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Chen, Weiwei. "Characterization of the movement of a circadian protein in the temperature-dependent root synchronization of Arabidopsis thaliana." Doctoral thesis, Universitat Autònoma de Barcelona, 2020. http://hdl.handle.net/10803/670449.
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El rellotge circadià està sincronitzat per senyals mediambientals externes, principalment la llum i la temperatura. Entendre com respon el rellotge circadià de la planta a les oscil·lacions de temperatura és crucial per comprendre la capacitat de resposta de la planta a l'entorn. En aquesta tesi doctoral, trobem una funció prevalent depenent de la temperatura de l'component de el rellotge d'Arabidopsis EARLY Flowering 4 (ELF4) en el rellotge circadià de l'arrel. En plantes en les quals l'àpex aeri s'ha eliminat, el rellotge pot funcionar en les arrels, tot i que exhibeix un període més curt i una fase avançada en comparació amb les arrels de plantes completes. Els assajos de microempelt mostren que ELF4 es mou des de l'àpex aeri per regular els ritmes en les arrels. El moviment de la proteïna ELF4 no transmet informació fotoperiòdica, sinó que és essencial per controlar el període de el rellotge circadià en l'arrel d'una manera depenent de la temperatura. Les baixes temperatures afavoreixen la mobilitat de ELF4, el que resulta en un rellotge de de ritme lent, mentre que les altes temperatures disminueixen el moviment, el que porta a un rellotge més ràpid. Per tant, el moviment de la proteïna ELF4 mòbil proporciona informació sobre la temperatura i ajuda a establir un diàleg entre l'àpex aeri i l'arrel de la planta per controlar el ritme circadià en l'arrel.El reloj circadiano está sincronizado por señales medioambientales externas, principalmente la luz y la temperatura. Entender cómo responde el reloj circadiano de la planta a las oscilaciones de temperatura es crucial para comprender la capacidad de respuesta de la planta al medio ambiente. En esta Tesis Doctoral, encontramos una función prevalente dependiente de la temperatura del componente del reloj de Arabidopsis EARLY FLOWERING 4 (ELF4) en el reloj circadiano de la raíz. En plantas en las que el ápice aéreo se ha eliminado, el reloj puede funcionar correctamente en las raíces, aunque exhibe un período más corto y una fase avanzada en comparación con las raíces de plantas completas. Los ensayos de microinjerto muestran que ELF4 se mueve desde el ápice aéreo para regular los ritmos en las raíces. El movimiento de la proteína ELF4 no transmite información fotoperiódica, sino que es esencial para controlar el período del reloj circadiano en la raíz de una manera dependiente de la temperatura. Las bajas temperaturas favorecen la movilidad de ELF4, lo que resulta en un reloj de de ritmo lento, mientras que las altas temperaturas disminuyen el movimiento, lo que lleva a un reloj más rápido. Por lo tanto, el movimiento de la proteína ELF4 móvil proporciona información sobre la temperatura y ayuda a establecer un diálogo entre el ápice aéreo y la raíz de la planta para controlar el ritmo circadiano en la raíz.
The circadian clock is synchronized by external environment cues, mostly through light and temperature. Explaining how the plant circadian clock responds to temperature oscillations is crucial to understanding plant responsiveness to the environment. In this thesis, we found a prevalent temperature-dependent function of the Arabidopsis clock component EARLY FLOWERING 4 (ELF4) in the root clock. The clocks in roots are able to run properly in the absence of shoots although shoot excision leads to a shorter period and advanced phase in excised roots compared to entire roots. Micrografting assays show that ELF4 moves from shoots to regulate rhythms in roots. ELF4 movement does not convey photoperiodic information, but trafficking is essential for controlling the period of the root clock in a temperature-dependent manner. Low temperatures favour ELF4 mobility, resulting in a slow paced root clock, whereas high temperatures decrease movement, leading to a faster clock. Hence, the mobile ELF4 delivers temperature information and establishes a shoot-to-root dialogue that sets the pace of the clock in roots.
Universitat Autònoma de Barcelona. Programa de Doctorat en Biologia i Biotecnologia Vegetal
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Wallach, Thomas. "A dynamic circadian protein-protein interaction network." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2012. http://dx.doi.org/10.18452/16604.
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Die dynamische Regulation von Protein-Protein Interaktionen (PPIs) ist wichtig für den Ablauf von biologischen Prozessen. Die circadiane Uhr, die einen ~24 Stunden Rhythmus generiert und eine Vielzahl von physiologischen Parametern steuert kann auch die Dynamik von PPIs regulieren. Um neue Erkenntnisse über regulatorische Mechanismen innerhalb des molekularen Oszillators zu gewinnen, habe ich zunächst alle möglichen PPIs zwischen 46 circadianen Komponenten mittels eines systematischen yeast-two-hybid (Y2H) Screens bestimmt. Dabei habe ich 109 bis dahin noch unbekannte PPIs identifiziert und einen repräsentativen Anteil mittels Co-Immunopräzipitationsexperimenten in humanen Zellen validiert. Unter den neuen PPIs habe ich bis dahin unbekannte Modulatoren der CLOCK/BMAL1 Transaktivierung identifiziert und dabei die Rolle der Proteinphosphatase 1 (PP1) als dynamischen Regulator der BMAL1 Stabilität funktionell charakterisiert. Das experimentelle PPI Netzwerk wurde mit bereits aus der Literatur bekannten PPIs und Interaktionspartnern ergänzt. Eine systematische RNAi Studie belegte außerdem die Relevanz der aus der Literatur stammenden Interaktoren für die ~24 Stunden Periodizität. Um eine Aussage über die Dynamik der PPIs im Netzwerk treffen zu können, wurden circadiane mRNA Expressionsdaten in das PPI Netzwerk integriert. Systematische Perturbationsstudien, in denen alle Komponenten des experimentellen Netzwerkes mittels RNAi herunterreguliert oder überexprimiert wurden, zeigten eine essentielle Bedeutung für die dynamischen PPIs innerhalb des circadianen Oszillators auf. Desweiteren wurden im circadianen PPI Netzwerk funktionelle Module identifiziert, welche dynamisch organsiert sind. Durch eine systemweite Analyse des humanen Proteoms wurden viele dynamische PPIs identifiziert, die biologische Prozesse wie z.B. Signaltransduktion und Zellzyklus miteinander verbinden. Rhythmische PPIs sind daher von Bedeutung für die zeitliche Organisation zellulärer Physiologie.Essentially all biological processes depend on protein-protein interactions (PPIs). Timing of such interactions is crucial for regulatory function. Although circadian (~24 hrs) clocks constitute fundamental cellular timing mechanisms regulating important physiological processes PPI dynamics on this timescale are largely unknown. To elucidate so far unknown regulatory mechanisms within the circadian clockwork, I have systematically mapped PPIs among 46 circadian components using high-throughput yeast-two-hybrid (Y2H) interaction experiments. I have identified 109 so far uncharacterized interactions and successfully validated a sub-fraction via co-immunoprecipitation experiments in human cells. Among the novel PPIs, I have identified modulators of CLOCK/BMAL1 function and further characterized the role of protein phosphatase 1 (PP1) in the dynamic regulation of BMAL1 abundance. Furthermore, to generate a more comprehensive circadian PPI network, the experimental network was enriched and extended with additional interactions and interaction partners from literature, some of which turned out to be essential for normal circadian dynamics. The integration of circadian mRNA expression profiles allowed us to determine the interaction dynamics within our network. Systematic genetic perturbation studies (RNAi and overexpression in oscillating human cells) revealed a crucial role of dynamic regulation (via rhythmic PPIs) for the molecular clockwork. Furthermore, dynamic modular organization as a pervasive circadian network feature likely contributes to time-of-day dependent control of many cellular processes. Global analysis of the proteome regarding circadian regulation of biological processes via rhythmic PPIs revealed time-of-day dependent organization of the human interactome. Circadian PPIs dynamically connect many important cellular processes like signal transduction and cell cycle, which contribute to temporal organization of cellular physiology.
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Han, Linqu. "Molecular and genetic analysis of a novel F-box protein, ZEITLUPE, in the Arabidopsis circadian clock." Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1155569207.
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Geng, Ruishuang. "Characterization and functional analysis of ZEITLUPE protein in the regulation of the circadian clock and plant development." Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1149013919.
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Kunisue, Sumihiro. "Roles of the Orphan Receptor Gpr176-mediated G-protein Signaling in the Central Circadian Clock." Kyoto University, 2019. http://hdl.handle.net/2433/242672.
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Kim, Kevin Dae Keon. "The Translationally Controlled Tumor Protein (TCTP) associates to and destabilizes the Circadian Factor Period 2 (Per2)." Thesis, Virginia Tech, 2010. http://hdl.handle.net/10919/76848.
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Period 2 (Per2) is a core circadian factor responsible for its own negative regulation. It operates in the circadian clock, which affects multiple biological functions such as metabolic rate, hormone release, and core body temperature. The Per2 protein functions directly with factors in other biological functions such as tumor suppression, immune system, and metabolism. In many cases, the Per2 deficiency caused by disrupted expression is sufficient to create severe abnormalities in many of the mentioned functions. The sequence contains several domains and motifs in Per2 that are traditionally involved in protein interactions which suggests that Per2 serving a regulatory role by effecting downstream biological roles dependent on Per2 stability.
In this work, we perform a two-hybrid screening assay using the C-terminal region of human Per2 and identified an extensive number of interactors. Utilizing a genetic ontology program, we assorted the list of clones into groups of proteins that are biologically relevant or operated in similar function. Through this program, we validated the two-hybrid screening by the clusters of biological function already attributed to hPer2 and identified new putative biological functions. We use the new putative interactors to gain further insight on the regulatory roles that hPer2 performs, in conjunction with operating as a core factor in circadian rhythmicity.
We also show that Translationally Controlled Tumor Protein (TCTP) is capable of binding to hPer2 and is a novel interaction. When a sufficient amount of TCTP (1:1 molar stoichiometric ratio) is present in a system, a cleavage of hPer2 is observed in vitro. This cleavage occurs in reactions independent of ATP, ubiquitin, and the proteasome. The data points towards a method of cleavage similar to that of the archael lon-tk (Thermococcus kodakaraensis) that preferentially cleaved unstructured substrates in ATP-independent reactions.Master of Science
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Han, Linqu. "Molecular and genetic analysis of a novel f-box protein, seitlupe, in the arabidopsis circadian clock." The Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=osu1155569207.
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Butcher, Gregory Quinn. "The mitogen-activated protein kinase (MAPK) pathway a signaling conduit for photic entrainment of the central mammalian circadian clock /." Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1147206998.
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Kalive, Madhavi. "An investigation of complex formation by the Drosophila circadian clock protein double-time and the effects of the double-time[superscript s] mutation on complex formation." Morgantown, W. Va. : [West Virginia University Libraries], 1999. http://etd.wvu.edu/templates/showETD.cfm?recnum=886.
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Thesis (M.S.)--West Virginia University, 1999.Title from document title page. Document formatted into pages; contains v, 65 p. : ill. (some col.) Vita. Includes abstract. Includes bibliographical references (p. [36]-45).
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Vakonakis, Ioannis. "Structure and function of circadian clock proteins and deuterium isotope effects in nucleic acid hydrogen bonds." Diss., Texas A&M University, 2003. http://hdl.handle.net/1969.1/2195.
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Circadian oscillators or clocks are a widespread, endogenous class of oscillatory mechanisms that control the ~24h temporal pattern of diverse organism functions. In cyanobacteria this mechanism is formed by three proteins, KaiA, KaiB and KaiC. KaiA is shown here to be a two domain protein that directly interacts with KaiC and enhances the KaiC autokinase activity. The amino-terminal domain of KaiA can be structurally categorized as a pseudo-receiver, a class of proteins used in signaling cascades and activated by direct protein??protein interactions. The carboxy-terminal domain interacts directly with KaiC, is sufficient to enhance the KaiC autokinase activity in a manner similar to full-length KaiA, and adopts a unique, all α-helical dimeric fold. The structure of this domain raises interesting probabilities regarding the mode of KaiA??KaiC interaction. The two KaiA domains are shown to directly interact with each other, which suggests a possible mechanism of signal transfer from the amino to carboxy-terminal domain. Hydrogen bonds are of paramount importance in nucleic acid structure and function. Here we show that changes in the width and anharmonicity of vibrational potential energy wells of hydrogen bonded groups can be measured in nucleic acids and can possibly be correlated to structural properties, such as length. Deuterium/protium fractionation factors, which are sensitive to the vibrational potential well width, were measured for the imino sites of thymidine residues involved in A:T base pairs or free in solution, and a correlation was established between decreasing fractionation factors and increasing imino proton chemical shift, δH3. Similarly, a correlation was observed between δH3and deuterium isotope effects (DIE) on chemical shift of thymidine carbon atoms. Combined these results indicate that as hydrogen-bond strength increases the vibrational potential wells of imino protons widen with a corresponding increase in anharmonicity. However, trans-hydrogen bond DIE on carbon chemical shifts of A:T base-paired adenosine residues do not correlate with those measured on thymidine residues. We propose that this lack of correlation is due to DIE dependence on base-pair geometry, which is not easily measured by traditional NMR experiments.
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Maric, Aida. "Characterization of the functional connection between the BROMODOMAIN and EXTRATERMINAL DOMAIN PROTEIN 9 (BET9) and the circadian clock in Arabidopsis thaliana." Doctoral thesis, Universitat de Barcelona, 2021. http://hdl.handle.net/10803/673868.
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The circadian clock is an endogenously generated timekeeping mechanism that generates 24-hour rhythms in multiple biological processes. The rhythmic oscillations provide an adaptive advantage, allowing organisms to anticipate and adjust to the environmental changes that occur during the day and night cycle. The generation of the rhythms rely on the oscillations in gene expression and protein function at the core of the oscillator. Over the last years, changes in chromatin marks have been identified as an important mechanism contributing to the rhythmic oscillations. However, we are still far from the identification of the chromatin-related components that are responsible for the rhythmic regulation of these chromatin changes. In this Doctoral Thesis, we have characterized the function of the BROMODOMAIN AND EXTRA- TERMINAL DOMAIN 9 (BET9) protein within the Arabidopsis thaliana circadian clock. We have identified the rhythmic oscillation of BET9 expression, which is controlled by the binding of the clock activator known as REVEILLE 8 (RVE8) to the BET9 promoter. Characterization of bet9 mutant plants and lines over- expressing BET9 (BET9-ox) showed that BET9 functions as an activator of clock gene expression. Chromatin immunoprecipitation assays (ChIP) also showed that BET9 directly binds to the promoters of essential clock genes such as TIMING OF CAB2 EXPRESSION 1 (TOC1) and PSEUDO
RESPONSE REGULATOR 5 (PRR5). Increased binding of BET9 to these promoters in BET9-ox plants correlated with increased HISTONE 3 acetylation at the TOC1 promoter, and conversely, with hypoacetylation in the bet9 mutant. Genetic interaction studies showed that BET9 requires a functional RVE8, as the BET9-ox gene expression phenotypes and BET9 binding to the gene target promoters were abolished in the rve8 mutant background. Our studies have thus uncovered a chromatin-related protein that together with RVE8 contributes to the activation of clock gene expression.El reloj circadiano es un mecanismo endógeno que genera ritmos de 24 horas en múltiples procesos biológicos. Las oscilaciones rítmicas proporcionan una ventaja adaptativa, permitiendo a los organismos anticipar y ajustarse a los cambios ambientales que ocurren en el ciclo de día-noche. La generación de los ritmos se basa en las oscilaciones rítmicas de expresión génica y función de proteínas del oscilador circadiano. En los últimos años, los cambios en marcas de cromatina se han identificado como un mecanismo importante que contribuye a las oscilaciones rítmicas. Sin embargo, estamos aún lejos de la identificación de todos los componentes relacionados con la cromatina que son responsables de la regulación rítmica de estos cambios de cromatina. En la presente Tesis Doctoral, se describe la caracterización de la función de la proteína BROMODOMAIN AND EXTRA-TERMINAL DOMAIN 9 (BET9) en el reloj circadiano de Arabidopsis thaliana. Hemos identificado la oscilación rítmica de la expresión BET9, que está controlada por la unión del activador de reloj denominado REVEILLE 8 (RVE8) al promotor de BET9. La caracterización de plantas mutantes de bet9 y líneas que sobre-expresan BET9 (BET9-ox) mostró que BET9 funciona como un activador de la expresión génica del reloj. Los ensayos de inmunoprecipitación de cromatina (ChIP) también mostraron que BET9 se une directamente a los promotores de genes de reloj esenciales como TIMING OF CAB2 EXPRESSION 1 (TOC1) y PSEUDO RESPONSE REGULATOR 5 (PRR5). Observamos que el aumento de la unión de BET9 a estos promotores en plantas BET9-ox se correlacionaba con el aumento de la acetilación de la HISTONA 3 en el promotor de TOC1 y, a la inversa, con la hipoacetilación en el mutante bet9. Los estudios de interacción genética mostraron que BET9 requiere un RVE8 funcional, ya que los fenotipos de expresión génica de plantas BET9-ox y la unión de BET9 a los promotores diana quedaban abolidos en el fondo mutante rve8. Por tanto, nuestros estudios han descubierto una proteína relacionada con la cromatina que junto con RVE8 contribuyen a la activación de la expresión génica del reloj.
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Gammash, Mohammed Matuq. "Novel roles of the circadian clock protein neuronal PAS domain protein 2 (NPAS2) in the response to oxidative and heat stress." Thesis, Bangor University, 2017. https://research.bangor.ac.uk/portal/en/theses/novel-roles-of-the-circadian-clock-protein-neuronal-pas-domain-protein-2-npas2-in-the-response-to-oxidative-andheat-stress(a5d513b0-84c5-486e-add9-afea7eeed9e2).html.
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Human NPAS2 is a basic-helix-loop-helix (BHLH) transcription factor with two N-terminal PAS domains that forms a hetero-dimeric complex with BMAL1/ARNTL to regulate the transcription of key circadian clock genes like Period and Cryptochrome. NPAS2 is unique in higher eukaryotic cells as both PAS domains contain one heme group each, which bind carbon monoxide. Although NPAS2 has been linked to cancer and neurological disorders, very little is known about its cellular roles. The results obtained in this study support a model in which NPAS2 responds to oxidative and heat stress in distinct manners. While oxidative stress results in the post-translational modification of NPAS2 and the formation of high molecular weight complexes, heat stress reduces the protein levels of NPAS2. These differences are also reflected in the changes to the NPAS2 mRNA levels. While oxidative stress increases expression of NPAS2, heat stress causes a drop in mRNA levels. The responses are cell line specific as, for example, the drop in NPAS2 protein levels after a heat shock was observed in MCF-7 breast cancer cells and HeLa cervical cancer cells, but not in non-malignant HEK-293 cells which may be of neuronal origin. Since both stress types are known to affect the peripheral clocks in human tissues in a manner which may be regulated by the MAP kinase p38β, it is proposed that NPAS2 is a novel target of this kinase. Why NPAS2 is regulated differently depending on the type of stress, could be linked with the different ways the p38 MAP kinase pathway responds to heat and oxidative stress. While heat activation is dependent on upstream kinases, heat activation may be dependent on the heat-induced increase in carbon monoxide in human cells. The works shows also that NPAS2 mRNA levels drop in the response to serum starvation and that the neuroblastoma KELLY cell line does not, or only at a very reduced level, express NPAS2 although an analysis of a large panel of human tissues shows high NPAS2 expression in the brain. A bioinformatical analysis of missense single nucleotide polymorphisms (SNPs) located the N-terminal domain of NPAS2 identifies two SNPs (S204P, rs750277651 and T252N, rs555063320) which may affect a p38 (S202) and EYRK1A (T252) kinase modification site, respectively. This is of interest as p38β may regulate NPAS2 and the dual-specific DYRK1A kinase is known to regulate the protein turnover of Cryptochrome. In summary, the work makes a strong case for NPAS2 as a novel target of the p38β MAP kinase pathway in the cellular response to oxidative and heat stress.
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Tulsian, Richa. "Circadian Clock as the mechanism of Caloric Restriction in regulating mTOR Signaling and Glucose Homeostasis." Cleveland State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=csu1539971252367088.
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Bao, Shu. "An investigation of the effects of the Drosophila circadian clock mutation double-time[superscript s] on double-time protein levels, nuclear localization of PER and temperature compensation." Morgantown, W. Va. : [West Virginia University Libraries], 1999. http://etd.wvu.edu/templates/showETD.cfm?recnum=883.
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Thesis (M.S.)--West Virginia University, 1999.Title from document title page. Document formatted into pages; contains vi, 62 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 35-42).
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Karlgren, Anna. "Genetic Control of Annual Growth Rhythm in the Conifer Norway Spruce (Picea Abies L. Karst)." Doctoral thesis, Uppsala universitet, Växtekologi och evolution, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-192180.
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Norway spruce (Picea abies L. Karst) is a conifer belonging to the group gymnosperms and is an ecologically and economically important species in several parts of Europe. It is crucial for trees like Norway spruce to adapt timing of events such as bud set and growth cessation to the local environment in order to maximize the growth period while avoiding frost damage. This thesis aims at widening the knowledge about genetic control of annual growth rhythm in Norway spruce and particularly the control of bud set. Using spruce transformants ectopically expressing PaFT/TFL1-LIKE 2 (PaFTL2) the prior hypothesis that PaFTL2 induces bud set is confirmed. This is further supported by spatial and temporal expression patterns in seedlings and adult trees. It is further shown that gymnosperms possess at least two FLOWERING LOCUS T/TERMINAL FLOWER 1 (FT/TFL1)-like genes with TFL1-like function, suggesting the ancestor of FT and TFL1 to be more TFL1-like. PaFTL1 appears to have complementary expression patterns to that of PaFTL2 both spatially and temporally indicating they may act together to control growth in Norway spruce. Since bud set is controlled by photoperiod and circadian clock genes are implicated in this process, putative clock homologs were studied to gain insight into the circadian clock in gymnosperms. Several clock homologs were identified and their expression showed a diurnal pattern but the expression was rapidly damped in constant conditions. Transgenic Arabidopsis expressing putative core clock genes from spruce indicate that at least three genes, PaCCA1, PaGI and PaZTL, appear to have a conserved function between angiosperms and gymnosperms. Taken together these results suggest that gymnosperms have a similar core clock structure as angiosperms even though fundamental differences might exist since the cycling of the clock genes were rapidly damped in free-running conditions. The studies presented in this thesis support substantial conservation of pathway components controlling photoperiodic responses in angiosperms and gymnosperms and identify PaFTL2 as a component of growth rhythm control. However, important changes in these processes are also evident. The results provide a solid basis for future research on molecular mechanisms controlling an adaptive trait in an important non-model organism.
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Klemz, Sabrina. "Protein Phosphatase 4 ist ein neuer Regulator der circadianen Uhr in Säugern." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2014. http://dx.doi.org/10.18452/17026.
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Circadiane Uhren sind endogene Oszillatoren, die tägliche Rhythmen in Physiologie, Metabolismus und Verhalten steuern. Auf molekularem Level wird die Dynamik der circadianen Oszillation über ein genregulatorisches Netzwerk aus transkriptionellen-translationalen Rückkopplungsschleifen gesteuert. Posttranslationale Modifikationen von Uhrproteinen sind für eine präzise Justierung der circadianen Periode essentiell. Dabei spielt die Phosphorylierung von Uhrproteinen für die Regulation von Aktivität, Stabilität und intrazellulärer Lokalisation eine wichtige Rolle. Bisher sind verschiedene Kinasen als Modulatoren der circadianen Uhr charakterisiert worden, jedoch ist eine funktionale Rolle von Protein Phosphatasen bisher nur unzureichend untersucht. In dieser Arbeit wurde mittels eines RNAi-basierten Screenings in oszillierenden humanen Zellen untersucht, ob sich die gezielte Depletion katalytischer Untereinheiten der Serin/Threonin-Phosphatasen auf die normale Oszillationsdynamik auswirkt und welche Rolle ausgewählte Phosphatase-Kandidaten für die posttranslationale Kontrolle des molekularen Oszillators spielen. Die RNAi vermittelte Depletion von Protein Phosphatase 4 führte gewebe- und speziesübergreifend zu einer signifikant kurzen circadianen Periode, während die Überexpression von wildtypischer Pp4c in einer stark reprimierten Amplitude resultierte. Mechanistische Untersuchungen zur funktionellen Relevanz von PP4c für die Regulation der circadianen Uhr zeigten, dass PP4c womöglich eine duale Rolle spielt: Einerseits ist PP4c in die direkte Aktivierung des Bmal1-Promotors über RRE-Elemente involviert. Anderseits wirkt PP4c inhibierend auf die CLOCK/BMAL1-vermittelte, E-Box getriebene Genexpression. Ein favorisiertes Modell fundiert auf der Vermutung, dass eine durch PP4c induzierte Modulation des Phosphorylierungsstatus von BMAL1 zu einem stabilen, aber transktiptionsinaktiven BMAL1 und damit zu einer verstärkten Repression der Uhrgentranskription führt.Circadian clocks are endogenous oscillators that drive daily rhythms in physiology, metabolism and behavior. On the molecular level the dynamics of circadian oscillations are regulated by a transcriptional-translational gene-regulatory network. Posttranslational modifications of clock proteins are essential for the precise timing of an about 24 hour-period. Among these modifications, protein phosphorylation plays an important role in regulating activity, stability and intracellular localization of clock proteins. Several kinases were characterized as regulators of the circadian clock. However, the function of protein phosphatases, which balance phosphorylation events, in the mammalian clock mechanism is less well understood. By using a systematic RNAi-based approach in oscillating human cells, this work aimed to study the impact of catalytic subunits of Serine/Threonin-phosphatases on normal circadian dynamics and the functional role of potential candidates in the posttranslational control of the mammalian molecular oscillator. This study demonstrates, that genetic depletion of the catalytic subunit of protein phosphatase 4 results independently from tissue and species in a significant shorter period, whereas overexpression of wildtype PP4c results in a severely reduced amplitude rhythm. Mechanistic experiments to uncover the functional relevance of PP4c in the regulation of the circadian clock showed, that PP4c plays a dual role: Firstly, PP4 is involved in the direct activation of the Bmal1-promotor via RRE elements. Secondly, PP4c is inhibiting the CLOCK/BMAL1-mediated gene expression. A favored model is based on the assumption, that PP4c-induced modulation of the phosphorylation status of BMAL1 leads to a more stable and transcriptional inactive protein and thereby to a repression of the transcription of clock genes.
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Haaf, Erik. "Qualitative und quantitative Analyse der Phosphorylierung von Proteinen der circadianen Uhr." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2012. http://dx.doi.org/10.18452/16578.
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Die Phosphorylierung als posttranslationale Modifikation von Proteinen spielt bei Signalwegen in Zellen eine große Rolle. Für die Entschlüsselung des molekularen Mechanismus der circadianen Uhr ist es daher von Interesse, die Phosphorylierungsstellen beteiligter Proteine zu identifizieren. Im Rahmen dieser Arbeit wurden die Proteine Period I und II mittels Flüssigkeitschromatographie und Tandemmassenspektrometrie (LC-MS/MS) auf Phosphorylierungsstellen untersucht. Hierbei lag der Fokus auf der Verbesserung bestehender Methoden, um eine bessere und umfassendere Identifizierung der Phosphorylierungsstellen, insbesondere in Bezug auf mehrfach phosphorylierte Peptide, zu erreichen. Der Arbeitsablauf beinhaltete die Verwendung mehrerer Proteasen, um eine hohe Sequenzabdeckung des Proteins zu erreichen. Nach der Proteolyse wurden die Phosphopeptide mittels Titandioxid angereichert. Hierbei und bei der LC-MS/MS-Analyse wurde Citrat als Additiv verwendet, welches eine bessere Chromatographie multiphosphorylierter Peptide ermöglicht. Bei der MS/MS-Analyse wurden CID und ETD als Fragmentierungsmethoden eingesetzt. Es konnten durch diese Methodik 30 bzw. 42 Phosphorylierungsstellen an den Proteinen Period I und II identifiziert werden, von denen 26 bzw. 14 zuvor nicht beschrieben waren. Nach der qualitativen Identifizierung wurden quantitative Varianten der optimierten Analytik untersucht, um die biologische Funktion der gefundenen Phosphorylierungsstellen untersuchen zu können. Hierbei wurden das metabolische Labeling der Zellen mit 15N-stickstoffhaltigen Aminosäuren und die säurekatalysierte Isotopenmarkierung auf Peptidebene mit 18O-Sauerstoff untersucht. Mit einer optimierten Variante der säurekatalysierten Isotopenmarkierung mit 18O-Sauerstoff lassen sich die Carboxygruppen der Peptide in 5h 30min mit einer Rate von >97% 18O-Sauerstoff markieren. Mit dieser Methode können weitere funktionelle Untersuchungen der Phosphorylierung an den Period-Proteinen durchgeführt werden.Protein phosphorylation, a posttranslational modification, plays an important role in signal cascades in cells. In order to understand the molecular mechanism of the circadian clock, it is thus of interest to identify the phosphorylation sites on proteins contributing to the system. During the work for this thesis, the proteins Period I and II were analyzed for phosphorylation sites with liquid chromatography and tandem mass spectrometry (LC-MS/MS). Hereby the focus was on improving existing methods in order to better identify multi-phosphorylated peptides. In the workflow, the Period proteins were digested with several proteases in order to archive a high sequence coverage for analysis. After proteolysis the phosphopeptides were subsequently enriched with titanium dioxide. During phosphopeptide enrichment and reversed phase chromatography, citrate was used as an additive for a better chromatography and recovery of multiphosphorylated peptides. During LC-MS/MS analysis, CID and ETD were used as fragmentation mechanisms in the mass spectrometer. Using these methods, 30 and 42 phosphorylation sites could be identified on the proteins Period I and II, respectively, including 26 and 14 which were previously unpublished. In order to unravel the biological function of these phosphorylation sites, quantitative methods for the optimized LC-MS approach were investigated. This included the metabolic labeling of cells with amino acids containing 15N-nitrogen as well as acid catalyzed 18O-oxygen labeling on peptide level. The developed optimized variant of acid catalyzed 18O-oxygen labeling achieves an inclusion of 18O-oxygen at the peptide carboxy groups with a rate of >97% in 5h 30min. This method can be used for further investigation of the biological function of the phosphorylation on the Period proteins.
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Zhang, Yuan. "Circadian clocks and cancer : The implication of BMAL1 (brain and muscle Arnt-like protein-1) in colorectal and breast carcinoma development and treatment." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS422.
Full textAbstract:
BMAL1, une protéine centrale de l'horloge circadienne.L’inactivation de BMAL1 (BMAL1-KO) entraîne une perte complète de la rythmicité dans les horloges central et périphérique. Le travail de ma thèse se concentre sur le rôle du gène BMAL1 dans la développement et le traitement des cancers du sein et du côlon.1. Pharmacodynamique in vitro de l’Everolimus en fonction du temps d’administration malgré une horloge circadienne défectueuse ((Zhang et al., 2018) (Zhang, Levi and Chang, 2018)L’everolimus (EV) est un inhibiteur de la mTOR chez les mammifères et il est utilisé pour traiter le cancer du sein positif aux oestrogènes (ER+). Nous avons focalisé nos recherches sur la chronopharmacologie de l’Everolimus administré sur des cellules MCF-7 (ER+). Les MCF-7 présentent une oscillation circadienne de l’activité de mTOR sans mise en évidence d’une oscillation des gènes d’horloge. L’oscillation d’activité de mTOR induirait une oscillation de synthèse et/ou de phosphorylation de protéines importantes dans la progression de la phase G1, notamment la Cycline D1 et RB phosphorylée. Ces variations rythmiques des MCF-7 synchronisées expliquent la chrono-efficacité de l’Everolimus selon des temps différents d’administration.Ce travail a révélé que même dans un système de cellules cancéreuses dont l’horloge était perturbée, l'intégration d'autres rythmes cellulaires dans la chronothérapie pouvait augmenter l'efficacité du médicament. Ce principe peut être appliqué à des traitements du cancer pour optimiser la chronothérapie du cancer.2. Le Knockdown BMAL1 a déclenché différents destins de cellules du carcinome du côlon (CRC) en modifiant l'équilibre délicat entre les voies AKT / mTOR et P21 / P53 (Article soumis)Premièrement, nos résultats ont révélé que le knockdown BMAL1 par le shRNA (BMAL1-KD) avait déclenché une activation plus évidente de l’AKT / mTOR dans deux lignées cellulaires primaires (HCT116 et SW480) que une lignée métastatique de CRC, SW620. De plus, bien que les deux lignées cellulaires primaires de CRC aient présenté une augmentation significative de l'activité de l'AKT/mTOR, elles avaient des statuts différents de P53 (WT ou mutant). Dans ce contexte, les cellules SW480 BMAL1-KD avec P53 mutant présentaient une sénescence accrue, mais les cellules HCT116 BMAL1-KD avec P53 WT présentaient d’abord une apoptose transitoire, puis un taux de prolifération plus élevé.Ainsi, nos travaux ont révélé le rôle crucial de BMAL1 pour équilibrer un régulateur central du métabolisme AKT / mTOR et une voie de réponse au stress P53 / P21 dans des lignées cellulaires de CRC, ce qui met en évidence l’importance de BMAL1 dans le développement de CRC et la progression du vieillissement.3. BMAL1 renforce les propriétés épithéliales et diminue la chimiorésistance des cellules du CRC (article en préparation)La transition épithélo-mésenchymateuse (EMT) est un événement critique dans l'invasion et la métastase des carcinomes, y compris le CRC.Dans ce travail, nous avons étudié comment BMAL1 knockdown (Bmal1-KD) altère l’équilibre délicat entre les propriétés épithéliales et mésenchymateuse de trois lignées cellulaires de CRC (HCT116, SW480 et SW620).Après BMAL1-KD, la diminution de l’expression Twist, un facteur de transcription favorisé l’EMT et des marqueurs mésenchymateux (N-Cadhérine, Vimentine) étaient associées à une expression accrue des marqueurs épithéliaux (E-cadhérine, CK20 et EpCAM). De manière constante, l'augmentation de l'expression de l’E-cadhérine après BMAL1-KD était accompagnée d'une co-localisation membranaire accrue de la β-caténine avec l'E-cadhérine, ainsi que d'une diminution de la localisation nucléaire de la β-caténine, suggérant une diminution de l'activation de la voie Wnt. De plus, les cellules BMAL1-KD ont montré une diminution des capacités de migration et de la résistance aux médicaments.Au total, ces données soulignent l’importance de BMAL1 dans l’EMT des cellules de CRCBMAL1 is a core circadian clock protein, forming a heterodimer with CLOCK to initiate the transcription of circadian and output genes. Among canonical clock genes, only BMAL1 knockout results in complete loss of rhythmicity in both the SCN and peripheral tissues. My thesis work focuses on exploring the important role of BMAL1 in human breast and colon cancer progression and treatment. My work is divided into three main parts:1. Dosing time dependent in vitro pharmacodynamics of Everolimus despite a defective circadian clock (Zhang et al., 2018)(Zhang, Levi and Chang, 2018) Everolimus (EV) is an inhibitor of mammalian target of Rapamycin (mTOR) and is used to treat estrogen positive (ER+) breast cancer. Here, we investigated whether EV efficacy varied according to administration timing by using the ER+ breast cancer cell line MCF-7 as a model system. Serum shock synchronization induced a circadian oscillation in mTOR activity in MCF-7 cells, which rhythmically regulated the synthesis or phosphorylation of key G1 progression proteins, such as Cyclin D1 and phosphorylated RB, ultimately resulting in different G0/G1 blockage efficiency according to different EV administration timing. Thus, the different delivery schedule of EV presented different efficacy in G0/G1 phase blockage in serum shocked MCF-7 cells.This investigation revealed that, even in a breast cancer cell system with disrupted circadian organization, modulating drug administration according to other protein rhythms could still increase drug efficacy. This principle may be applied to many other cancer systems and treatment types to optimize cancer chronotherapy.2. Knockdown BMAL1 triggered different colon carcinoma cells fates by altering the delicate equilibrium between AKT/mTOR and P21/P53 pathways (Article in preparation)We tried to evaluate in vitro how knockdown BMAL1 (BMAL1-KD) by shRNA influences human colorectal cancer cell (CRC) behavior.The results revealed that BMAL1-KD triggered different CRC cell fates based on distinct p53 status in different cell lines. First, after BMAL1 knockdown, two primary CRC cell lines (HCT116 and SW480) presented a more evident AKT/mTOR activation than the metastatic colon carcinoma cell line, SW620. Furthermore, although both primary CRC cell lines presented a significant increase of AKT/mTOR activity, they had different P53 status (WT or mutant) and activation pattern. Under these context, SW480 BMAL1-KD cells exhibited increased senescence but HCT116 BMAL1-KD cells showed firstly a transient apoptosis and then higher proliferation rate.Thus, our work uncovered the crucial role of BMAL1 to balance a central metabolism regulator AKT/mTOR and a stress response pathway P53/P21 in CRC cell lines, which highlighted the importance of BMAL1 in CRC development and aging progression.3. BMAL1 knockdown leans epithelial–mesenchymal balance toward epithelial properties and decreased the chemoresistance of colon carcinoma cell (Article in preparation)Epithelial-mesenchymal transition (EMT) is a critical early event in the invasion and metastasis of carcinoma, including colorectal cancer (CRC). In this work, we studied how BMAL1-KD alters the delicate equilibrium between epithelial and mesenchymal properties of three colon carcinoma cell lines (HCT116, SW480 and SW620).The results showed the molecular alterations after BMAL1-KD promote mesenchymal-to-epithelial transition-like changes mostly appeared in two primary CRC cell lines (HCT116 and SW480) compared to the metastatic cell line SW620. Subsequently, BMAL1-KD HCT116 and SW480 cells harbored a decreased migration, invasiveness and drug resistance capacities relative to their scramble counterpart cells. All these data suggested the importance of BMAL1 on EMT inducing in colon carcinoma cells
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Nayak, Aditya. "Mécanismes moléculaires de la perception de la température ambiante chez les plantes." Thesis, Université Grenoble Alpes (ComUE), 2019. http://www.theses.fr/2019GREAV009.
Full textAbstract:
La température ambiante joue un rôle direct dans le fonctionnement et le développement de la plante. L'augmentation des températures ambiantes mondiales pose un défi important aux espèces de plantes sauvages et cultivées. La plupart des espèces de plantes ajustent leur cycle de reproduction et leur développement pour optimiser leur survie et leur forme par temps ambiant élevé (Barnabás et al., 2008; Fitter et Fitter, 2002; Willis et al., 2008). Ces adaptations conduisent généralement à des hypocotyles allongés, à une réduction du nombre de feuilles au moment de la floraison, à une transition accélérée des phases de croissance végétative à reproductive, à une réduction du nombre de graines, à des gousses plus petites et à une diminution de la surface foliaire. Face au changement climatique rapide, en particulier à l'augmentation de la température permissive à la croissance ambiante, il est urgent de régler la thermoréponse des usines pour adapter les installations aux changements climatiques et garantir la production alimentaire future.L'expression de PIF4 est contrôlée par le complexe du soir (EC), un complexe à 3 protéines comprenant ELF3, ELF4 et LUX, en fonction de la température. La CE est capable de réprimer l'expression de PIF4 en se liant à des motifs spécifiques sur le promoteur de PIF4 à une température de croissance ambiante inférieure. Cependant, cette répression de l'expression de PIF4 est éliminée à une température ambiante de croissance plus élevée, ce qui entraîne un vieillissement prématuré des plantes.RésultatsLes trois protéines de la CE ont été produites en utilisant une stratégie d’expression différente, ELF4 et LUX dans E. coli, et ELF3 dans des cellules d’insecte. Les méthodes de purification des protéines et les tampons ont été optimisés pour obtenir des ELF3 et LUX stables. Une chromatographie d'exclusion de taille a été réalisée pour vérifier les états oligomères des protéines. LUX étant la seule protéine de la CE à posséder un domaine de liaison à l'ADN connu, elle a été utilisée pour effectuer des tests de déplacement de la mobilité afin de comprendre l'affinité de liaison de LUX pour ses motifs ADN cibles obtenus à partir de tests de puces de liaison de protéines. D'après les tests de décalage de mobilité, il a été observé que le domaine de liaison à l'ADN (DBD) seul pouvait se lier aux motifs d'ADN de son substrat à des concentrations molaires nano alors qu'une quantité de protéine excédentaire de 10 fois était nécessaire pour que la longueur totale de LUX obtienne la même quantité de liaison. Des pistes de cristallisation à haut débit ont été réalisées pour LUX et LUX-DBD pleine longueur avec deux motifs de liaison différents. Aucun cristal n'a été obtenu pour le LUX complet, alors que des cristaux ont été obtenus pour le LUX DBD avec les deux motifs d'ADN. La structure de LUX DBD en complexe avec ses motifs cibles a été résolue par diffraction aux rayons X. À partir de la structure Crystal, il a été constaté que LUX DBD avait adopté une structure à 3 hélices, la seconde hélice étant responsable de la lecture de la base. Fait intéressant, il a été observé qu'un résidu d'arginine présent au niveau de l'hélice n-terminale servait de pince pour interagir avec le motif ADN cible.En utilisant les tests de mutagenèse dirigée et de décalage de mobilité, il a été confirmé que cette arginine présente à la position 146 de la protéine est essentielle pour déterminer l'affinité. Il a été constaté que l'affinité de liaison était réduite d'un facteur 5 lorsque cet acide aminé était modifié. En outre pour comprendre son effet in planta. Les lignes de lux mutantes ont été complétées par une longueur totale de type sauvage et une version substituée par Arg14Ala de la longueur totale de LUX. Grâce à ces expériences, nous avons pu montrer que la complémentation complète du mutant R146A n’était pas observée, alors que la version de type sauvage était capable de compléter complètement le phénotype du mutantAmbient temperature plays a direct role in plant functioning and development. Increase in global ambient temperatures poses a significant challenge to wild and cultivated plant species. Most plant species adjust reproductive timing and development to optimize survival and fitness in higher ambient temperatures (Barnabás et al., 2008; Fitter and Fitter, 2002; Willis et al., 2008). These adaptations generally lead to elongated hypocotyls, fewer leaves at time of flowering, accelerated transition from vegetative to reproductive growth phases, fewer seeds, smaller seed pods and decreased leaf area. In the face of rapid climate change, specifically increased ambient growth permissive temperatures, tuning plant thermoresponse is urgently needed to engineer plants for adaptation to climate change and for securing future food production.PIF4 expression is controlled by evening complex (EC), a 3 protein complex comprising of ELF3, ELF4 and LUX, in a temperature dependent manner. The EC is able to repress PIF4 expression by binding to specific motifs at the promoter of PIF4 at lower ambient growth temperature. However this repression of PIF4 expression is removed at higher ambient growth temperature leading to premature ageing of plantsResultsAll three proteins of EC were produced using different expression strategy, ELF4 and LUX in E. coli while ELF3 in insect cells. Protein purification methods and buffers were optimized to obtain stable ELF3 ELF4 and LUX. Size exclusion chromatography was done to verify oligomeric states of the proteins. Since LUX is the only protein in the EC which has known DNA binding domain, it was used for doing mobility shift assays to understand the binding affinity of LUX for its target DNA Motifs which were obtained from protein binding microarrays assays. From the mobility shift assays, it was observed that the DNA binding domain (DBD) alone could bind to its substrate DNA motifs at Nano molar concentrations while 10 fold excesses amount of protein was required for the full length LUX to obtain same amount of binding. High throughput crystallization trails were carried out for full length LUX and LUX- DBD with two different binding motifs. No crystals were obtained for full length LUX while Crystals were obtained for LUX DBD with both the DNA Motifs. Structure for LUX DBD in complex with its target motifs were solved through X-Ray diffraction. Using site directed mutagenesis and Mobility shift assays it was confirmed that this Arginine present at the 146 position of the protein is critical for determining affinity. It was found that the binding affinity was reduced by a factor of 5 when this amino acid was changed. Further to understand its effect in planta.. With this experiments we were able to show that with the R146A mutant full complementation wasn’t observed while the wildtype version was able to completely complement the mutant phenotype.To understand temperature based dynamics of the complex, ELF3, which is the most intrinsically disordered protein of the three proteins that constitute the complex, was studied for structural variation through CD spectroscopy and DLS experiments. From these experiments it was found that ELF3 attains a β-sheet like confirmation at higher temperature while a more globular confirmation at lower temperatures. It was found that this activity of ELF is reversible allowing for flexibility of the whole complex. We found that there were prion like domains in ELF3 protein which were primarily responsible for transition to β-sheet structure at higher temperature.In Order to engineer plants that could survive at higher ambient temperature, we decided to mutate promoter elements of PIF4 through CRISPR/Cas9 to obtain plants that can survive higher temperature
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Andreazza, Simonetta. "Analysis of new genes controlling Drosophila melanogaster rest-activity rhythms." Thesis, Paris 11, 2013. http://www.theses.fr/2013PA112314.
Full textAbstract:
Les mécanismes moléculaires contrôlant les rythmes circadiens sont conservés parmi les organismes des différents règnes (plantes, animaux et champignons). Ils se composent de boucles de rétroaction où un complexe d’activation transcriptionnelle, l’hétérodimère CLK/CYC chez la drosophile, entraîne l'expression des répresseurs de son activité, les gènes et protéines PER et TIM chez la mouche. De manière importante, la période de l'oscillateur dépend en grande partie par des mécanismes post-transcriptionnels qui régulent l’accumulation et l'activité des composantes positifs et négatifs de la boucle. Bien que de nombreux partenaires d'interaction modifiant les composants d'horloge de base ont déjà pu être isolés, le schéma reste encore incomplet. Dans le cadre de la recherche de nouveaux composants de cette horloge, nous avons réalisé un crible comportemental basé sur l'expression ciblée de transgènes ARNi dirigés contre la moitié du génome de Drosophila melanogaster. Cinquante-quatre nouveaux gènes putatifs ont pu être identifiés. Au cours de ce travail, j'ai étudié le rôle de deux d’entre eux, sélectionnés pour les forts défauts comportementaux de l'expression de leur transgène ARNi. Le gène CG12082 de la drosophile est l’orthologue de l’Ubiquitin-specific protéase 5 (USP5) chez l’homme. La dérégulation d’Usp5 retarde les oscillations de la protéine PER dans les neurones d'horloge et allonge la période d'activité locomotrice des mouches. Chez les mouches ARNi Usp5, des formes à haut poids moléculaire des protéines PER et TIM s'accumulent pendant le matin, alors qu’elles sont normalement dégradées chez les contrôles. On a pu montrer que Usp5 participe directement à la dégradation de la protéine PER, indépendamment de TIM. En accord avec le rôle décrit pour l’orthologue humaine, Usp5 serait susceptible de contrôler la dégradation des protéines par son activité de démontage des chaînes libres de polyubiquitine présents dans la cellule, qui peuvent entrer en compétition avec les protéines ubiquitinylées pour la reconnaissance au niveau du protéasome, bloquant leur dégradation. La majorité des travaux ont porté sur un gène isolé au cours de notre crible, Strip, dont les fonctions étaient encore inconnues. Strip interagit avec Cka, une nouvelle sous-unité régulatrice de l’enzyme phosphatase PP2A. La dérégulation à la fois de Strip et/ou de Cka amène à des phénotypes comportementaux de période longue. D’un point de vue moléculaire, des formes hyper-phosphorylées de la protéine CLK s’accumulent dans la matinée quand Cka et/ou Strip sont perturbées. La dérégulation des activités générales de PP2A produit également une hyper-phosphorylation de CLK le matin, indiquant que, grâce à Cka/Strip, les complexes PP2A contrôlent la déphosphorylation de CLK à la fin du cycle. Il est connu que les formes hyper-phosphorylés de CLK sont transcriptionnellement inactives. En effet, la transcription des gènes tim et vrille, cibles de CLK, est fortement réduite dans les mouches ARNi Cka. En plus de PP2A/Cka, des complexes PP2A contenant une autre sous-unité régulatrice, Wdb, ont été montré pour déstabiliser CLK en culture des cellules (Kim et Edery, 2006). Nous montrons que la dérégulation de Wdb affecte la stabilité du CLK également dans la mouche adulte, sans toutefois induire aucun effet apparent sur sa phosphorylation. En conclusion, deux complexes PP2A différents agissent sur la protéine CLK : le complexe PP2A/Cka/Strip contrôle la déphosphorylation de CLK et sa réactivation, tandis que PP2A/Wdb affecte la stabilité de CLK indépendamment ou après PP2A/Cka. Ces résultats enrichissent l’étude de la régulation post-traductionnelle de la protéine CLK, qui était largement mal connue.Pour conclure, cette étude a permis de décrire deux nouveaux composants de la boucle moléculaire qui contrôle les rythmes circadiens chez la mouche du vinaigre, Drosophila melanogasterThe molecular mechanism underlying circadian rhythms is conserved among organisms and consists of feedback loops where a transcriptional activating complex (the CLOCK (CLK)/CYCLE (CYC) heterodimer in Drosophila) drives the expression of the repressors of its activity (the period (per) and timeless (tim) genes and proteins in Drosophila). Importantly, the pace of the oscillator largely depends on post-transcriptional mechanisms that regulate the accumulation and activity of both the positive and negative components of the loop. A number of interacting partners that modify core clock components have already been isolated, but more are expected. Looking for new clock components, we set up a behavioral screen based on targeted expression of RNAi transgenes directed to half of the Drosophila genome. 54 putative new clock genes have been identified. Among them, some were independently reported to function within the fruit fly molecular clock, thus validating the screen. In this work, I investigated the circadian role of additional “positive” genes, selected for the strong behavioral defect induced by the expression of the corresponding RNAi. The CG12082 gene codes for the fruit fly ortholog of the human Ubiquitin-specific protease 5 (USP5). Downregulation of USP5 in clock cells lengthens the period of locomotor activity of flies as well as PER protein oscillations in clock neurons. High molecular weight forms of PER and TIM proteins accumulate during the morning after USP5 knockdown, while these forms are degraded in controls. In addition, TIM is not stabilized in the absence of PER, while PER still accumulate in the absence of TIM. Therefore, USP5 directly participates in the degradation of the PER protein and, later, of the TIM protein at the end of the cycle. Being a deubiquitinylase enzyme, USP5 may directly deubiquitinate PER. However, accordingly to the role described for the human ortholog, USP5 likely controls protein degradation through the disassembling of the unanchored polyubiquitin chains present in the cell that could compete with ubiquitinated-PER for proteasome recognition and subsequent breakdown.The majority of the work has focused on an unknown gene isolated in the screen, that, accordingly to the human homolog, we named STRIP. We show that STRIP interacts with Connector of Kinase to AP-1 (CKA), a novel regulatory subunit for the PP2A phosphatase holoenzyme, both in insect S2 cells and in fly head extracts. Downregulation of both STRIP and/or CKA causes long-period behavioral phenotypes and high molecular weight forms of the CLK protein to accumulate in the morning. Perturbation of general PP2A activities also produces hyper-phosphorylated CLK in the morning indicating that, through CKA/STRIP, PP2A complexes controls CLK dephosphorylation at the end of the cycle. Hyper-phosphorylated CLK forms are transcriptionally inactive. Accordingly, transcription of the tim and vrille (vri) CLK targets is strongly reduced in Cka-RNAi fly head extracts. PP2A complexes containing the Widerborst (WDB) regulatory subunits were already shown to affect CLK stability in insect S2 cells (Kim and Edery, 2006). We show that WDB downregulation also affects the stability of CLK in fly head extracts, but has no apparent effects on CLK phosphorylation. Therefore, we could describe two different PP2A complexes acting on the CLK protein: PP2A/CKA/STRIP complex controls CLK dephosphorylation and reactivation, while PP2A/WDB affects CLK stability independently or after PP2A/CKA functions. Moreover, STRIP, but not CKA, downregulation affects the stability of PER, indicating that STRIP possesses some functions unrelated to CKA. In conclusion, this work has allowed the isolation of new components of the Drosophila molecular clock. In particular, we give evidence for a double role for the PP2A phosphatase in modulating the activity and stability of the CLK protein, the regulation of which is not well understood yet
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Yuan, Quan. "The Circadian Clock in Monarch Butterfly: A Tale of Two CRYs: A Dissertation." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/429.
Full textAbstract:
Every fall, Northeastern America monarch butterflies (Danaus plexippus) undergo an extraordinary migration to their overwintering site in Central Mexico. During their long migration, monarch migrants use sun compass to navigate. To maintain a southward flying direction, monarch migrants compensate for the continuously changing position of the sun by providing timing information to the compass using their circadian clock.
Animal circadian clocks depend primarily on a negative transcriptional feedback loop to track time. I started my work to re-construct the monarch butterfly circadian clock negative feedback loop in cell culture, focusing on homologs of Drosophila clock genes. It turned out that in addition to a Drosophila-like cryptochrome (cry1) gene, a second mammalian-like cry2 gene exists in monarch butterflies and many other insects, except in Drosophila. The two CRYs showed distinct functions in our initial assays in cultured Drosophila Schneider 2 (S2) cells. CRY2 functions as a potent transcriptional repressor, while CRY1 is light sensitive but shows no obvious transcriptional activity. The existence of two cry genes in insects changed the Drosophila-centric view of insect circadian clock.
During the course of my study, our lab obtained a monarch cell line called DpN1 cells. These cells possess a light-driven clock and contributed tremendously to the research on monarch circadian clock. Using this cell line, I provided strong evidence supporting monarch CRY2’s role as a major circadian clock repressor and identified a protein-protein protective interaction cascade underlying the CRY1-mediated resetting of the molecular oscillator in DpN1 cells.
I continued my work trying to understand how insect CRY2 inhibits transcription. I provided evidence suggesting the involvement of monarch PER in promoting CRY2 nuclear entry in both S2 cells and DpN1 cells. Finally, I mapped CRY2’s transcriptional inhibitory activity onto its N-terminal domain.
Collectively, my research helped to change our view of insect clocks from a Drosophila-centric standpoint to a much more diverse picture. My studies also advanced the understanding of monarch circadian clock mechanism, and provides a foundation for further studies.
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Krahmer, Johanna. "Circadian abundance and modification of proteins in Arabidopsis." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/15969.
Full textAbstract:
Circadian clocks are endogenous pacemakers found in many organisms including plants, generating approximately 24h rhythms. Knowledge about the plant circadian clock plays a role for crop improvement. The plant circadian clock and its downstream outputs have been studied in detail by transcriptomics, however post-transcriptional and post-translational aspects are still to be researched. In addition, it has recently been shown that a protein modification remains rhythmic when rhythmic transcription is absent. This gives evidence for the existence of two oscillators: a transcription-translation feedback loop and a non-transcriptional oscillator. The aim of this PhD is to gain knowledge about circadian changes in abundance and phosphorylation of proteins as well as protein-protein interaction using the model plant Arabidopsis thaliana. I used high-throughput proteomics and phosphoproteomics methods to identify hundreds of phosposites that change in abundance in WT plants as well as dozens of proteins that exhibit circadian changes in their abundance. I also found significant temporal changes in protein phosphorylation in the transcriptionally arrhythmic mutant CCA1-Ox, albeit with dynamics different from the WT, demonstrating that without transcriptional rhythms, protein modification can still undergo rhythmic changes to some extent. In addition, I found reproducibly that the majority of changing phosphopeptides are most abundant at dawn and this is independent of the presence of a functional transcriptional oscillator. Roles of different kinases and affected phosphoproteins are discussed. I chose one of the rhythmically phosphorylated proteins, the bifunctional enzyme F2KP, for further functional experiments. In vitro experiments demonstrate that the rhythmic phosphosite is important for the activity of the enzyme. This is discussed in the light of circadian regulation of carbon metabolism. In addition to these studies on circadian protein abundance and modification, I investigated time-of-day dependent protein-protein interaction of the clock protein GIGANTEA (GI). Using an interaction proteomics timecourse, I identified about 100 potential new interactors of GI, some of which are candidates for links between diel timing and carbon metabolism. These results will help to generate hypotheses for explaining the surprising pleiotrophy of gi mutants.
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Korge, Sandra [Verfasser]. "A non-conventional nuclear import pathway for circadian clock proteins / Sandra Korge." Berlin : Freie Universität Berlin, 2016. http://d-nb.info/1122111142/34.
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Mei, Qiming, and 梅启明. "Molecular evolution of cryptochrome (CRY) and PAS-containing proteins in eukaryotic circadian clock." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/198833.
Full textAbstract:
Circadian rhythmsare biochemical, physiological, and behavioral processes display oscillations of oughly 24-hour, which existing in both prokaryotes and eukaryotes. Circadian rhythms improve fitness of organisms in both constant and changing environments. The cryptochrome (CRY)and PAS-containing proteins are light sensors and key elements of the circadian system in eukaryotic organisms.
Photolyases and cryptochromes are evolutionarily related flavoproteins which perform distinct physiological functions. Photolyases are evolutionarily ancient enzymes that activated by light and repairing UV-induced DNA damage. Although cryptochromes share structural similarity with the DNA photolyases, they lack the DNA repair activity. CRYs are key elements of mammal circadian system, and play roles in light sensing in insects and plants to entrain circadian rhythms.
The PAS domains are widely distributed in proteins across all kingdoms of life and act as signal modules. They are common in photoreceptors and transcriptional regulators of eukaryotic circadian clock components including bHLH-PAS proteins (BMAL, CYC,CLK and NPAS2) and PER in animals, PHY and ZTL in plants, WC-1, 2and VVD in fungi. They are mainly involved in protein-protein interaction and light sensing functions.
The CRY/PHR superfamily consists of 7 major subfamilies: CPD class I and CPD class II PHRs, (6-4) PHR, CRY-DASH, plant PHR2, plant CRY and animal CRY. Although the superfamily evolved primarily under strong purifying selection (average ω = 0.0178), it experienced strong episodic positive selection at some periods of evolution. The level of variation is subfamily-and domain-specific. The homologs with apparent circadian functions (i.e., plant and animal CRY) are significantly more conserved than the other photolyases. Photolyases were lost in eukaryotic groups like placental mammals, suggesting that natural selection apparently became weaker in the late stage of evolutionary history.
The phylogenetic trees of fish Cry features two major clusters, which correspond to Cry1and Cry2. Teleost species possess extra copies of Cry1 due to fish-specific genome duplication (FSGD), and formed 3 clades of Cry1. Clade1B of Cry1(π= 0.129 ±0.062) is more conserved than the other paralogs (πrange from 0.173to 0.195). Test of positive selection revealed that fish cryptochromes evolved under strong purifying selection (average ω= 0.0066).Different fishes preserved different Cry duplicates that associated with reciprocal gene loss, thus generated the diverse circadian molecular mechanisms.
The level of DNA variation in the PAS-containing proteins appears to be subfamily-specific. The animal PAS-containing homologs are more polymorphic than the plant and fungal homologs. Although the whole superfamily evolved primarily under strong purifying selection (average ω range from 0.0030to 0.1164), it experienced strong positive selection at some periods of the evolution. Although the PAS domains from different proteins vary in sequence and length, they maintain a fairly conserved 3D structure. The 3D fold of PAS domains is determined by only 8 conserved residues which shared by all subfamilies.
The evolutionary time estimates showed that plant and animal Cry, WC-1& 2, bHLH-PAS proteins and Per originated in the Neoproterozoic Era (~1000 –542 Mya), plant Phy and ZTL evolved in the Paleozoic (541 –252 Mya), which might be a result of adaptation to the global climate and light regime changes.published_or_final_version
Biological Sciences
Doctoral
Doctor of Philosophy
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Karlgren, Anna, Niclas Gyllenstrand, Thomas Källman, and Ulf Lagercrantz. "Conserved function of core clock proteins in the gymnosperm Norway spruce (Picea abies L. Karst)." Uppsala universitet, Växtekologi och evolution, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-192151.
Full textAbstract:
From studies of the circadian clock in the plant model species Arabidopsis (Arabidopsis thaliana), a number of important properties and components have emerged. These include the genes CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), GIGANTEA (GI), ZEITLUPE (ZTL) and TIMING OF CAB EXPRESSION 1 (TOC1 also known as PSEUDO-RESPONSE REGULATOR 1 (PRR1)) that via gene expression feedback loops participate in the circadian clock. Here, we present results from ectopic expression of four Norway spruce (Picea abies) putative homologs (PaCCA1, PaGI, PaZTL and PaPRR1) in Arabidopsis, their flowering time, circadian period length, red light response phenotypes and their effect on endogenous clock genes were assessed. For PaCCA1-ox and PaZTL-ox the results were consistent with Arabidopsis lines overexpressing the corresponding Arabidopsis genes. For PaGI consistent results were obtained when expressed in the gi2 mutant, while PaGI and PaPRR1 expressed in wild type did not display the expected phenotypes. These results suggest that protein function of PaCCA1, PaGI and PaZTL are at least partlyconserved compared to Arabidopsis homologs, however further studies are needed to reveal the protein function of PaPRR1. Our data suggest that components of thethree-loop network typical of the circadian clock in angiosperms were present beforethe split of gymnosperms and angiosperms.
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Sarma, Ashapurna. "Circadian Timing of Curcumin Efficacy and Nuclear Transport Properties of Cancer Cells." Bowling Green State University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1447971823.
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Wiegard, Anika. "Zeitliche Koordination in Cyanobakterien." Doctoral thesis, Humboldt-Universität zu Berlin, Lebenswissenschaftliche Fakultät, 2015. http://dx.doi.org/10.18452/17252.
Full textAbstract:
Das Cyanobakterium Synechococcus elongatus PCC 7942 besitzt eine circadiane Uhr, die aus nur drei Proteinen besteht: KaiA, KaiB und KaiC. Durch 24stündige Phosphorylierungs- und ATPase-Zyklen des KaiC wird u. a. die globale Genaktivität gesteuert. Der Anteil circadian regulierter Gene sowie die Zahl und Organisation der kai-Gene scheinen in Cyanobakterien stark zu variieren. Um die Komponenten eines potenziell komplexeren Kai-Systems zu untersuchen, wurde in der vorliegenden Arbeit Synechocystis sp. PCC 6803 als Modell ausgewählt. In dessen Genom werden ein KaiA- sowie jeweils drei divergierte KaiB- und KaiC-Proteine kodiert. Durch in vitro Studien konnte die Aktivität von KaiC1 und KaiC3 erstmals charakterisiert werden: KaiC1 zeigte eine KaiA-abhängige Kinase-Aktivität und bildet mit KaiA und KaiB1 vermutlich einen „Standard-Oszillator“. KaiC3 wies die typischen Kinase-, ATP-Synthase- und ATPase-Aktivitäten des KaiC aus Synechococcus auf. Deren Ausprägung erschien jedoch modifiziert. Ferner wurde die zeitliche und räumliche intrazelluläre Verteilung des KaiA sowie der KaiC-Proteine aufgeklärt. Die Kai-Proteine verhielten sich insgesamt abweichend von den Homologen aus Synechococcus, was das Fehlen einer circadianen Rhythmik unter den gewählten Wachstumsbedingungen erklärt. Angesichts kontroverser Diskussionen über die molekularen Details der Assemblierung von KaiC und KaiB aus Synechococcus wurde in einem ergänzenden Projekt demonstriert, dass die gesteigerte Phosphorylierung des KaiC bei 4°C zur Bildung stabiler KaiC-KaiB-Komplexe führt. Die dabei etablierte Methode erlaubt Untersuchungen der KaiC-KaiB-Interaktion unter Verwendung der Wildtyp-Proteine.The cyanobacterium Synechococcus elongatus PCC 7942 harbors a circadian clock consisting of only three proteins: KaiA, KaiB and KaiC. 24hour phosphorylation and ATPase cycles of KaiC control global gene activity. The number of circadian regulated genes as well as the number and organization of kai-genes seem to vary strongly among cyanobacteria. To analyze the components of a probably more complex Kai-system, Synechocystis sp. PCC 6803 was chosen as a model in the present study. Its genome encodes one KaiA- and each three KaiB and KaiC proteins. The activity of KaiC1 and KaiC3 was – for the first time - characterized by in vitro studies: KaiC1 displayed a KaiA-dependent kinase activity and builds a ,standard oscillator‘ together with KaiA and KaiB1. KaiC3 displayed the typical kinase, ATP synthase and ATPase activities of KaiC from Synechococcus. However, the characteristics of the activities appeared to be modified. Moreover, the temporal and spatial intracellular distribution of KaiA and the KaiC proteins was elucidated. Altogether, the Kai proteins performed different from their Synechococcus homologs, explaining the lack of circadian rhythms under the chosen growth conditions. In view of the controversial discussions about the assembly of KaiC and KaiB from Synechococcus, an additional project was set up to demonstrate that increased auto-phosphorylation of KaiC at 4 °C leads to the formation of stable KaiC-KaiB-complexes. In this context, a protocol was established that allows to analyse KaiC-KaiB interactions using wild-type proteins.
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Yu, Elizabeth A. "Investigating Age-Dependent Arthropathy in a Circadian Mutant Mouse Model: A Dissertation." eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/544.
Full textAbstract:
Ectopic calcification can cause pain and limit mobility. Studies suggest that circadian genes may play a role in the calcification process. Core circadian genes Clock, Npas2, and Bmal1 are transcription factors that form CLOCK:BMAL1 or NPAS2:BMAL1 transactivator complexes that drive the rhythmic expression of circadian oscillator genes and output genes. Circadian oscillator genes Period1-3 and Cryptochrome1-2 encode proteins that form transcription repressor complexes that feedback to inhibit CLOCK/NPAS2:BMAL1 activity, thus completing the feedback loop that is the basis of the molecular circadian clockwork. Arrhythmic Bmal1-/- mice exhibit site-specific, age-dependent arthropathy. While studying the circadian phenotype of Clock-/-;Npas2m/m double mutant mice, we discovered that these double mutant mice develop site-specific arthropathy similar to the arthropathy described in Bmal1-/- mice. Based on the circadian clockwork mechanism, we hypothesized that CLOCK/NPAS2:BMAL1 transactivator complexes drive the expression of a gene (or genes) that prevents age-dependent arthropathy. To investigate Clock-/-;Npas2m/m double mutant mouse arthropathy, we evaluated mutant mice using X-ray, micro-computed tomography, and histology, and found that Clock-/-;Npas2m/m double mutant mice exhibit age-dependent, site-specific arthropathy that phenocopies that of Bmal1-/- mice. The costosternal junction and calcaneal tendon are most prominently affected, in that calcification of those tissues is detectable as early as 4-5 weeks and 11-12 weeks, respectively. The arthropathic lesions in these tissues consist of calcium phosphate vii deposits, and in Bmal1-/- costosternal junction calcifications, the deposits contain calcium pyrophosphate dihydrate crystals. Mechanical stress, disregulation of centrally-regulated circadian rhythms, and systemic serum mineral imbalances likely do not contribute to this pathology. In vitro micromass cultures generated from Clock-/-;Npas2m/m double mutant mouse embryonic fibroblasts do not exhibit irregular chondrocyte differentiation compared to wild-type cultures, suggesting that chondrocyte cell-autonomous mechanisms are insufficient to induce this arthropathy. Analysis of Clock-/-;Npas2m/m double mutant intersternebral tissue RNA did not reveal significant changes in chondrocyte or calcification-related gene expression. Histological stains showed an absence of osteoblasts and osteoclasts around costosternal junction calcifications, suggesting that these cell types are not contributing to this pathology. Instead, chondrocytes are localized to the costosternal junction but there were no significant changes in the distribution of chondrocyte markers in this tissue, as evaluated by immunohistochemistry. These findings suggest that Clock or Npas2, and Bmal1, regulate ectopic calcification through a combination of systemic and local factors, and that the cells affected by Clock and Npas2, or Bmal1, disruption are a subset of the cells distributed in specific tissues that develop age-dependent arthropathy. The significance of these findings is that “circadian genes” play a role in the regulation of ectopic calcification in a non-oscillator capacity. Understanding this new mechanism by which ectopic calcification is controlled could lead to novel approaches for the treatment of some human calcification diseases.
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Schöning, Jan Christoph [Verfasser]. "Molecular and functional characterisation of circadian clock regulated RNA-binding proteins from Arabidopsis thaliana / Jan Christoph Schöning." Bielefeld : Universitätsbibliothek Bielefeld, 2021. http://d-nb.info/1235664236/34.
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Murad, Alejandro D. "Molecular and Neuronal Analysis of Circadian Photoresponses in Drosophila: A Dissertation." eScholarship@UMMS, 2007. https://escholarship.umassmed.edu/gsbs_diss/357.
Full textAbstract:
Most organisms, from cyanobacteria to humans are equipped with circadian clocks. These endogenous and self-sustained pacemakers allow organisms to adapt their physiology and behavior to daily environmental variations, and to anticipate them. The circadian clock is synchronized by environmental cues (i.e. light and temperature fluctuations).
The fruit fly, Drosophila melanogaster, is well established as a model for the study of circadian rhythms. Molecular mechanisms of the Drosophilacircadian clock are conserved in mammals. Using genetic screens, several essential clock proteins (PER, TIM, CLK, CYC, DBT, SGG and CK-II) were identified in flies. Homologs of most of these proteins are also involved in generating mammalian circadian rhythms. In addition, there are only six neuronal groups in the adult fly brain (comprising about 75 pairs of cells) that express high levels of clock genes. The simplicity of this system is ideal for the study of the neural circuitry underlying behavior.
The first half of this dissertation focuses on a genetic screen designed to identify novel genes involved in the circadian light input pathway. The screen was based on previous observations that a mutation in the circadian photoreceptor CRYPTOCHROME (CRY) allows flies to remain rhythmic in constant light (LL), while wild type flies are usually arrhythmic under this condition. 2000 genes were overexpressed and those that showed a rhythmic behavior in LL (like crymutants) were isolated. The candidate genes isolated in the screen present a wide variety of biological functions. These include genes involved in protein degradation, signaling pathways, regulation of transcription, and even a pacemaker gene. In this dissertation, I describe work done in order to validate and characterize such candidates.
The second part of this dissertation focuses on identifying the pacemaker neurons that drive circadian rhythms in constant light (LL) when the pacemaker gene period is overexpressed. We found that a subset of pacemaker neurons, the DN1s, is responsible for driving rhythms in constant light. This attractive finding reveals a novel role for the DN1s in driving behavioral rhythms under constant conditions and suggests a mechanism for seasonal adaptation in Drosophila.
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TWINKLE. "IN SILICO EXPLORATION FOR REPURPOSING BREAST CANCER MEDICATIONS IN SLEEP AND NEURODEVELOPMENTAL DISORDERS." Thesis, 2023. http://dspace.dtu.ac.in:8080/jspui/handle/repository/19922.
Full textAbstract:
Sleep is crucial neurobiological behavior that serves a significant part in maintaining
brain homeostasis and functioning. It is controlled by two main mechanisms:
Homeostatic regulation, which involves the accumulation and dissipation of sleep
pressure based on prior wakefulness, and Circadian regulation, which follows a 24-hour
cycle synchronized with environmental cues. This article employs bioinformatics
software and utilizes databases commonly used in computer science and biology to
investigate the potential interaction between CRY1, CLOCK, and PER1/PER2 in drug
binding. CRY1 (Cryptochrome 1) and PER1/PER2 (Period 1 and Period 2) are genes
involved in the molecular circadian clock. Understanding their interaction and potential
drug binding can provide valuable insights into the molecular mechanisms underlying
sleep regulation.
The bioinformatics software and databases aid in predicting and analyzing the structural
and functional aspects of CRY1, CLOCK, and PER1/PER2 interactions. By utilizing
computational approaches, this research aims to uncover potential binding sites, identify
key residues involved in the interaction, and predict the binding affinity between drugs
and these proteins. By exploring the drug-binding potential of CRY1, CLOCK, and
PER1/PER2, this study seeks to contribute to understanding the intricate molecular
processes underlying sleep regulation. The findings have the potential to inform the
development of targeted therapeutics and interventions aimed at modulating sleep
patterns and addressing sleep-related disorders.
vi
Cyto Hubba, Bio via, Open babel, Drug bank, Avogadro, Auto dock, PyRx, and Protein protein interaction clustering String are several computational techniques implemented
in the investigation. The CRY1/CRY2 cytochrome genes are crucial for the circadian
rhythm. CRY1 is a stronger repressor of clock: BMAL1 as compared to CRY2. It has
been recently discovered CRY1 by creating a transcriptional inhibitor enhances its
repressive function that results from the lengthening of Circadian rhythm in humans.
The Association of PER2 with CRY2 forms a stable complex with CLOCK: BMAL1
whereas in the case of CRY1, there is no need for PER1/PER2 to bind with CLOCK:
BMAL1. Methylation in PER1/PER2 promoter is one of the variables that increase the
chance of breast cancer. Trastuzumab is used for the treatment of breast cancer by
repairing cell division and repair. The present investigation used Molecular Docking of
Trastuzumab as a control to analyze the interaction between CRY1 and PER1/PER2
applying Cyto Hubba as well as PPI clustering relationships or other breast cancer FDA approved drugs i.e., (Dichloroplatinum, Anastrozole, Exemestane, Fluvestrant,
Lapatinib Ditosylate, Letrozole, Olaparib, Palbociclib, and Talazoparib Tosylate) and
Circadian clock protein complex. Exemestane can be seen as a possible medication to
treat sleep disturbances because of its highest binding energy as compared to others. The
results suggest that Exemestane and Circadian clock protein complex laboratory trials
determine its inhibitory potential on CLOCK to minimize sleep problems.
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Voytsekh, Olga [Verfasser]. "The function of the RNA-Binding protein CHLAMY1 in the circadian clock and its temperature integration process / von Olga Voytsekh." 2008. http://d-nb.info/992336732/34.
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Al-Safadi, Sherin. "The regulation of stress-induced changes in the expression of the circadian clock protein PERIOD1, in the mammalian limbic forebrain and hypothalamus." Thesis, 2014. http://spectrum.library.concordia.ca/978510/1/Al%2DSafadi_PhD_S2014.pdf.
Full textAbstract:
Most organisms have developed internal mechanisms, including the circadian and stress systems, to allow for anticipation of and adaption to regular and unpredictable changes in the environment. The circadian and stress systems communicate constantly with one another; the circadian control of the release of effectors of the stress system, such as glucocorticoid hormones, is well documented, but the processes that govern how stressful events disrupt circadian rhythmicity are less understood. Here, we sought to elucidate these cross-talk mechanisms, by demonstrating that the expression of the circadian clock protein PER1, in the mammalian forebrain is strongly modifiable by stress. Throughout our work, the light-sensitive master pacemaker, the suprachiasmatic nucleus (SCN), remained immune to the effects of all stress manipulations. We first established that categorically different acute stressors distinctively modulate the expression of PER1 and the neuronal activity marker FOS. Systemic stressors increased protein expression in the piriform cortex, paraventricular and dorsomedial nuclei, as well as in the central extended amygdala. Contrastingly, processive stressors increased protein levels in all regions except for in the central extended amygdala, where protein expression was uniquely suppressed. Interestingly, the emotional state of fear, a complex processive stressor, increased PER1 expression in this region, an effect characteristic of systemic stress. Furthermore, we determined that the time of day and modality of stress exposure are vital factors that influence PER1 activity. We then explored the role of glucocorticoids and glucocorticoid receptors (GR) in the modulation of stress-induced PER1, using manipulations that included adrenalectomy and pharmacological blockade of GR. We found that stress-induced PER1 expression in all regions studied, aside from the piriform cortex and SCN, are dependent on glucocorticoid signaling. In summary, the results demonstrate that stress, through the modulatory action of glucocorticoids and GR, can alter circadian clock protein expression in select forebrain and hypothalamic nuclei, possibly leading to their functional dysregulation and subsequent disturbances in circadian physiology and behavior. Our findings allude to a novel functional role for the circadian protein PER1 as an intermediary between the circadian system and systems that dictate emotional states, in the mammalian brain.
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Dong, Guogang. "Cellular Function and Localization of Circadian Clock Proteins in Cyanobacteria." Thesis, 2008. http://hdl.handle.net/1969.1/ETD-TAMU-2008-12-197.
Full textAbstract:
The cyanobacterium Synechococcus elongatus builds a circadian clock on an
oscillator comprised of three proteins, KaiA, KaiB, and KaiC, which can
recapitulate a circadian rhythm of KaiC phosphorylation in vitro. The molecular
structures of all three proteins are known, and the phosphorylation steps of
KaiC, the interaction dynamics among the three Kai proteins, and a weak
ATPase activity of KaiC have all been characterized. A mutant of a clock gene in
the input pathway, cikA, has a cell division defect, and the circadian clock
inhibits the cell cycle for a short period of time during each cycle. However, the
interaction between the circadian cycle and the cell cycle and the molecular
mechanisms underlying it have been poorly understood. In addition, the
subcellular localization of clock proteins and possible localization dynamics,
which are critical in the timing circuit of eukaryotic clock systems and might also
shed light on the interaction between circadian cycle and cell cycle, have remained largely unknown. A combination of genetics, cell biology, and
microscopy techniques has been employed to investigate both questions.
This work showed that the cell division defect of a cikA mutant is a function of
the circadian clock. High ATPase activity of KaiC coincides with the inhibition of
cytokinesis by the circadian clock. CikA likely represses KaiC's ATPase activity
through an unknown protein, which in cikA's absence stimulates both the
ATPase and autokinase activities independently of KaiA or KaiB. SasA-RpaA
acts as an output in the control of cell division, and the localization of FtsZ is the
target, although it still remains to be seen how RpaA, directly or indirectly,
inhibits FtsZ localization.
The project also showed that clock proteins are localized to the cell poles.
KaiC is targeted to the cell pole in a phosphorylation-dependent manner. KaiB
and CikA are also found at the poles independently of KaiC. KaiA likely only
localizes to the cell pole during the dephosphorylation phase, which is
dependent on both KaiB and KaiC, specifically on the phosphorylation of KaiC at
S431.
Overall, significant progress was made in both areas and this project sheds
light on how the circadian oscillator operates in cyanobacterial cells and
interacts with another fundamental cellular function.
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岩崎, 秀雄, and Hideo Iwasaki. "Identification and Functional Analyses of the Circadian Clock Proteins in Cyanobacteria." Thesis, 1999. http://hdl.handle.net/2237/17078.
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Schalie, Ellena A. van der. "Structure/function analyses of the cryptochrome proteins in the molecular circadian clock /." 2008. http://wwwlib.umi.com/dissertations/fullcit/3322484.
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Wang, Ying, and 王瑛. "Molecular studies of clock proteins, LWD1 and LWD2, in Arabidopsis circadian rhythm." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/31229142866848053018.
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博士國防醫學院
生命科學研究所
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In plants, circadian clock could control day-length-dependent developmental processes such as photoperiodic flowering. The Arabidopsis circadian clock is formed by several negative feedback loops composed of oscillator genes expressing at specific time during a day. The identification of additional clock genes will help to better dissect the complex nature of the circadian clock. Here we show light-regulated WD repeats protein 1 (LWD1) and LWD2 are new clock genes regulating photoperiodic flowering and circadian period length. LWD1 and LWD2 proteins share 91.4 % identity in amino acid sequence. The lwd1 lwd2 double mutant has an early flowering phenotype, and a short period length for its internal clock under free running condition. Analysis of the lwd1 lwd2 double mutant also revealed that LWD1/2 plays dual functions in the light input pathway and the regulation of the central oscillator. Promoter:luciferase fusion studies showed that transcriptional activities of LWD1/2 are rhythmic and depend on functional PRR9 and PRR7. LWD1/2 is also needed for the expression of PRR9, PRR7 and PRR5. LWD1 is preferentially localized within the nucleus and associates with promoters of PRR9, PRR5 and TOC1 in vivo. Our results support the existence of a positive feedback loop within the Arabidopsis circadian clock. Further mechanistic studies of this positive feedback loop and its regulatory effects on the other clock components will further elucidate the complex nature of the Arabidopsis circadian clock.
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Mou, Xiang. "Fibrinolytic Proteins and Brain-Derived Neurotrophic Factor Modulation of Suprachiasmatic Nucleus Circadian Clock." 2010. http://trace.tennessee.edu/utk_graddiss/830.
Full textAbstract:
Mammalian circadian rhythms are controlled by a clock located in the suprachiasmatic nucleus (SCN). The mechanisms through which light phase-shifts the SCN circadian clock are similar to those underlying memory formation and long-term potentiation (LTP). Several secreted proteins, including tissue-type plasminogen activator (tPA), plasminogen, and brain-derived neurotrophic factor (BDNF), have been implicated in this process. These same proteins are important for photic phase-shifts of the SCN circadian clock. Early night glutamate application to SCN containing brain slices resets the circadian clock. Our experiments find that the endogenous tPA inhibitor, plasminogen activator inhibitor 1(PAI-1), blocked these shifts in slices from wildtype mice but not mice lacking its stabilizing protein, vitronectin (VN). Plasmin, but not plasminogen, prevented inhibition by PAI-1. Both plasmin and active BDNF reversed alpha2-antiplasmin inhibition of glutamate-induced shifts. alpha2-Antiplasmin decreased the conversion of inactive to active BDNF in the SCN. Both tPA and BDNF allowed daytime glutamate-induced phase-resetting. Together, these data are the first to demonstrate expression of these proteases in the SCN, their involvement in modulating photic phase-shifts, and their activation of BDNF in the SCN, a potential ‘gating’ mechanism for photic phase-resetting.
Using western-blot analyses of SCN tissue maintained in vitro, we find higher tPA, plasmin and mBDNF levels in the SCN at night vs. the day. Also, in vitro glutamate treatment of SCN tissue during early night increases tPA levels to ~2.5 times control levels, while similar treatments during late night and mid-day do not alter tPA expression. Glutamate treatment in the early night does not alter PAI-1, plasmin and BDNF levels. Co-treatment with glutamate and PAI-1 decreases plasmin levels (vs. glutamate treatment alone), while co-treatment with glutamate and alpha2-antiplasmin decreases the amount of pro- and mBDNF in the SCN relative to glutamate treatment alone. We also show that mBDNF levels are significantly lower in tPA knockout mice during both day and night. Together, these results support circadian clock modulation of BDNF and fibrinolytic protein levels in the SCN. They also suggest that glutamate modulates tPA expression in the SCN, while tPA and plasmin modulate BDNF expression.
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Nadeef, Seba S. "Polycomb PRC2-Ezh1 cell memory system in circadian clock and diet induced cellular stress regulation in mammalian skeletal muscle." Diss., 2019. http://hdl.handle.net/10754/660378.
Full textAbstract:
The majority of our physiological and metabolic processes are coordinated by an internal clock, which has evolved as an adaptive response to the daily light-dark cycles. Thus, several physiological and behavioral activities display an oscillatory rhythmic period of 24 hours. This highly conserved molecular mechanism is achieved through a specific program of gene expression, characterized by a complex interaction between clock-core proteins, chromatin remodelers and epigenetic events associated with the oscillatory nature of circadian transcriptional activity in the genome. Clock disruption leads to a wide spectrum of severe health problems including chronic metabolic disorders, muscle waste and cardiopathies. Previous studies revealed that each cell and organ possess an intrinsic clock and that coordination between central versus peripheral clocks is key for health. Furthermore, it has been found that under nutritional challenge such as High Fat Diet (HFD), the circadian transcriptome and metabolome are rapidly remodeled in the mouse model. Surprisingly, metabolome and gene expression analysis on various tissues revealed that skeletal muscle is the most affected under HFD.
Mechanisms that regulate circadian cycle and stress induced rapid adaptation and in particular metabolic stress at the chromatin level are largely unknown. In this study, we investigated the role of Polycomb proteins group (PcG) mediate cell memory system by maintaining transcriptional gene silencing, in particular the PRC2-Ezh1. We hypothesized that Ezh1 could play an important role in circadian clock regulation in post-mitotic skeletal muscle, and this pathway has never been explored in this context. We explored the circadian role of PRC2-Ezh1 in the mouse skeletal muscle. Intriguingly, we found that the oscillatory profile of a novel isoform of Ezh1 (Ezh1beta), localized specifically in the cytoplasm and controlling stress induced nuclear PRC2 activity, was completely disrupted under HFD. More interestingly, the circadian pattern of core clock components was impaired in Ezh1 depleted cells. Our data unveils an interesting physiological role of the PcG memory system, from cytoplasm to chromatin, which could indicate a new link between the chromatin remodeler Polycomb proteins and the endogenous clock in adaptation mechanism in skeletal muscle.
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Winter, Sherry Lynn. "Genetic and functional characterization of the interaction of BRCA1 with the serine/threonine phosphatase, PP1, and the circadian clock proteins, Per1 and Per2 /." 2006. http://link.library.utoronto.ca/eir/EIRdetail.cfm?Resources__ID=442638&T=F.
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Conte, Francesca Erika. "Biochemical studies of the clock proteins BMAL1 and CRYs in Xenopus : regulation of the CRYs subcellular localization and implications on their role in the modulation of the circadian loop /." 2005. http://wwwlib.umi.com/dissertations/fullcit/3169665.
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Hsu, Po-Hung, and 徐鉑泓. "Use Proximity-dependent biotinylation to identify C-terminal interacting proteins of mouse Cryptochrome 1 and to characterize their roles in the regulation of transcription-translation feedback loop of circadian clock." Thesis, 2019. http://ndltd.ncl.edu.tw/cgi-bin/gs32/gsweb.cgi/login?o=dnclcdr&s=id=%22107NCHU5107005%22.&searchmode=basic.
Full textAbstract:
碩士國立中興大學
生物化學研究所
107
CRY is an important transcriptional regulator in the transcription-translation feedback loop of circadian rhythm. In mammalian cells, CRY transcription is activated by the CLOCK::BMAL1 transcription factor, and the production protein CRY can alone or form a heterodimer with another protein PER to repress the transcriptional activty of the circadian rhythm core proteins CLOCK and BMAL1 to achieve the feedback mechanism. CRY is one of a photolyase/cryptochrome family protein. The main difference from other member proteins is the sequence and length of the C-terminus. The major different from CRY1 and CRY2 in mouse and human is also the sequence at the C-terminus. It is known that the deletion of the C-terminal sequence does not affect the ability of CRY to repress CLOCK::BMAL1 complex, but its research is very limited. We hope to utilize the identification C-terminal interaction proteins to explore their effects on CRY1. The Biotin Labeling System (BioID) uses engineered biotin ligase to activate biotin to label surrounding proteins. The advantage is that the affinity of biotin and avidin enables the interacting proteins to be purified under the stronger conditions. In HEK293T cells, we expressed this biotin ligase at the C-terminus of CRY1 and Confirm the biotin ligase activity of the fusion protein. Then using the Dual-Luciferase Assay to confirm the CRY1 maintained the activity of repressing CLOCK::BMAL1 after BioID binding to C-terminus. And then using this fusion protein to express in the CPN_KO cells which deficient in CRY, PER and NR1D genes to rule out possible indirect affects. By using the detection of biotin-labeled proteins and qPCR to analysis the CLOCK: BMAL downstream gene Dbp expression to confirmed that the mark of the intracellular fusion protein and the activity of the transcriptional repressor still exist. At present, it is possible to label peripheral proteins near to CRY1 using BioID method by cell nuclear fraction treatment, and to distinguish many different kinds and sizes of proteins with a certain binding ability. And then, this protein will be further analyzed and purified as a target, and analyzed by mass spectrometry to study the interaction of its protein with CRY1 and its identity.