enhanced auxin production in the pistil suggest that NtCDKG;2 is transcriptionally regulated by this hormone. The transient expression of the fusion protein NtCDKG;2-GFP, in N. tabacum leaves, evidentiated the nuclear localization of the studied protein. Stable transgenic plants overexpressing and silencing NtCDKG;2 by RNAi were also generated. Despite the high transcript levels in the plants overexpressing NtCDKG;2 and the low transcript levels in the silencing plants, macroscopic phenotypic alterations were not observed on these plants. Additionally, the expression of the NtCDKG;2 protein, with a histidine tag fused in its N-terminal, was obtained in Escherichia coli BL21(DE3)CodonPlusRP cells. Through studies performed on this work and literature analyses, it is possible to propose that NtCDKG;2 encodes a protein that is involved in the control of cell cycle at the N. tabacum stigmas/styles.
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Brundin, Carl. "Alternative energy concepts for Swedish wastewater treatment plants to meet demands of a sustainable society." Thesis, Umeå universitet, Institutionen för tillämpad fysik och elektronik, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-146831.
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This report travels through multiple disciplines to seek innovative and sustainable energy solutions for wastewater treatment plants. The first subject is a report about increased global temperatures and an over-exploitation of natural resources that threatens ecosystems worldwide. The situation is urgent where the current trend is a 2°C increase of global temperatures already in 2040. Furthermore, the energy-land nexus becomes increasingly apparent where the world is going from a dependence on easily accessible fossil resources to renewables limited by land allocation. A direction of the required transition is suggested where all actors of the society must contribute to quickly construct a new carbon-neutral resource and energy system. Wastewater treatment is as required today as it is in the future, but it may move towards a more emphasized role where resource management and energy recovery will be increasingly important. This report is a master’s thesis in energy engineering with an ambition to provide some clues, with a focus on energy, to how wastewater treatment plants can be successfully integrated within the future society. A background check is conducted in the cross section between science, society, politics and wastewater treatment. Above this, a layer of technological insights is applied, from where accessible energy pathways can be identified and evaluated. A not so distant step for wastewater treatment plants would be to absorb surplus renewable electricity and store it in chemical storage mediums, since biogas is already commonly produced and many times also refined to vehicle fuel. Such extra steps could be excellent ways of improving the integration of wastewater treatment plants into the society. New and innovative electric grid-connected energy storage technologies are required when large synchronous electric generators are being replaced by ‘smaller’ wind turbines and solar cells which are intermittent (variable) by nature. A transition of the society requires energy storages, balancing of electric grids, waste-resource utilization, energy efficiency measures etcetera… This interdisciplinary approach aims to identify relevant energy technologies for wastewater treatment plants that could represent decisive steps towards sustainability.
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Koch, Garance. "Effet du stress hydrique sur la croissance de la tomate : une étude multi-échelle : de la cellule à la plante entière pour une meilleure compréhension des interactions entre les différentes échelles." Thesis, Avignon, 2018. http://www.theses.fr/2018AVIG0347.
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Comme pour les autres organes multicellulaires, la croissance et le développement de la feuille et du fruit sont caractérisés par la coordination de la division et de l'expansion des cellules qui sont des processus majeurs de la croissance. Les cellules du péricarpe du fruitcharnu connaissent également des endocycles successifs entrainant ainsi une augmentationimportante de la ploïdie des cellules. Il existe un lien évident entre la croissance cellulaire etl'endoréduplication, cependant, celui-ci est encore mal connu du point de vue fonctionnel. Lesprocessus cellulaires interagissent fortement durant le développement de l’organe et sont liésaux flux de carbone et d'eau dans la plante. L’objectif de ce travail de thèse est de mieuxcomprendre le contrôle multi-échelles de la croissance des feuilles et des fruits chez la tomate(Solanum lycopersicum Mill.) et de la plasticité de leur croissance en réponse à des stresshydriques du sol.L’étude a essentiellement porté sur le génotype de tomate cerise Solanum lycopersicum, cv.West Virginia 106 (WVa 106) qui a été cultivé dans différentes conditions d’irrigation grâce àdes systèmes automatisés développés pour cette étude. La réponse au déficit hydrique du sol aété étudiée à différentes échelles d’observation, (tissu, organe, plante entière) et à différentsstades de croissance de la plante en adaptant des protocoles utilisés jusque-là pour des plantesà croissance déterminée et des feuilles simples. Deux génotypes transgéniques modifiés sur ungène de régulation du cycle cellulaire ont aussi été cultivés afin de faire varier les traits liés àla croissance cellulaire et mieux comprendre leurs liens. Les cinétiques de croissance desorganes source et puits que sont la feuille et le fruit aux échelles cellulaire et tissulaire ontaussi été décrites. Les résultats ont apporté des éléments nouveaux sur les coordinations entreles différents processus étudiés et conforté des hypothèses déjà présentes dans la littérature.Ces travaux ont permis de fournir un jeu de données original sur les effets du stress hydriquesur les processus cellulaires (division, expansion, endoréduplication) impliqués dans lacroissance de la feuille et du fruit chez la tomate et, de mieux comprendre leur interactions àplus large échelle, la plante dans sa globalité. En perspectives, ce jeu de données pourrapermettre de faire évoluer un modèle de développement du fruit charnu en condition optimaleet tester sa généricité sur un autre organe, la feuille. Il ouvre des pistes sur la réflexion autourde la modélisation de la plasticité de la plante en réponse au stress hydriqueAs for other multicellular organs, growth and development of leaves and fruits arecharacterized by cell division and expansion. Cell division and expansion are two maingrowth processes. Fleshy fruit pericarp cells also include successive endocycles that providean important increase in cell ploidy. There is a clear link between cell growth andendoreduplication. However, this link is still unclear from a functional point of view. Cellularprocesses interact during organ development and are related to plant water and carbon flows.The objective of this thesis is to give insights into the multi-scale control of leaves and fruitsgrowth in tomato (Solanum lycopersicum Mill.) and the plasticity of growth-related traits inresponse to soil water stresses.This study mainly focused on cherry tomato Solanum lycopersicum, cv. West Virginia 106(WVa 106). This genotype was cultivated in different conditions of watering regimes withautomated systems developed for this study. Soil water deficit response was studied atdifferent observation scales (tissue, organ, whole plant) and at different plant growth stagesthanks to protocols that were used until now on plants with determinate growth and simpleleaves that were modified for this study. Two transgenic genotypes modified on a cell cycleregulation gene were also cultivated to create variations on growth related traits for a betterunderstanding of their relationships. Multi-scale growth kinetics of source and sink organs(leaf and fruit) were also analyzed. Results have brought new elements about growth-relatedtraits coordination and have reinforced a few hypotheses already presented in scientificpapers. This work has supplied an original dataset on water stress effects on cellular processes(division, expansion, endoreduplication) related to leaf and fruit growth in tomato in thecontext of the plant as a whole. In perspectives, this dataset may allow to further develop anexisting model of fleshy fruit development which was first developed for fruits of plantsgrowing optimal condition. Genericity of this model will be tested on another organ, the leaf.This work also opens some tracks about how the model could be modified when growth islimited by water stress
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Weinl, Christina [Verfasser]. "Challenging the plant cell cycle : analysis of key cell cycle regulators in Arabidopsis thaliana / vorgelegt von Christina Weinl." 2005. http://d-nb.info/97680610X/34.
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Chang, Kerong. "Regulatory proteins and genes in plant cell division and differentiation." Phd thesis, 1993. http://hdl.handle.net/1885/142233.
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Chen, Yi-Lun, and 陳宜倫. "Study on a Plant Product, THKM76: Cytotoxicity and Cell Cycle Effect." Thesis, 2000. http://ndltd.ncl.edu.tw/handle/04684269170185130156.
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碩士國立清華大學
生命科學系
88
Many anticancer agents are currently available for clinical therapy, but very few agents are effective against some types of cancer. Fortunately, we have obtained a potential anticancer agent THKM76 from a plant product. In this purpose, we studied the effects of THKM76 on the morphology, cytotoxicity, and cell cycle progression in a human colon adenocarcinoma cell line (RKO) and a human fibroblast cell line (HF). For RKO cells, characteristic morphological changes and drug-induced apoptosis, related to the administration of the cytotoxic agent, were interpreted as degenerative in nature, but drug-treated HF cells revealed less characteristic features of damage. Cytotoxicity was evaluated by clonogenic survival assay showed that RKO was more sensitive to THKM76 than HF. In addition, by MTT assay, we found that treating the cells with THKM76 resulted in a decrease of cell viability in a dose- and a time-dependent manner. However, there were differences in survival rate between using clonogenic survival assay (assayed after release 7 days) and MTT assay (assayed immediately after exposure). According to the results and some evidences indicated that the cell growth delayed after release from treatment, we demonstrated that there were two major cytotoxic effects for inducing cell killing. (i) Direct effect: Cells were killed first during THKM76 treatment. (ii) Persisted effect: After removal of THKM76, drug-treated viable cells lost reproductive ability further and even died. Finally, flow cytometry analysis of the DNA content revealed G1 block, slight G2/M arrest, and the presence of a ‘sub-G2’ region in RKO cells after THKM76 treatment with indicated concentrations. Besides, after release from treatment, there was a significant increase of RKO cells arrested in G2/M phase. The results of combining the arrest status with irradiation supported a role for THKM76 as a radiosensitizer.
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Badelt, Kim. "Location and enzyme activity of the key cell cycle protein p34 ͨ ᵈ ͨ ² in maize." Phd thesis, 1996. http://hdl.handle.net/1885/145964.
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Sakuanrungsirikul, Suchirat. "Cyclic AMP and the Chlamydomonas reinhardtii cell division cycle." Phd thesis, 1991. http://hdl.handle.net/1885/142214.
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Wu, Li-Ping. "Mutational and biochemical analysis of the cell cycle in Chlamydomonas reinhardtii." Phd thesis, 1993. http://hdl.handle.net/1885/142311.
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"Active fraction of licorice inhibits proliferation of lung cancer cells A549 via inducing cell cycle arrest and apoptosis." 2012. http://library.cuhk.edu.hk/record=b5549045.
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肺癌是導致男性死亡的最常見原因以及是排在乳腺癌和結腸癌之後的導致女性死亡的第三大原因。雖然肺癌如此嚴重，但是如今治疗肺癌仍然是一个挑战。現今對肺癌的治療主要集中在化學治療和靶點藥物治療，但是由於這些治療有著很大的副作用和低治愈率，尋找其他的醫學替代方法十分迫切。甘草是其中最常用的中藥，它常常用作食品工業中的甜味劑。以往的研究表明，甘草具有多種的生物活性。但是甘草提取物對於肺癌的治療卻是十分匱乏的。本論文主要目的是評價甘草提取物以及其中的有效成份對非小型肺癌細胞株A549 的影響，以及其作用的機理。我們的數據表明，甘草的乙酸乙酯（EAL）成份比甘草的乙醇提取物有著比較強的抑制癌細胞的作用。另外，對甘草的五個單體進行的測試中發現lico-3 是最具有抑制肺癌作用的。利用高效液相色譜法對甘草活性成份分析表明，lico-3 是EAL中的其中一個單體。
乳酸脫氫酶滲漏（LDH）的檢測結果以及异硫氰酸荧光素-碘化丙啶（FITC-PI）雙染的結果表明，EAL 能夠引起肺癌細胞的凋亡現象而非壞死現象。實驗結果表明由EAL引起的A549細胞凋亡是跟Bcl-2家族及Caspase家族有關係，同時EAL還能夠抑制Akt途徑從而導致細胞的死亡。
致肺癌細胞死亡的原因進行進一步研究表明，EAL還能夠引起抑制細胞週期的運作，停留在G2/M 時期。這可能是由於EAL引發了p53與p21的上調作用從而抑制了細胞的生長與增殖。
實驗結果說明了EAL引起的肺癌細胞株A549的凋亡作用是跟多重細胞通路有關， 同時表明了EAL是具有抗擊肺癌作用的潛能，能夠作為治療肺癌的藥物。
Lung cancer is the most common cause of cancer death in men and third in women followed by breast cancer and colon cancer, yet treatment of lung cancer remains a challenge. Current treatments including chemotherapy and targeted drug treatment come with side-effects and low successful rate. Alternative medicine for treatment of lung cancer is warranted. Glycyrrhiza uralensis (Gan-Cao), commonly called “licorice, is one of the most commonly used herbs in traditional Chinese medicine (TCM). It is also used as flavoring and sweetening agents in many of food products. Previous studies have indicated that licorice exhibits a variety of biological activities. However, anticancer effects of licorice extract on lung cancer remain unclear.
In this study, we evaluated effects of licorice extract and its chemical components on human lung cancer cell line A549, and studied its mode of action. Our results showed the ethyl acetate fraction of licorice (EAL) was more effective in inhibition of A549 cell growth followed by ETL (IC₅₀: 50μg/mL). Moreover, among the five compounds tested, lico-3 was more potent compound. The HPLC analysis of the active fraction indicated that lico-3 was one of the compounds distributed in the EA fraction.
The results of LDH assay and FITC-PI co-staining method suggested low concentration of EAL can trigger apoptosis but not necrosis. The experimental findings show that EAL induce apoptosis in A549 cell lines involved in Bcl-2 family and caspase cascade. Also, EAL can arrest the Akt survival pathway in A549. Furthermore, the results indicate that EAL triggered G2/M phase arrest. The studies suggest EAL can up-regulate p53 and p21 to promote cell cycle arrest resulting in inhibition of proliferation.
Experimental results indicate that EAL is involved in multiple signal pathways to induce lung cancer cell death. The result suggests EAL is a potential candidate for lung cancer therapy.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Zhou, Yanling.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves 99-110).
Abstracts in Chinese.
Abstract --- p.III
論文摘要 --- p.V
Acknowledgement --- p.VII
List of Contents --- p.VIII
List of Figures --- p.X
List of Tables --- p.XI
List of Abbreviations --- p.XII
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- Lung cancer --- p.1
Chapter 1.1.1 --- Overview --- p.1
Chapter 1.1.2 --- Risk factors --- p.2
Chapter 1.1.3 --- Types of lung cancer --- p.4
Chapter 1.1.4 --- Stages and treatment of lung cancer --- p.5
Chapter 1.1.5 --- Chemotherapy for lung cancer treatment --- p.8
Chapter 1.2 --- Traditional Chinese Medicines --- p.11
Chapter 1.2.1 --- Overview --- p.11
Chapter 1.2.2 --- Licorice --- p.14
Chapter 1.2.3 --- Chemical study of licorice --- p.16
Chapter 1.2.4 --- Pharmacological activities of licorice --- p.16
Chapter 1.3 --- Molecular mechanism of apoptosis --- p.21
Chapter 1.3.1 --- Overview --- p.21
Chapter 1.3.2 --- Bcl2 family --- p.21
Chapter 1.3.3 --- Caspase pathway --- p.23
Chapter 1.3.4 --- Akt pathway --- p.24
Chapter 1.3.5 --- p53 protein --- p.26
Chapter 1.3.6 --- Apoptosis and cancer --- p.27
Chapter 1.4 --- Cell cycle --- p.29
Chapter 1.4.1 --- Overview --- p.29
Chapter 1.4.2 --- Cell cycle and p53 --- p.29
Chapter 1.4.3 --- Cell cycle and cancer --- p.30
Chapter 1.5 --- Aims of study --- p.32
Chapter Chapter 2 --- Materials and Methods --- p.33
Chapter 2.1 --- Cell culture and treatment --- p.33
Chapter 2.1.1 --- Cell line --- p.33
Chapter 2.1.2 --- Chemicals and reagents --- p.34
Chapter 2.1.3 --- Preparation of solutions --- p.34
Chapter 2.2 --- Preparation of Licorice sample --- p.35
Chapter 2.3 --- HPLC analysis --- p.35
Chapter 2.3.1 --- Chemical and materials --- p.35
Chapter 2.3.2 --- Instrumentation --- p.36
Chapter 2.3.3 --- Preparation of Standard solutions --- p.36
Chapter 2.3.4 --- Preparation of samples --- p.37
Chapter 2.3.5 --- HPLC conditions --- p.37
Chapter 2.3.6 --- Method validation --- p.37
Chapter 2.4 --- Cell viable assay --- p.38
Chapter 2.4.1 --- Samples preparation --- p.39
Chapter 2.4.2 --- Procedure --- p.39
Chapter 2.5 --- LDH assay --- p.40
Chapter 2.5.1 --- Reagent preparation --- p.40
Chapter 2.5.2 --- Procedure --- p.41
Chapter 2.6 --- Annexin V assay --- p.41
Chapter 2.6.1 --- Reagent --- p.42
Chapter 2.6.2 --- Procedure --- p.42
Chapter 2.7 --- Cell cycle study --- p.43
Chapter 2.7.1 --- Chemicals and reagent --- p.43
Chapter 2.7.2 --- Procedure --- p.44
Chapter 2.8 --- Caspase3/7 Assay --- p.44
Chapter 2.8.1 --- Reagent preparation --- p.45
Chapter 2.8.2 --- Procedure --- p.46
Chapter 2.9 --- Western blotting --- p.46
Chapter 2.9.1 --- Reagent and antibodies --- p.46
Chapter 2.9.2 --- Procedure --- p.50
Chapter 2.9.3 --- Determination of protein concentration --- p.51
Chapter 2.10 --- Data analysis --- p.51
Chapter Chapter 3 --- Results --- p.52
Chapter 3.1 --- Chromatographic conditions and HPLC identity conformation --- p.52
Chapter 3.1.1 --- Linearity, limits of detection and quantification --- p.56
Chapter 3.1.2 --- Reproducibility --- p.56
Chapter 3.1.3 --- Analysis of ethyl acetate of licorice (EAL) using the validated method --- p.56
Chapter 3.2 --- Licorice induces apoptosis in nonsmall cell lung carcinoma --- p.61
Chapter 3.2.1 --- Cell viability assay --- p.61
Chapter 3.2.2 --- LDH leakage assay --- p.71
Chapter 3.2.3 --- Annexin V and PI staining --- p.73
Chapter 3.3 --- Protein expression in EALinduced apoptotic cells --- p.75
Chapter 3.3.1 --- Bcl2 family --- p.75
Chapter 3.3.2 --- Activation of caspases by EAL treatment --- p.77
Chapter 3.4 --- EAL could block Akt survival pathway --- p.79
Chapter 3.5 --- EAL induces cell cycle arrest in nonsmall cell lung carcinoma --- p.83
Chapter Chapter 4 --- Discussion --- p.85
Chapter 4.1 --- Chemical analysis of licorice --- p.85
Chapter 4.2 --- Licorice induced apoptosis but not necrosis on lung cancer cell A549 --- p.86
Chapter 4.2.1 --- Licorice exhibits specific cytotoxicity to different cancer cells in vitro --- p.86
Chapter 4.2.2 --- EAL induces cell death via apoptosis but not necrosis --- p.87
Chapter 4.3 --- Growth inhibition by EAL inducing apoptosis --- p.89
Chapter 4.3.1 --- EAL induces apoptotic cell death through modification of Bcl2 family --- p.89
Chapter 4.3.2 --- EAL activate the caspase proteins --- p.90
Chapter 4.4 --- Growth inhibition by EAL inducing survival pathway arrest --- p.92
Chapter 4.5 --- Growth inhibition by EAL inducing cellcycle arrest --- p.94
Chapter 4.6 --- General discussion --- p.96
Reference --- p.99
40
Liu, Bo. "Cell-Cycle Engineering And Gfp-Online Monitoring Of Heterologous Protein Production In High Density Plant Suspension Cultures." Thesis, 2005. http://hdl.handle.net/10125/10513.
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Jordan, Chad Victor. "Geminivirus-induced gene silencing as a method to determine the role of essential cell cycle genes in plant development." 2005. http://www.lib.ncsu.edu/theses/available/etd-05182005-011034/unrestricted/etd.pdf.
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Nadal, Marina. "Exploring the role of autophagy and cell wall degrading enzymes in the life cycle and pathogenic development of the basidiomycete fungal plant pathogen Ustilago maydis." 2009. http://purl.galileo.usg.edu/uga%5Fetd/nadal%5Fmarina%5F200912%5Fphd.
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Carmichael, Jeremy Paddon. "Strategies for cloning cell division cycle genes in plants." Phd thesis, 1990. http://hdl.handle.net/1885/141097.
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DiasBaltazar, Miguel Ângelo. "Cytogenetic and biochemical characterization of the progeny of plants of bread wheat ‘Jordão’ biofortified with Iron and/or Zinc." Master's thesis, 2020. http://hdl.handle.net/10348/9980.
Full textAbstract:
Dissertação de Mestrado em Genética Molecular Comparativa e TecnológicaSeed priming with micronutrients (nutripriming) has several agronomic advantages if performed with suitable dosages. Nutripriming has been commonly used in the biofortification of crops such as bread wheat (Triticum aestivum L. em Thell.). However, the usage of excessive amounts of micronutrients like Iron (Fe) and Zinc (Zn) can induce cytotoxicity (traceable by both cytogenetic and biochemical analyses) and phytotoxicity (detectable upon the characterization of yield-related components). Nutripriming is performed in each generation, but the benefits and/or the cytotoxicity-related stress memory of nutripriming in the next unprimed generation/offspring is unknown. Therefore, this work evaluated how hydropriming and nutripriming performed in the parental S0 seeds influenced the germination, mitosis, biochemical profile and yield-related components of the first generation of unprimed bread wheat cv. ‘Jordão’ seeds (S1 seeds). The S0 seeds were previously primed with distilled water (hydropriming) and nutriprimed with 4 mg.L-1 and/or 8 mg.L-1 of Fe and/or Zn. These concentrations induced cytotoxicity, nucleolar stress and increased the total soluble protein content. Unprimed S1 seeds were used as control (control S1). In the whole wheat flour samples of each S1 offspring, sixteen free amino acids (a.a.) and five soluble sugars were identified and quantified using HPLC-FLD and HPLC-PAD, respectively. In most of the S1, the content of each a.a. increased relative to the control being glutamic acid and glutamine, proline and glycine the most abundant. Glucose, ash content and crude protein (CP) of the S1 offspring increased relative to the control. However, only the ash content was significantly different (p ˂ 0.05) among the control and remaining offspring. Sucrose, fructose, raffinose, maltose and total starch showed a significant decrease (p ˂ 0.05) in few S1 offspring relative to the control. The protein amount previously determined in the whole wheat flour of S0 seeds was significantly higher (p ˂ 0.05) in those primed with 4 mg.L-1 or 8 mg.L-1 of Fe + Zn. Similar results were observed in their S1. The overall biochemical data revealed that the Fe and/or Zn nutripriming improved the grain nutritional status and the stress tolerance in the S0 seeds and plants, and these advantages were transmitted to the unprimed S1 offspring. The cytotoxicity generated by nutripriming with 4 mg.L-1 and/or 8 mg.L-1 of Fe and/or Zn in the S0 seeds resulted in longer mean germination time (MT) values and high frequencies of cell cycle and chromosomal anomalies. However, their respective S1 offspring showed a higher germination rate, shorter MT values and higher mitotic index (MI). Nonetheless, despite the higher percentages of dividing cells with anomalies (%DCA) observed in the S1, revealing the inheritance of a stress memory, a lower number of anomalies was detected in this offspring. In sum, the germination and cytogenetic data of the S1 suggested an attenuation of the cytotoxicity detected in the S0. Furthermore, the S1 plants surpassed the average values of seven yield-related components characterized previously in the S0 plants. The biochemical, cytogenetic and the yield-related components characterizations performed in this work evidenced the transmission of an attenuated cytotoxicity-related stress memory from the S0 to the S1. A higher intergenerational inheritance of benefits at the biochemical, germination, mitotic and yield level was also detected. Our results demonstrated that the repetition of nutripriming in each generation is unnecessary converting this method on an even more affordable biofortification approach.
O priming de sementes com micronutrientes (nutripriming) tem vantagens agronómicas quando realizado com dosagens adequadas e é frequentemente usado na biofortificação de trigo mole (Triticum aestivum L. em Thell.). Porém, dosagens excessivas de micronutrientes como o Ferro (Fe) e Zinco (Zn) induzem citotoxidade (detetável através de análises citogenéticas e bioquímicas) e fitotoxicidade (observável pela caracterização de componentes relacionados com o rendimento). Como o priming de sementes é realizado em cada geração, desconhece-se se ocorre transmissão de benefícios e/ou memória de stress relacionada com a citotoxicidade para a descendência não-tratada. Assim, este trabalho avaliou a influência do priming de sementes na germinação, mitose, perfil bioquímico e componentes relacionados com o rendimento da primeira geração de sementes não-tratadas da cultivar ‘Jordão’ de trigo mole (sementes S1). Esta descendência teve origem em sementes S0 tratadas com água destilada (hydropriming) e com 4 mg.L-1 e/ou 8 mg.L-1 de Fe e/ou Zn. Estas concentrações induziram citotoxicidade, stress nucleolar e aumento do conteúdo proteico. Uma descendência S1 de sementes não-tratadas foi usada como controlo (controlo S1). Nas amostras de farinha de trigo integral de cada descendência S1, identificaram-se e quantificaram-se dezasseis aminoácidos (a.a.) e cinco açúcares solúveis usando HPLC-FLD e HPLC-PAD, respetivamente. Na maioria das descendências S1, o conteúdo de cada a.a. aumentou relativamente ao controlo. Os mais abundantes foram o ácido glutâmico + glutamina, prolina e glicina. A glucose, o conteúdo de cinza e a proteína bruta (PB) das descendências S1 aumentaram relativamente ao controlo. Contudo, apenas o conteúdo de cinza foi significativamente diferente (p ˂ 0.05) entre o controlo e as restantes descendências. A concentração de sacarose, frutose, rafinose, maltose e amido apresentaram uma diminuição significativa (p ˂ 0.05) num reduzido número de descendências relativamente ao controlo. O conteúdo proteico previamente determinado em farinha integral das sementes S0 foi significativamente (p ˂ 0.05) mais elevado nos tratamentos com 4 mg.L-1 ou 8 mg.L-1 de Fe + Zn. Resultados semelhantes foram encontrados nas suas descendências S1. Os dados bioquímicos revelaram que o nutripriming com Fe e/ou Zn terá melhorado o valor nutricional e a tolerância ao stress nas sementes e plantas S0, e que estas vantagens foram transmitidas à descendência S1 não-tratada. Sementes S0 tratadas com 4 mg.L-1 e/ou 8 mg.L-1 de Fe e/ou Zn apresentaram tempos médios de germinação (TM) longos e elevadas frequências de anomalias no ciclo celular. Contudo, as respetivas descendências S1 apresentaram uma taxa de germinação superior, menores valores de TM, e índice mitótico (IM) superior à S0. Embora na S1 se tenham observado percentagens de células em divisão com anomalias (%CDA) superiores às da S0, revelando a transmissão de uma memória de stress, o número de anomalias foi mais reduzido. Globalmente, os dados de germinação e citogenéticos da S1 sugeriram uma atenuação da citotoxicidade detetada na S0. Adicionalmente, as plantas S1 ultrapassaram as médias relativas a sete componentes relacionados com o rendimento previamente caracterizados nas plantas S0. As caracterizações realizadas neste trabalho evidenciaram a transmissão da S0 para a S1 de uma memória de stress relacionada com a citotoxicidade, embora atenuada, e de benefícios ao nível bioquímico, de germinação, mitótico e de rendimento. Este trabalho demonstrou que não é necessário repetir o priming de sementes em cada geração, tornando este método de biofortificação ainda mais económico.
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Taniwiryono, Darmono. "Analyses of haploid cells in the life cycle of the plant pathogenic fungus Armillaria mellea." 1993. http://catalog.hathitrust.org/api/volumes/oclc/29968851.html.
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Abraham, Ambily. "Chimeric Virus Like Particles as Nanocarriers for Antibody Delivery in Mammalian Cells & Role of Groundnut Bud Necrosis Virus NSs in Viral Life Cycle." Thesis, 2015. http://etd.iisc.ernet.in/2005/3920.
Full textAbstract:
Knowledge of the dissociation constants of the ionizable protons of weak acids in aqueous media is of fundamental importance in many areas of chemistry and biochemistry. The pKa value, or equilibrium dissociation constant, of a molecule determines the relative concentration of its protonated and deprotonated forms at a specified pH and is therefore an important descriptor of its chemical reactivity. Considerable efforts have been devoted to the determination of pKa values by different experimental techniques. Although in most cases the determination of pKa values from experimental is straightforward, there are situations where interpretation is difficult and the results ambiguous. It is, therefore, not surprising that the capability to provide accurate estimates of the pKa value has been a central goal in theoretical chemistry and there has been a large effort in developing methodologies for predicting pKa values for a variety of chemical systems by differing quantum chemical techniques. A prediction accuracy within 0.5 pKa units of experiment is the desirable level of accuracy. This is a non-trivial exercise, for an error of 1 kcal/mol in estimates of the free energy value would result in an error of 0.74 pKa units.
In this thesis ab initio Car-Parrinello molecular dynamics (CPMD) has been used for investigating the Brϕnsted acid-base chemistry of weak acids in aqueous solution. A key issue in any dissociation event is how the solvating water molecules arrange themselves spatially and dynamically around the neutral and dissociated acid molecule. Ab initio methods have the advantage that all solvent water molecules can, in principle, be con- sidered explicitly. One of the factors that has inhibited the widespread use of ab initio MD methods to study the dissociation reaction is that dissociation of weak acids are rare events that require extremely long simulation times before one is observed. The metady- namics formalism provides a solution to this conundrum by preventing the system from revisiting regions of configuration space where it has been in the past. The formalism allows the system to escape the free-energy minima by biasing the dynamics with a history dependent potential (or force) that acts on select degrees of freedom, referred to as collective variables. The bias potentials, modeled by repulsive inverted Gaussians that are dropped during propagation, drive the system out of any free-energy minima and allow it to explore the configurational space by a relatively quick and efficient sampling. The the- sis deals with a detailed investigation of the Brϕnsted acid-base chemistry of weak acids in aqueous solutions by the CPMD-metadynamics procedure. In Chapter 1, current approaches for the theoretical estimation of pKa values are summarized while in Chapter 2 the simulation methodology and the metadynamics sampling techniques used in this
study are described.
The potential of the CPMD-metadynamics procedure to provide estimates of the acid dissociation constant (pKa) is explored in Chapter 3, using acetic acid as a test sys- tem. Using the bond-distance dependent coordination number of protons bound to the dissociating carboxylic groups as the collective variable, the free-energy profile for the dissociation reaction of acetic acid in water was computed. Convergence of the free-energy profiles and barriers for the simulations parameters is demonstrated. The free-energy profiles exhibit two distinct minima corresponding to the dissociated and neutral states of the acid and the deterrence in their values provides the estimate for pKa. The estimated value of pKa for acetic acid from the simulations, 4.80, is in good agreement with the experiment at value of 4.76. It is shown that the good agreement with experiment is a consequence of the cancellation of errors, as the pKa values are computed as the divergence in the free energy values at the minima corresponding to the neutral and dissociated state. The chapter further explores the critical factors required for obtaining accurate estimates of the pKa values by the CPMD-metadynamics procedure. It is shown that having water molecules sufficient to complete three hydration shells as well as maintaining water density in the simulation cell as close to unity is important.
In Chapter 4, the CPMD-metadynamics procedure described in Chapter-3 has been used to investigate the dissociation of a series of weak organic acids in aqueous solutions. The acids studied were chosen to highlight some of the major factors that influence the dissociation constant. These include the influence of the inductive effect, the stabilization of the dissociated anion by H-bonding as well as the presence of multiple ionizable groups. The acids investigated were aliphatic carboxylic acids, chlorine-substituted carboxylic acids, cis- and trans-butenedioic, the isomers of hydroxybenzoic acid and ophthalmic acids and its isomers. It was found that in each of these examples the CPMD-metadynamics procedure correctly estimates the pKa values, indicating that the formulism is capable of capturing these influences and equally importantly indicating that the cancellation of errors is indeed universal. Further, it is shown that the procedure can provide accurate estimates of the successive pKa values of polypro tic acids as well as the subtle difference in their values for different isomers of the acid molecule.
Changes in protonation-deprotonation of amino acid residues in proteins play a key role in many biological processes and pathways. It is shown that CPMD simulations in conjunction with metadynamics calculations of the free energy profile of the protonation- deprotonation reaction can provide estimates of the multiple pKa values of the 20 canonical α-amino acids in aqueous solutions in good agreement with experiment (Chapter 5). The distance-dependent coordination number of the protons bound to the hydroxyl oxygen of the carboxylic and the amine groups is used as the collective variable to explore the free energy profiles of the Brϕnsted acid-base chemistry of amino acids in aqueous solutions. Water molecules, sufficient to complete three hydration shells surrounding the acid molecule were included explicitly in the computation procedure. The method works equally well for amino acids with neutral, acidic and basic side chains and provides estimates of the multiple pKa values with a mean relative error with respect to experimental results, of 0.2 pKa units.
The tripeptide Glutathione (GSH) is one of the most abundant peptides and the major repository for non-protein sulfur in both animal and plant cells. It plays a critical role in intracellular oxidative stress management by the reversible formation of glutathione disulfide with the thioldisulfide pair acting as a redox buffer. The state of charge of the ionizable groups of GSH can influences the redox couple and hence the pKa value of the cysteine residue of GSH is critical to its functioning. In Chapter 6, it has been reported that ab initio Car-Parrinello Molecular Dynamics simulations of glutathione solvated by 200 water molecules, all of which are considered in the simulation. It is shown that the free-energy landscape for the protonation - deprotonation reaction of the cysteine residue of GSH computed using metadynamics sampling provides accurate estimates of the pKa and correctly predicts the shift in the dissociation constant values as compared to the isolated cysteine amino acid.
The dissociation constants of weak acids are commonly determined from pH-titration
curves. For simple acids the determination of the pKa from the titration curves using the Henderson-Hasselbalch equation is relatively straightforward. There are situations, however, especially in polyprotic acids with closely spaced dissociation constants, where titration curves do not exhibit clear inflexion and equivalence stages and consequently the estimation of multiple pKa values from a single titration curve is no longer straightfor-
ward resulting in uncertainties in the determined pKa values. In Chapter 7, the multiple dissociation constant of the hexapeptide glutathione disulfide (GSSG) with six ionizable groups and six associated dissociation constants has been investigated. The six pKa values of GSSG were estimated using the CPMD-metadynamics procedure from the free-energy profiles for each dissociation reaction computed using the appropriate collective variable. The six pKa values of GSSG were estimated and the theoretical pH-titration curve was then compared with the experimentally measured pH-titration curve and found to be in excellent agreement. The object of the exercise was to establish whether interpretation of pH-titration curves of complex molecules with multiple ionizable groups could be facilitated using results of ab initio molecular dynamics simulations.