Light and phytohormones, such as cytokinins, have been known to play a pivotal role in numerous physiological processes in plant cells. Previous work in our laboratory has revealed the light- and cytokinin- modulated changes both in the levels of specific tRNA species and their modified nucleotide contents, in addition to the characterization of specific tRNAs and tRNA genes from higher plants. The plant hormone cytokinin, which is of particular interest to us has been implicated to be involved in processes such as induction of cell division, plastid biogenesis and delay of senescence. Ongoing work in our laboratory also points towards the role of Ca2+ as a second messenger in cytokinin mediated gene expression. With the objective of isolation of specific tRNA genes which could then be used as probes to study the light- and phytohormone- induced changes in the levels of respective functional mature tRNAs, a previously isolated clone containing a 6.6kb insert that hybridized with 3 end labeled cucumber total cellular tRNA was sequenced by the dideoxy chain termination method. Sequence analysis of the 6.6 kb DNA fragment has revealed a chloroplast genome DNA fragment containing the trnNGUU and trnRACG genes in addition to the genes coding for the ribosomal RNAs 4.5S, 5S and 23S as well as the protein coding genes ccsA (cytochrome c-synthesis) and ndhD(NADH plastoquinone oxidoreductase).These genes were found to be arranged in the order-23S-4.5S-5S-trnRACG-trnNGUU-ccsA-ndhD. This shows a divergence from the gene organization in the completely sequenced chloroplast genomes of other higher plant species such as tobacco, maize, rice and Arabidopsis, especially with regard to the absence of a highly conserved trnLUAG gene that has been shown to be present in the trnNGUU-ndhD intergenic region. The cucumber chloroplast trnNGUU and trnRACG genes have shown very high homology (>90%) whereas ccsA and ndhD show 50-61% similarity to corresponding genes from chloroplast genomes of other plant species. The relative levels of tRNAArg and tRNAAsn were determined by Northern analysis using the tRNA gene probes, in etiolated excised cucumber cotyledons treated with light or phytohormones, such as cytokinin (BA) and auxin (2,4-D). Light and phytohormones were found to significantly increase the levels of tRNAArg unlike in the case tRNAAsn where no significant changes in the levels were observed. This result points towards the regulation of relative levels of specific tRNA species by light and cytokinin so as to match the codon usage of the mRNA population during light- and cytokinin- induced plant development in cucumber. Northern analyses were also performed to monitor the relative transcript levels of the plastid encoded ccsA and ndhD in etiolated excised cucumber cotyledons treated with light or phytohormones. ccsA transcript levels were found to be significantly reduced in auxin treated cucumber cotyledons where as exogenous application of cytokinin to either dark-grown or light exposed cotyledons did not seem to have any pronounced effect. ndhD transcripts were found to be up-regulated by cytokinin treatment or light exposure in comparison to un-treated controls probably indicating a point of overlap in the light/ cytokinin mediated signal transduction pathways. Auxin treatment on the other hand was found to down-regulate ndhD transcript levels also. Recent studies from our laboratory have demonstrated the involvement of a calcium-dependent protein kinase(CDPK) in the cytokinin-signal transduction pathway associated with the induction of pathogenesis-related proteins (chitinase and β 1-3 modulation of nuclear-encoded CDPK transcripts in response to light and exogenously added phytohormones such as cytokinins and auxin. Towards this end, partial CDPK cDNAs were generated from Cucumis Sativus by RT-PCR using degenerate primers designed based on the conserved regions of the known CDPK proteins available in the database, cloned in pGEM-T and sequenced. Sequence analysis of twenty partial cDNA clones revealed the presence of at least four CDPK isoforms in Cucumis sativus (CuCDPK 1-4). Of the four partial CDPK cDNAs, the tissue-specific expression level of CuCDPK3 was studied using the highly sensitive Taqman Analysis (Quantitative RT-PCR). The results obtained indicate that, in excised dark-grown cucumber cotyledons light and cytokinin were found to up-regulate the levels of CuCDPK3 unlike auxin, which was found to have no significant effect. In cucumber hypocotyls, which had the highest levels of CuCDPK3, light was found to have a down-regulatory effect whereas cytokinin and auxin did not bring about any significant changes in the levels of CuCDPK3. In cucumber root tissue, both light and cytokinin were found to have a down-regulatory effect on the levels of CuCDPK3, unlike auxin. The southern analysis of cucumber genomic DNA revealed a CDPK multi-gene family in cucumber. Since cytokinins have been known to play a role in both etioplast and chloroplast biogenesis and since various groups have recently reported the presence of higher plant homologues of bacterial cell-division protein FtsZ and the requirement of plant nuclear-encoded FtsZs for plastid division, efforts were also made to isolate and to study the expression of cucumber FtsZ in dark-grown cucumber cotyledon tissue treated exogenously with light/phytohormones. Towards this end, a partial FtsZ cDNA was generated from cucumber by RT-PCR using degenerate primers designed based on conserved regions of known plant FtsZ proteins. Results of the Taqman Analysis indicate that cytokinin, unlike auxin, mimics the action of light by increasing the levels of CuFtsZ transcripts in dark-grown cotyledon tissue suggesting the involvement of FtsZ in cytokinin-induced plastid-biogenesis.

Calcium ion plays a pivotal role as second messenger during signal/response coupling in plant cells (Trewavas, 1999). Elevations of cytosolic Ca2+ occur in plants as a consequence of abiotic and biotic stresses, environmental and hormonal stimuli. However, the molecular mechanism by which changes in cytosolic calcium are sensed and transduced in the plant cell has not been completely elucidated. The detection of Ca2+-binding proteins, especially Ca2+-dependent protein kinases (CDPKs) in plants led to drawing analogy with animal systems wherein the Ca2+-message is perceived and transduced by proteins that bind Ca2+. CDPKs are stimulated by the direct binding of Ca2+ to their endogenous calmodulin (CaM) -like domain (Harper et al, 1991). CDPKs exist as multiple isoforms in a single species, and show tissue-specific and developmentally regulated expression. Furthermore, the diversity among different CDPK isoforms with respect to Ca2+-binding properties, activation, substrate specificity, regulatory mechanisms and other kinetic properties suggest their specialization in the regulation of distinct signaling pathways. These observations therefore have led to the speculation that most of the Ca2+-mediated signal transduction in plants occurs via the mediation of CDPKs (Harmon et al, 2000). Over the last 15 years there has been a dramatic unfolding of information on Ca2+-mediated signaling in plants. Nevertheless, little is known about the environmental/hormonal signals and the signaling events that regulate early plant developmental processes such as embryogenesis, seed development and germination. The present investigation was initiated with the objectives 1) to determine the role of Ca2+ during embryogenesis, 2) to examine the involvement of a CDPK during early developmental processes in sandalwood plant (Santalum album L.) and 3) to purify and biochemically characterize this CDPK. The study initially investigated the possible involvement of calcium-mediated signaling in the induction/regulation of somatic embryogenesis from proembryogenic cells of sandalwood. 45 Ca + uptake studies and fura-2 fluorescence ratio photometry were used to measure changes in [Ca2+]cyt of proembryogenic cells in response to culture conditions conducive for embryo development. Sandalwood proembryogenic cell masses (PEMs) were obtained in the callus proliferation medium that contains the auxin 2,4-D. Subculture of PEMs into the embryo differentiation medium which lacks 2,4-D and has higher osmoticum resulted in a 4-fold higher 45Ca2+ incorporation into the symplast. Fura-2 based ratiometric analysis also showed a 10-16- fold increase in the [Ca2+]cyt of PEMs under identical culture conditions, increasing from a resting concentration of 30-50 nM to 650-800 nM. Chelation of exogenous Ca2+ with EGTA arrested such an elevation in [Ca2+]cyt. Exogenous Ca2+ when chelated or deprived also arrested embryo development and inhibited the accumulation of a Ca2+-dependent protein kinase (swCDPK) in embryogenic cultures. However, such culture conditions did not cause cell death as the PEMs continued to proliferate to form larger cell clumps. Culture treatment with W7 reduced embryogenic frequency by 85%, indicating that blockage of Ca2+-mediated signaling pathway(s) involving swCDPK and/or CaM caused inhibition of embryogenesis. These observations suggest a second messenger role for exogenous Ca2+ and the existence of Ca2+-mediated signaling pathway(s) during sandalwood somatic embryogenesis. The detection of a 55 kD protein showing cross reactivity with polyclonal antisoybean CDPK and the detection of Ca2+-dependent protein kinase activity in protein extracts from somatic embryos, prompted investigation on the spatio-temporal accumulation and activity of a CDPK in different developmental stages of sandalwood. Western blot analysis and protein kinase assays identified a Ca2+-dependent protein kinase (swCDPK) of 55 kD in soluble protein extracts of different developmental stages of sandalwood somatic embryos. However, swCDPK was not detected in plantlets regenerated from somatic embryos. swCDPK exhibited differential expression and activity in the developmental stages of sandalwood. Zygotic embryos, endosperm and seedlings showed high accumulation of swCDPK. However, the enzyme was not detected in the soluble proteins of shoots and flowers of sandalwood tree. swCDPK exhibited a temporal pattern of expression in endosperm, showing high accumulation and activity in mature fruit and germinating stages, the enzyme being localized strongly in the storage bodies of the endosperm cells. Interestingly, these storage bodies were thereafter identified as oil bodies, suggesting that a Ca2+-mediated regulation of oil hydrolysis and/or mobilization might be operative during seed germination. swCDPK in the zygotic embryo was found to be inactive during seed dormancy and early stages of germination, indicating a possible post-translational hibition/inactivation of the enzyme during these stages. The temporal expression of swCDPK during somatic/zygotic embryogenesis, seed maturation and germination thus suggests involvement of the enzyme in these early developmental processes. In view of the diversity exhibited by members of the CDPK family, characterization of swCDPK, the early development specific CDPK from sandalwood was undertaken. Purification of swCDPK was achieved by chromatography on DEAE-cellulose, hydroxyapatite and Blue-Sepharose. The purified enzyme resolved into a single band on 10 % polyacrylamide gels, both under denaturing and non-denaturing conditions. swCDPK was strictly dependent on Ca2+, K0.5 (apparent binding constant) for Ca2+-activation of substrate phosphorylation activity being 0.7 μM and for autophosphorylation activity —50 nM. Ca2+-dependence for activation, CaM-independence, inhibition by CaM-antagonist (IC50 for W7 = 6 μM, for W5 = 46 μM) and cross-reaction with polyclonal antibodies directed against the CaM-like domain of soybean CDPK, confirmed the presence of an endogenous CaM-like domain in the purified enzyme. Kinetic studies revealed a Km value of 13 mg/mL for histone III-S and a Vmax of 0.1 nmolmin-1rng-1. The enzyme exhibited high specificity for ATP with a Km value of 10 nM. Titration with Ca2+ resulted in enhancement of the intrinsic emission fluorescence of swCDPK and a shift in the λmax emission from tryptophan residues. A reduction in the efficiency of non-radiative energy transfer from tyrosine to tryptophan residues was also observed. These are taken as evidence for the occurrence of Ca2+-induced conformational change in swCDPK. The emission spectral properties of swCDPK in conjunction with Ca2+ levels required for autophosphorylation and substrate phosphorylation help elucidate the possible mode of Ca2+ activation of this enzyme.

No description available.

Les protéines accomplissent leur fonction dans la cellule grâce à leur faculté de changer de forme. Chaque classe de protéines peut se caractériser par une structure spécia- lisée partagée par ses membres avec un certain degré de variabilité. Tel est le cas des protéines à motifs mains-EF, qui se transforment en liant et déliant l ’ion calcium. Ce motif permet à la Troponin C de s’ouvrir et se refermer afin de moduler le mécanisme de contraction des fibres musculaires. Un mécanisme similaire permet à la Calmoduline de gérer l’activité de divers canaux cellulaires. Les techniques de simulations numériques peuvent aider à comprendre les trajectoires de ces transformations. Le projet principal de cette thèse consistait à développer une méthode informatique multi-échelle permettant de simuler des mouvements complexes à l’intérieur d ’une protéine. La représentation multi-échelle développée peut changer et s’adapter en cours de simulation. La méthode, ART holographique, explore l’espace en générant des basculements d’ensembles atomiques, selon des champs de force atomistiques non biaisés indiquant à tout moment comment les ensembles doivent pivoter. La méthode réduit le calcul des fluctuations locales mais conserve une représentation spatiale complète. La représentation multi-échelle est combinée à une technique de recherche de passages de transition énergétiquement favorables, ART nouveau, qui conduit la trajectoire moléculaire d ’étape en étape. Appliquée à plusieurs protéines, dont la Calmodulin et la Troponin C, ART holographique génère des trajectoires de transformation entre des conformations distantes de celles-ci, déjà connues grâce aux techniques de RMN ou de cristallographie. L’usage d ’une représentation spatiale complète tout au long de la simulation favorise le discernement de certains détails des mécanismes. Le rôle, l’ordre d ’intervention, ainsi que la coopérativité de certains résidus et structures impliqués dans le mécanisme des paires main-EF ont été explorés plus en détail et un état intermédiaire est proposé. / Proteins accomplish their function inside cells by means of conformational changes. Each protein class may be characterized by a specialized structure shared by its members with some variability. EF-hands proteins present a special motif which transforms itself while binding or unbinding the calcium ion. This structure allows Troponin C domains to open and close as it modulates the muscular fibers contraction. A similar mechanism allow Calmodulin to manage the activity of a diversity of protein channels. Computational techniques may help discover how these transformations occur. The main project of this thesis was the development of a multi-scale computational method for the simulation of complex motions inside a protein. The multi-scale approach is designed to adapt and change all along the simulation. The method, holographic ART, explore conformational space by generating swiveling and rotation of atomic ensembles, leaded by non biased atomistic forcefields. This determines at each step the overall motion, keeping a complete spatial representation, but with minimal local fluctuations computation. The multi-scale representation is combined with a unbiased open ended algorithm for identifying transitions states, ART nouveau, which guides the molecular trajectory from state to state. Applied to several proteins, the method was able to generate transforma- tion trajectories between distant conformations known from NMR and crystallography techniques. The use of a complete spatial representation throughout the simulation allows the method to capture atomistic details of each event. The purpose, the intervention order, as well as cooperativity between some residues and sub-structures involved in the EF-hand pair mechanism have been explored more in detail and an intermediate state is proposed. / Les films de simulations qui accompagnent le document ont été réalisés avec Pymol.

Many molecules and the complex interactions between them underlie plasticity in the cerebellum. However, the exact relationship between cerebellar plasticity and the different signalling cascades remains unclear. Calcium-calmodulin dependent protein kinase II (CaMKII) regulates many forms of synaptic plasticity, but very little is known about its function during plasticity induction in the cerebellum. The aim of this thesis is to contribute to a better understanding of the molecular mechanisms that regulate the induction of synaptic plasticity in cerebellar Purkinje cells (PCs). The focus of the thesis is to investigate the role of CaMKII isoforms in the bidirectional modulation of plasticity induction at parallel fibre (PF)-PC synapses. For this investigation, computational models that represent the CaMKII activation and the signalling network that mediates plasticity induction at these synapses were constructed. The model of CaMKII activation by calcium-calmodulin developed by Dupont et al (2003) replicates the experiments by De Koninck and Schulman (1998). Both theoretical and experimental studies have argued that the phosphorylation and activation of CaMKII depends on the frequency of calcium oscillations. Using a simplified version of the Dupont model, it was demonstrated that the CaMKII phosphorylation is mostly determined by the average calcium-calmodulin concentration, and therefore depends only indirectly on the actual frequency of calcium oscillations. I have shown that a pulsed application of calcium-calmodulin is, in fact, not required at all. These findings strongly indicate that the activation of CaMKII depends on the average calcium-calmodulin concentration and not on the oscillation frequency per se as asserted in those studies. This thesis also presents the first model of AMPA receptor phosphorylation that simulates the induction of long-term depression (LTD) and potentiation (LTP) at the PF-PC synapse. The results of computer simulations of a simple mathematical model suggest that the balance of CaMKII-mediated phosphorylation and protein phosphatase 2B (PP2B)-mediated dephosphorylation of AMPA receptors determines whether LTD or LTP occurs in cerebellar PCs. This model replicates the experimental observations by Van Woerden et al (2009) that indicate that CaMKII controls the direction of plasticity at PF-PC synapses. My computer simulations support Van Woerden et al’s original suggestion that filamentous actin binding can enable CaMKII to regulate bidirectional plasticity at these synapses. The computational models of intracellular signalling constructed in this thesis advance the understanding of the mechanisms of synaptic plasticity induction in the cerebellum. These simple models are significant tools for future research by the scientific community.

Regenerative medicine is a rapidly developing field, merging engineering and biological life sciences to create biological replacements for damaged tissue and organ function. Development of cellular based therapies has the potential of curing present untreatable diseases and conditions, such as diabetes. The identification of protein expression patterns, that guide undifferentiated cells to different lineages, can provide important information about the progression of cellular differentiation at various stages. This research project utilizes proteomics and in vitro live-cell microscopy to investigate two distinct cellular systems: (1) the signaling pathways of calmodulin (CaM) in the differentiation of a human glioblastoma cell line; and (2) the effect of islet neogenesis associated protein (INGAP) on human islet-derived progenitor cells (hIPCs). Using a proteomic readout with a long term live-cell imagining approach, it was hypothesized that highly specific binding proteins of a CaM-mutant, and proteins in hIPCs perturbed by INGAP, could be identified and studied in vitro, characterizing specific signaling pathways which control the function of CaM in brain tumour cells and the mechanism(s) of INGAP in islet-derived progenitor cells. This thesis presents the utility of a proteomics and an in vitro cell microscopy approach to investigate therapeutic proteins, such as INGAP, on cell culture systems. The results have established the limitations and the utility of DIGE, differential binding of a CaM-mutant versus calcium-CaM, and the cell specific uptake feasibility of using the TAT-binding domain. In the hIPC system, proteomic, phenotypic, motility, proliferation and nuclear effects of INGAP were determined. Specifically, hIPCs exposed to INGAP had 50% decrease in average nuclear speed, the translocation of two identified proteins caldesmon and tropomyosin and INGAP was found to bind specifically to hIPCs. However, hIPCs had no changes in insulin specific hormone expression.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Nevada, Reno, 2002. / Includes bibliographical references. Online version available on the World Wide Web.

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 100-118).

La calmoduline (CaM) est une protéine modulatrice de la réponse cellulaire chez les eucaryotes composée de quatre domaines de liaison au calcium et d'une hélice centrale flexible. Elle peut interagir avec d'autres protéines en présence de calcium, entraînant l'activation et l'inhibition d'enzymes, l'ouverture de canaux membranaires et modulant le trafic intracellulaire. L'identification de protéines parternaires de la CaM requière la mise au point de techniques permettant de mesurer les paramètres de la liaison pour un grand nombre de protéines dans des conditions variables mimant l'environnement cellulaire (par exemple en présence de ligands ou d'autres protéines). Le premier objectif de cette thèse a été de développer une technique de mesure des interactions CaM-parternaire reposant sur des mesures d'anisotropie de fluorescence. Les tests ont été ensuite utilisés pour caractériser de manière quantitative l'interaction préalablement mise en évidence de deux protéines chloroplastiques (NADK2 et Tic32) avec la CaM. Afin d'identifier d'autres cibles chloroplastiques de la CaM nous avons alors effectué une analyse à haut-débit en couplant une purification par affinité à des analyses protéomiques. La validation des interactions a été réalisée grâce à l'utilisation de méthodes biochimiques complémentaires. Nous avons ensuite focalisé notre attention sur la protéine ceQORH dont la très forte affinité pour la CaM a pu être confirmée. Nos résultats fournissent par ailleurs de nouveaux éléments pour la compréhension de ces interactions. Afin de vérifier la présence de CaM ou de CaM-like (CML) dans le chloroplaste nous avons utilisé une approche biochimique et protéomique. Nous avons d'autre part étudié la localisation de CMLs potentiellement chloroplastiques fusionnées à la GFP dans des protoplastes d'Arabidopsis. A ce jour ces deux approches ne nous ont pas permis d'identifier ce type de protéines dans le chloroplaste. / Calmodulin (CaM) is an important modulator of cell responses of eukaryotes. This protein is composed of four calcium (Ca2+)-binding sites and a flexible central helix. CaM can interact with other proteins in a Ca2+-dependent way. This leads to a wide variety of effects, such as activation/inhibition of enzymes, opening of membrane channels and regulation of protein trafficking. The identification of high-affinity CaM targets requires techniques allowing the study of the CaM-binding parameters of a large number of protein, and in several conditions mimicking the cell environment (e.g. presence of ligands or other proteins). The first objective of this PhD was to develop flexible and quantitative assays of CaM-partners interactions based on measurements of fluorescence anisotropy. these tests were used to perform a quantitative characterization of the interaction between CaM and two previously identified targets located in Arabidopsis chloroplast (NADK2 and Tic32). We then performed a high-throughput analysis (CaM-affinity chromatography coupled with mass spectrometry) in order to detect new potential plastidial CaM targets. We validated our approach with several biochemical techniques. We finally focused our attention on the ceQORH protein, whose high CaM affinity was confirmed by several tests. Our results confirm the Ca2+-dependent CaM affinity of NADK2, Tic32 and ceQORH and provide new elements for understanding the effects of these interactions. In addition, in order to verify the presence of CaMs or CaM-like proteins in the chloroplast, we used a biochemical and proteomic approach. We also studied the intracellular localization of some putative plastidial CMLs tagged with GFP in Arabidopsis protoplasts. For the moment, these approaches did not allow identifying such proteins in the chloroplast.