During the process of lateral organ development after plant decapitation, cell division and differentiation occur in a balanced manner initiated by specific signaling, which triggers the reentrance into the cell cycle. Here, we investigated short-term variations in the content of some endogenous signals, such as auxin, cytokinins (Cks), and other mitogenic stimuli (sucrose and glutamate), which are likely correlated with the cell cycle reactivation in the axillary bud primordium of pineapple nodal segments. Transcript levels of cell cycle-associated genes, CycD2;1, and histone H2A were analyzed. Nodal segments containing the quiescent axillary meristem cells were cultivated in vitro during 24 h after the apex removal and de-rooting. From the moment of stem apex and root removal, decapitated nodal segment (DNS) explants showed a lower indol-3-acetic acid (IAA) concentration than control explants, and soon after, an increase of endogenous sucrose and iP-type Cks were detected. The decrease of IAA may be the primary signal for cell cycle control early in G1 phase, leading to the upregulation of CycD2;1 gene in the first h. Later, the iP-type Cks and sucrose could have triggered the progression to S-phase since there was an increase in H2A expression at the eighth h. DNS explants revealed substantial increase in Z-type Cks and glutamate from the 12th h...
A hemopoese é um processo dinâmico regulado pelo microambiente no qual se situa. O principal tecido hemopoético após o nascimento, a medula óssea, é constituído basicamente por substâncias solúveis, como fatores de crescimento, por uma matriz extracelular (MEC) e por células estromais, além das células hemopoéticas. Esse microambiente indutor íntegro é capaz de regular os processos de sobrevivência, proliferação e diferenciação celular, induzindo a célula a sair de um estado quiescente e entrar em ciclo celular. Contudo, na desnutrição protéica (DP) observa-se redução significativa da celularidade das células hemopoéticas, tanto no compartimento periférico quanto no central, a medula óssea. O comprometimento estrutural do microambinte medular decorrente da desnutrição pode prejudicar a sinalização de indução do ciclo celular, fato este que justificaria o quadro de pancitopenia. Portanto, no presente estudo nos propusemos avaliar o ciclo celular de células tronco/progenitoras hemopoéticas (CTPH) da medula óssea de camundongos desnutridos. Para tanto, utilizamos um modelo murino, sendo a desnutrição induzida a partir de uma ração hipoprotéica. As CTPH foram obtidas por método de depleção imunomagnética e utilizadas para a avaliação do ciclo celular a partir da incorporação de Iodeto de Propídeo (PI) e Laranja de Acridina (AO). Também...
A acidez do solo é um dos principais fatores limitantes à produção vegetal. Apesar da toxicidade por alumínio ter sido extensamente investigada, pouca atenção tem sido dada ao estresse causado pelo baixo pH em si. Existem diferenças marcantes entre células quanto à sensibilidade ao pH baixo que dependem do seu estado de crescimento e desenvolvimento celular e que devem ser exploradas para se entender o que determina a sensibilidade e tolerância a pH baixo. Em alguns casos, a suscetibilidade a pH baixo está relacionada a desarranjos na parede de células em crescimento, chegando a causar o rompimento da célula, como já foi demonstrado em pêlos radiculares em expansão. Por outro lado, o metabolismo oxidativo e a geração de espécies reativas de oxigênio (ROS) na parede podem influenciar neste processo por romper ou criar ligações dentro ou entre cadeias de polissacarídeos, modulando assim a extensibilidade da parede celular. Em células de tabaco (Nicotiana tabacum) cv. BY-2, há um aumento acentuado na sensibilidade ao pH baixo no final da fase lag da cultura, que ocorrre entre 12 e 24 h de cultivo. Os objetivos deste trabalho foram: a) Investigar se a mudança na sensibilidade pH baixo ocorre durante a retomada do ciclo celular e determinar...
Embryonic stem cells have the capacity for unlimited proliferation while retaining their potential to differentiate into a wide variety of cell types. Murine, primate and human embryonic stem cells (ESCs) exhibit a very unusual cell cycle structure, characterized by a short G1 phase and a high proportion of cells in S-phase. In the case of mESCs, this is associated with a unique mechanism of cell cycle regulation, underpinned by the precocious activity of cyclin dependent protein kinase (Cdk) activities. As ES cells differentiate, their cell cycle structure changes dramatically so as to incorporate a significantly longer G1 phase and their mechanism of cell cycle regulation changes to that typically seen in other mammalian cells. The unique cell cycle structure and mechanism of cell cycle control indicates that the cell cycle machinery plays a role in establishment or maintenance of the stem cell state. This idea is supported by the frequent involvement of cell cycle regulatory molecules in cell immortalization.; Josephine White and Stephen Dalton
OBJECTIVES: Tumour re-population during radiotherapy was identified as an important reason for treatment failure in head and neck cancers. The process of re-population is suggested to be caused by various mechanisms, one of the most plausible one being accelerated division of stem-cells (i.e. drastic shortening of cell cycle duration). However, the literature lacks quantitative data regarding the length of tumour stem-cell cycle time during irradiation. MATERIALS AND METHODS: The presented work suggests that if accelerated stem-cell division is indeed a key mechanism behind tumour re-population, the stem-cell cycle time can drop below 10 h during radiotherapy. To illustrate the possible implications, the mechanism of accelerated division was implemented into a Monte Carlo model of tumour growth and response to radiotherapy. Tumour response to radiotherapy was simulated with different stem-cell cycle times (between 2 and 10 h) after the initiation of radiotherapy. RESULTS: It was found that very short stem-cell cycle times lead to tumour re-population during treatment, which cannot be overcome by radiationinduced cell kill. Increasing the number of radiation dose fractions per week might be effective, but only for longer cell cycle times. CONCLUSION: It is of crucial importance to quantitatively assess the mechanisms responsible for tumour re-population...
The aim of this thesis is to develop mathematical models of cell cycle progression which can be used in conjunction with biological experiments. The thesis focusses on modelling processes which have biological relevance, and uses mathematics to investigate biological hypotheses about mechanisms which drive experimental results. In this thesis, we introduce a mathematical model of cell cycle progression and apply it to the MCF-7 breast cancer cell line. The model considers the three typical cell cycle phases, which we further break up into model phases in order to capture certain features such as cells remaining in phases for a minimum amount of time. This results in a unique system of delay differential equations which are solved numerically using MATLAB. The model is also able to capture a uniquely important part of the cell cycle, during which time cells are responsive to their environment. The model parameters are carefully chosen using data from various sources in the biological literature. The model is then validated against a variety of experiments, and the excellent fit with experimental results allows for insight into the mechanisms that influence observed biological phenomena. In particular, the model is used to question the common assumption that a ‘slow cycling population’ is necessary to explain some results. A model analysis is also performed...
Although traditionally little attention has
been paid to the interplay between neurotrophins and the
cell cycle, a number of recent findings suggest an
important role for these growth factors in the regulation
of this aspect of the cellular physiology.
In this article, we review the evidence from a
number of studies that neurotrophins can influence cell
cycle progression or mitotic cycle arrest both in the
nervous system as well as in other cell types. The
contrary response of different cells to neurotrophins in
terms of cell cycle regulation derives in part from the
fact that these factors use two different receptor types to
transmit their signals: members of the Trk family and the
p75 neurotrophin receptor (p75NTR). With this in mind,
we outline the current state of our knowledge regarding
the molecular basis underlying the control of cell cycle
progression by neurotrophins. We focus our interest on
the receptors that transduce these signals and, in
particular, the striking finding that p75NTR interacts with
proteins that can promote mitotic cycle arrest. Finally,
we discuss the mechanisms of cell death mediated by
p75NTR in the context of cell cycle regulation.
A fundamental property of cell populations is their growth rate as well as the time needed for cell division and its variance. The eukaryotic cell cycle progresses in an ordered sequence through the phases G1, S, G2, and M, and is regulated by environmental cues and by intracellular checkpoints. Reflecting this regulatory complexity, the length of each phase varies considerably in different kinds of cells but also among genetically and morphologically indistinguishable cells. This article addresses the question of how to describe and quantify the mean and variance of the cell cycle phase lengths. A phase-resolved cell cycle model is introduced assuming that phase completion times are distributed as delayed exponential functions, capturing the observations that each realization of a cycle phase is variable in length and requires a minimal time. In this model, the total cell cycle length is distributed as a delayed hypoexponential function that closely reproduces empirical distributions. Analytic solutions are derived for the proportions of cells in each cycle phase in a population growing under balanced growth and under specific non-stationary conditions. These solutions are then adapted to describe conventional cell cycle kinetic assays based on pulse labelling with nucleoside analogs. The model fits well to data obtained with two distinct proliferating cell lines labelled with a single bromodeoxiuridine pulse. However...
A biologia da reprodução de plantas é um campo de grande interesse, já que a maioria dos alimentos consumidos pelo homem é composta de partes reprodutivas das plantas (frutos e sementes). O pistilo é o órgão reprodutivo feminino, composto de estigma, estilete e ovário. Devido à importância central do pistilo no sucesso da reprodução de plantas, faz-se necessário um melhor conhecimento dos genes e processos que regulam seu desenvolvimento e funcionamento. Estudos comparativos da expressão gênica nos órgãos vegetativos e reprodutivos de Nicotiana tabacum revelaram genes de expressão preferencial nos órgãos reprodutivos, entre eles alguns codificando proteínas de função ainda desconhecida. Um destes genes foi caracterizado e denominado SCI1 (Stigma/style Cell-cycle Inhibitor 1), por apresentar um papel importante no desenvolvimento do estigma/estilete, atuando como um inibidor de ciclo celular tecido-específico (DePaoli et al., 2011). O presente trabalho teve como objetivo estudar os mecanismos moleculares pelos quais NtSCI1 regula o ciclo celular, investigando seus parceiros de interação. Em um ensaio de pull-down, utilizando-se extrato proteico nuclear de estigmas/estiletes de N. tabacum, vários putativos reguladores de ciclo celular foram identificados...
Les protéines sont les produits finaux de la machinerie génétique. Elles jouent des rôles essentiels dans la définition de la structure, de l'intégrité et de la dynamique de la cellule afin de promouvoir les diverses transformations chimiques requises dans le métabolisme et dans la transmission des signaux biochimique. Nous savons que la doctrine centrale de la biologie moléculaire: un gène = un ARN messager = une protéine, est une simplification grossière du système biologique. En effet, plusieurs ARN messagers peuvent provenir d’un seul gène grâce à l’épissage alternatif. De plus, une protéine peut adopter plusieurs fonctions au courant de sa vie selon son état de modification post-traductionelle, sa conformation et son interaction avec d’autres protéines. La formation de complexes protéiques peut, en elle-même, être déterminée par l’état de modifications des protéines influencées par le contexte génétique, les compartiments subcellulaires, les conditions environmentales ou être intrinsèque à la croissance et la division cellulaire. Les complexes protéiques impliqués dans la régulation du cycle cellulaire sont particulièrement difficiles à disséquer car ils ne se forment qu’au cours de phases spécifiques du cycle cellulaire...
Quelques évidences suggèrent que Bcl-xL, un membre anti-apoptotique de la famille Bcl-2, possède également des fonctions au niveau du cycle cellulaire et de ses points-contrôle. Pour étudier la régulation et fonction de Bcl-xL au cours du cycle cellulaire, nous avons généré et exprimé dans des cellules humaines une série de mutants de phosphorylation incluant Thr41Ala, Ser43Ala, Thr47Ala, Ser49Ala, Ser56Ala, Ser62Ala et Thr115Ala.
L'analyse de cette série de mutants révèle que les cellules exprimant Bcl-xL(Ser62Ala) sont moins stables au point-contrôle G2 du cycle cellulaire comparées aux cellules exprimant le type sauvage ou les autres mutants de phosphorylation incluant Thr41Ala, Ser43Ala, Thr47Ala, Ser56Ala et Thr115Ala. Les études de cinétiques de phosphorylation et de localisation de phospho-Bcl-xL(Ser62) dans des cellules synchronisées et suite à l'activation du point-contrôle en G2 médié par l'étoposide (VP16), nous indiquent que phospho-Bcl-xL(Ser62) migre dans les corps nucléolaires durant l'arrêt en G2 dans les cellules exposées au VP16. Une série d'expériences incluant des essais kinase in vitro, l'utilisation d'inhibiteurs pharmacologiques et d'ARN interférant, nous révèlent que Polo kinase 1 (PLK1) et MAPK9/JNK2 sont les protéines kinase impliquées dans la phosphorylation de Bcl-xL(Ser62)...
The eukaryotic cell cycle is one of the most studied biological processes.
However, variations of the canonical cell cycle have been discovered and
found to be more predominant than previously expected. Endoreplication
and endocycling - two such variants - produce polyploid cells, conferring
advantages in growth and genotoxic stress. Although some of the regulatory
principles of these cycles are being discovered, little is known about how
cells can transition from a mitotic cell cycle to them. Recently, it has been
proposed that methylation of histone 3 lysine 79 (H3K79me) might have a
function distinct from most histone modifications - a timer for a cell’s age
-, for methylation of all H3K79 states is performed in a distributive fashion
(unlike all other methylated lysines in histones) by Dot1 and no demethylase
is known. Functional studies on Dot1 have shown aberrant patterns of
replication, linking it directly to a cell’s ability to regulate its cell cycle. This
project aims to study the mechanisms of transition between different cell
cycle modes in Oikopleura dioica - a marine chordate employing endocycling
extensively through its life cycle. We performed ChIP-seq on two antagonist
transcription factors (E2F1 and E2F7) involved in the control of cell cycle
progression that have been shown to have differential patterns of regulation
in mitotic and endocycling cell cycle modes. Results reveal a high degree
of spatial and temporal occupancy of both transcription factors in close
proximity to transcription start sites...
12 pages, 7 figures.-- PMID: 19355788 [PubMed].-- Supporting information (Suppl. figures S1-S3, movies S1-S7) available at: http://biology.plosjournals.org/perlserv/?request=get-document&doi=10.1371/journal.pbio.1000079#toclink6; Regulation of cell proliferation has been extensively studied in cultured cell systems that are characterized by coordinated growth and cell-cycle progression and relatively uniform cell size distribution. During the development of multicellular organisms, however, growth and division can be temporally uncoupled, and the signaling pathways that regulate these growth programs are poorly understood. A good model for analyzing proliferation control in such systems is the morphogenesis of the Drosophila adult abdominal epidermis by histoblasts. These cells undergo a series of temporally regulated transitions during which neither cell size nor division rate is constant. The proliferation of histoblasts during metamorphosis is uniquely amenable to clonal analysis in combination with live imaging. Thereby, we show that abdominal histoblasts, which grow while in G2 arrest during larval stages, enter a proliferative stage in the pupal period that is initiated by ecdysone-dependent string/Cdc25 phosphatase transcription. The proliferating histoblasts have preaccumulated stores of Cyclin E...
Control of E2F transcription factor activity, regulated by the action of the retinoblastoma tumor suppressor, is critical for determining cell cycle entry and cell proliferation. However, an understanding of the precise determinants of this control, including the role of other cell cycle regulatory activities, has not been clearly defined.
Recognizing that the contributions of individual regulatory components could be masked by heterogeneity in populations of cells, we made use of an integrated system to follow E2F transcriptional dynamics at the single cell level and in real time. We measured and characterized E2F temporal dynamics in the first cell cycle where cells enter the cell cycle after a period of quiescence. Quantitative analyses revealed that crossing a threshold of amplitude of E2F transcriptional activity serves as the critical determinant of cell-cycle commitment and division.
By using a developed ordinary differential equation model for Rb/E2F network, we performed simulations and predicted that Myc and cyclin D/E activities have distinct roles in modulating E2F transcriptional dynamics. Myc is critical in modulating the amplitude whereas cyclin D/E activities have little effect on the amplitude but do contribute to the modulation of duration of E2F transcriptional activation. These predictions were validated through the analysis of E2F dynamics in single cells under the conditions that cyclin D/E or Myc activities are perturbed by small molecule inhibitors or RNA interference.
Cell division is a biological process fundamental to all life. One aspect of the process that is still under investigation is whether or not cells in a lineage are correlated in their cell-cycle progression. Data on cell-cycle progression is typically acquired either in lineages of single cells or in synchronized cell populations, and each source of data offers complementary information on cell division. To formally assess dependence in cell-cycle progression, I develop a hierarchical statistical model of single-cell measurements and extend a previously proposed model of population cell division in the budding yeast, Saccharomyces cerevisiae. Both models capture correlation and cell-to-cell heterogeneity in cell-cycle progression, and parameter inference is carried out in a fully Bayesian manner. The single-cell model is fit to three published time-lapse microscopy datasets and the population-based model is fit to simulated data for which the true model is known. Based on posterior inferences and formal model comparisons, the single-cell analysis demonstrates that budding yeast mother and daughter cells do not appear to correlate in their cell-cycle progression in two of the three experimental settings. In contrast...
The cell cycle is a series of ordered events that culminates in a single cell dividing into two daughter cells. These events must be properly coordinated to ensure the faithful passage of genetic material. How cell cycle events are carried out accurately remains a fundamental question in cell biology. In this dissertation, I investigate mechanisms orchestrating cell-cycle events in the yeast, Saccharomyces cerevisiae.
Cyclin dependent kinase (CDK) activity is thought to both form the fundamental cell-cycle oscillator and act as an effector of that oscillator, regulating cell-cycle events. By measuring transcript dynamics over time in cells lacking all CDK activity, I show that transcriptional oscillations are not dependent on CDK activity. This data indicates that CDKs do not form the underlying cell-cycle oscillator. I propose a model in which a transcription factor network rather than CDK activity forms the cell-cycle oscillator. In this model, CDKs are activated by the periodic transcription of cyclin genes and feedback on the network increasing the robustness of network oscillations in addition to regulating cell-cycle events.
I also investigate CDK-dependent and -independent mechanism regulating the duplication of the yeast centrosome...
Human germ cell tumors show a strong sensitivity to genetic background similar to Dnd1(Ter/Ter) mutant mice, where testicular teratomas arise only on the 129/SvJ genetic background. The introduction of the Bax mutation onto mixed background Dnd1(Ter/Ter) mutants, where teratomas do not typically develop, resulted in a high incidence of teratomas. However, when Dnd1(Ter/Ter); Bax(-/-) double mutants were backcrossed to C57BL/6J, no tumors arose. Dnd1(Ter/Ter) germ cells show a strong downregulation of male differentiation genes including Nanos2. In susceptible strains, where teratomas initiate around E15.5-E17.5, many mutant germ cells fail to enter mitotic arrest in G0 and do not downregulate the pluripotency markers NANOG, SOX2 and OCT4. We show that DND1 directly binds a group of transcripts that encode negative regulators of the cell cycle, including p27(Kip1) and p21(Cip1). P27(Kip1) and P21(Cip1) protein are both significantly decreased in Dnd1(Ter/Ter) germ cells on all strain backgrounds tested, strongly suggesting that DND1 regulates mitotic arrest in male germ cells through translational regulation of cell cycle genes. Nonetheless, in C57BL/6J mutants, germ cells arrest prior to M-phase of the cell cycle and downregulate NANOG...
The cell cycle is one of the fundamental processes in all living organisms, and all cells arise from the division of existing cells. To better understand the regulation of the cell cycle, synchrony experiments are widely used to monitor cellular dynamics during this process. In such experiments, a large population of cells is generally arrested or selected at one stage of the cycle, and then released to progress through subsequent division stages. Measurements are then taken in this population at a variety of time points after release to provide insight into the dynamics of the cell cycle. However, due to cell-to-cell variability and asymmetric cell division, cells in a synchronized population lose synchrony over time. As a result, the time-series measurements from the synchronized cell populations do not accurately reflect the underlying dynamics of cell-cycle processes.
In this thesis, we introduce a deconvolution algorithm that learns a more accurate view of cell-cycle dynamics, free from the convolution effects associated with imperfect cell synchronization. Through wavelet-basis regularization, our method sharpens signal without sharpening noise, and can remarkably increase both the dynamic range and the temporal resolution of time-series data. Though it can be applied to any such data...
The cell division cycle is the process in which the entirety of a cell's contents is duplicated completely and then equally segregated into two identical daughter cells. The order of the steps in the cell cycle must be followed with fidelity to guarantee two viable cells. Understanding the regulatory mechanisms that control cell-cycle events remains to be a fundamental question in cell biology. In this dissertation, I explore the mechanisms that coordinate and regulate cell-cycle progression in the budding yeast, Saccharomyces cerevisiae.
Cell-cycle events have been shown to be triggered by oscillations in the activity of cyclin dependent kinases (CDKs) when bound to cyclins. However, several studies have shown that some cell-cycle events, such as periodic transcription, can continue in the absence of CDK activity. How are periodic transcription and other cell-cycle events coupled to each other during a wild-type cell cycle? Currently, two models of cell-cycle regulation have been proposed. One model hypothesizes that oscillations in CDK activity controls the timing of cell-cycle events, including periodic transcription. The second model proposes that a transcription factor (TF) network oscillator controls the timing of cell-cycle events...
Within individual cells, the opposing processes of proliferation and apoptosis are precisely regulated. When this regulatory balance is interrupted, cells may become abnormal or even transformed. Understanding how to reverse or avoid these detrimental transformative processes begins with an intimate knowledge of the processes governing the cell cycle and apoptosis. Cell proliferation is governed by the cell cycle machinery. The cell cycle is driven by Cyclin-dependent kinase (Cdk) activity, which is dependent on the availability of specific Cyclin binding partners. The amount of available Cyclin is tightly controlled by a ubiquitin ligase protein complex called the anaphase promoting complex/cyclosome (APC/C.) This complex mediates the timely ubiquitylation and degradation of cell cycle regulators in order to control mitotic exit, the G1/S transition and to respond to signals emanating from spindle assembly checkpoint.
Given the importance of the APC/C, cells develop many ways to regulate APC/C activity. Post-translational modifications of the APC/C have been shown to alter its functionality, and many pseudosubstrate-based inhibitors have been discovered. Moreover, inhibitors such as Emi1 and Emi2, have been showed to inhibit the APC/C through their own intrinsic ubiquitin E3 ligase activities. Utilizing the Xenopus egg extract system...