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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
641

Regulation of tubulin heterodimer partitioning during interphase and mitosis

Holmfeldt, Per January 2008 (has links)
The microtubule cytoskeleton, which consists of dynamic polymers of alpha/beta tubulin heterodimers, organizes the cytoplasm and is essential for chromosome segregation during mitosis. My thesis addresses the significance and potential interplay between four distinct microtubule-regulatory proteins. The experimental approach included the development of a replicating vector system directing either constitutive expression of short hairpin RNAs or inducible ectopic expression, which allows stable depletion and/or conditional exchange of gene-products. Based on the originally observed activities in frog egg extracts, MCAK and TOGp have been viewed as major antagonistic proteins that regulate microtubule-dynamics throughout the cell cycle. Surprisingly, while my thesis work confirmed an essential role of these proteins to ensure mitotic fidelity, tubulin subunits partitioning is not controlled by the endogenous levels of MCAK and TOGp in human somatic cells. Our major discovery in these studies is that the activities of both CaMKII and TOGp are essential for spindle bipolarity through a mechanism involving protection of spindle microtubules against MCAK activity at the centrosome. In our search for the major antagonistic activities that regulates microtubule-dynamics in interphase cells, we found that the microtubule-destabilizing activity of Op18 is counteracted by MAP4. These studies also established Op18 and MAP4 as the predominant regulators of tubulin subunit partitioning in all three human cell model systems studied. Moreover, consistent with phosphorylation-inactivation of these two proteins during mitosis, we found that the microtubule-regulatory activities of both MAP4 and Op18 were only evident in interphase cells. Importantly, by employing a system for inducible gene product replacement, we found that site-specific phosphorylation-inactivation of Op18 is the direct cause of the demonstrated hyper-polymerization in response to T-cell antigen receptor triggering. This provides the first formally proven example of a signal transduction pathway for regulation of interphase microtubules. Op18 is frequently upregulated in various types of human malignancies. In addition, a somatic mutation of Op18 has recently been identified in an adenocarcinoma. This thesis work revealed that the mutant Op18 protein exerts increased microtubule-destabilizing activity. The mutant Op18 protein was also shown to be partially resistant to phosphorylation-inactivation during mitosis, which was associated with increased chromosome segregation aberrancies. Interestingly, we also observed the same phenotype by overexpressing the wild type Op18 protein. Thus, either excessive levels of wild type Op18 or normal levels of mutated hyper-active Op18 seems likely to contribute to tumor progression by exacerbating chromosomal instability.
642

Design and optimization of efficient microfluidic platforms for particle manipulation and cell stimulation in systems biology

Paul, Alison Marie 25 August 2011 (has links)
The overall goal of this research was to develop an efficient microfluidic system to study signal transduction in stimulation dynamics. This research applied reactive transport fundamentals in concert with biological systems knowledge to completely understand diffusion of soluble signals, fluid and particle flow properties, and dynamics of cellular responses. First, a device capable of parallel multi-time-point cell stimulation and lysis on-chip was developed in collaboration. Second, to understand flow of cells through complex 3-D flow schemes, a Single-field Three-dimensional Epifluorescence Particle (STEP) imaging technique was developed. Using the STEP imaging technique, we were able to determine particle distributions and track individual particles in complex flow geometries. Third, during the design of the stimulation device it was observed that the cells do not distribute across the channel in the same way as the fluids. Based on the observation that geometry and particle size were most influential factors on particle distribution, it was hypothesized that our earlier observation and all observed phenomena in our experimental range were due to the volume exclusion of particles of finite size near the wall of the complex flow geometry. Overall, this work contributed to the realization of microfluidic platforms as powerful tools for probing areas of biology and medicine that are difficult with existing technology. The high-throughput format enabled simple and fast generation of large sets of quantitative data, with consistent sample handling. We demonstrated the necessary first steps to designing efficient unit operations on cells in microfluidic devices. The model can be used for informed design of unit operations in many applications in the future.
643

MonoAminergic Receptors in the Stomatogastric Nervous System: Characterization and Localization in Panulirus Interruptus

Clark, Merry Christine 22 April 2008 (has links)
Neural circuit flexibility is fundamental to the production of adaptable behaviors. Invertebrate models offer relatively simple networks consisting of large, identifiable neurons that are useful for investigating the electrophysiological properties that contribute to circuit output. In particular, central pattern generating circuits within the crustacean stomatogastric nervous system have been well characterized with regard to their synaptic connectivities, cellular properties, and response to modulatory influences. Monoaminergic modulation is essential for the production of adaptable circuit output in most species. Monoamines, such as dopamine and serotonin, signal via metabotropic receptors, which activate intracellular signaling cascades. Many of the neuronal and network targets of monoaminergic modulation in the crustacean stomatogastric nervous system are known, but nothing is known of the signal transduction cascades that mediate the biophysical response. This work represents a thorough characterization of monoaminergic receptors in the crustacean stomatogastric nervous system. We took advantage of the close phylogenetic relationship between crustaceans and insects to clone monoaminergic receptors from the spiny lobster. Using a novel database mining strategy, we were able to identify several uncharacterized monoaminergic receptors in the Panulirus interruptus genome. We cloned one serotonin (5-HT2βPan) and three dopamine receptors (D1αPan, D1βPan, and D2αPan), and characterized them with regard to G protein coupling and signal transduction cascades. We used a heterologous expression system to show that G protein couplings and signaling properties of monoaminergic receptors are strongly conserved among vertebrate and invertebrate species. This work further shows that DAR-G protein couplings in the stomatogastric nervous system are unique for a given receptor subtype, and receptors can couple to multiple signaling pathways, similar to their mammalian homologs. Custom made antibodies were used to localize monoamine receptors in the stomatogastric ganglion, and in identified neurons. Pyloric neurons show unique receptor expression profiles, which supports the idea of receptor expression as an underlying mechanism for cell-type specific effects of a given modulator. Receptors are localized to the synaptic neuropil, but are not expressed in the membrane of large diameter processes or the soma. The localization of dopamine receptors in identified pyloric neurons suggests that they may respond to synaptic, paracrine or neurohormonal dopamine signals. This work also supports the idea that different types of signals can be generated by a single receptor.
644

Physicochemical properties of amino acid sequences of G-proteins for understanding GPCR-G-protein coupling

Ghimire, Ganga D., ギミレ, ガンガ D, Imai, Kenichiro, 今井, 賢一郎, Akazawa, Fumitsugu, 赤沢, 史嗣, Tsuji, Toshiyuki, 辻, 敏之, Sonoyama, Masashi, 園山, 正史, Mitaku, Shigeki, 美宅, 成樹 January 2006 (has links) (PDF)
No description available.
645

A Multielectrode Microcompartment Platform for Signal Transduction in the Nervous System

Ravula, Surendra Kumar 23 June 2006 (has links)
This dissertation presents the development of a multielectrode microcompartment platform for understanding signal transduction in the nervous system. The design and fabrication of the system and the characterization of the system for pharmacological and electrophysiological measurements of cultured neurons is presented in this work. The electrophysiological activity of cultured dorsal root ganglion (DRG) neurons and cortical neurons is shown on the MEA substrate. These recordings were measured and tied to the toxicological effects of the chemotherapeutic drug vincristine on DRGs. Conventional electrophysiological recordings (via a patch micropipette) are made routinely to record action potentials and ion channel activity in neurons. Moreover, Campenot chambers (traditional compartmented culture systems) have been used for the last thirty years to study the selective application of drugs to neurons. Both of these techniques are useful and well established; however they have their limitations. For instance, Campenot chambers cannot be used very well for small processs-producing neurons, since the barriers are difficult to tranverse. Moreover, conventional patch recordings are labor-intensive, especially when more than one microelectrode needs to be positioned. The developed system is composed of a two compartment divider, each compartment capable of housing axons or cell bodies. Underneath the divider, the substrate has 60 electrodes, arranged in several lines to accommodate several different neurite tracks. Neurons can be stimulated and their activity can be recorded in both of the compartments. The neurotoxin and chemotherapeutic drug vincristine was tested in the system on the DRGs. The drug caused length-dependent axonal degeneration in the DRGs when applied locally. Moreover, electrophysiological activity in both compartments showed that only the activity in the axonal compartment was affected, leading us to believe that the mechanism behind the degeneration is localized to the distal axon.
646

The role of protein-membrane interactions in modulation of signaling by bacterial chemoreceptors

Draheim, Roger Russell 15 May 2009 (has links)
Environmental signals are sensed by membrane-spanning receptors that communicate with the cell interior. Bacterial chemoreceptors modulate the activity of the CheA kinase in response to binding of small ligands or upon interaction with substrate-bound periplasmic-binding proteins. The mechanism of signal transduction across the membrane is a displacement of the second transmembrane domain (TM2) a few angstroms toward the cytoplasm. This movement repositions a dynamic transmembrane helix relative to the plane of the cell membrane. The research presented in this dissertation investigated the contribution of TM2-membrane interactions to signaling by the aspartate chemoreceptor (Tar) of Escherichia coli. Aromatic residues that reside at the cytoplasmic polar-hydrophobic membrane interface (Trp-209 and Tyr-210) were found to play a significant role in regulating signaling by Tar. These interactions were subsequently manipulated to modulate the signaling properties of Tar. The baseline signaling state was shown to be incrementally altered by repositioning the Trp-209/Tyr-210 pair. To our knowledge, this is the first example of harnessing membrane-protein interactions to modulate the signal output of a transmembrane receptor in a controlled and predictable manner. Potential long-term applications include the use of analogous mutations to elucidate two-component signaling pathways, to engineer the signaling parameters of biosensors that incorporate chemoreceptors, and to predict the movement of dynamic transmembrane helices in silico.
647

Sinec: Large Scale Signaling Network Topology Reconstruction Using Protein-protein Interactions And Rnai Data

Hashemikhabir, Seyedsasan 01 September 2012 (has links) (PDF)
Reconstructing the topology of a signaling network by means of RNA interference (RNAi) technology is an underdetermined problem especially when a single gene in the network is knocked down or observed. In addition, the exponential search space limits the existing methods to small signaling networks of size 10-15 genes. In this thesis, we propose integrating RNAi data with a reference physical interaction network. We formulate the problem of signaling network reconstruction as finding the minimum number of edit operations on a given reference network. The edit operations transform the reference network to a network that satisfy the RNAi observations. We show that using a reference network does not simplify the computational complexity of the problem. Therefore, we propose an approach that provides near optimal results and can scale well for reconstructing networks up to hundreds of components. We validate the proposed method on synthetic and real datasets. Comparison with the state of the art on real signaling networks shows that the proposed methodology can scale better and generates biologically significant results.
648

Studies On The Molecular Mechanism Of Cytokinin Action: Involvement Of Ca2+, Protein Kinase And Concurrent Protein Synthesis In Signaling Of Cytokinin-Induced Expression Of Pathogenesis-Related Enzymes In Cucumber

Barwe, Sonali P 11 1900 (has links)
Phytohormones act as signals to regulate plant growth and development by modulation of gene expression in response to internal developmental cues or external environmental stimuli, such as light and pathogen infection. There are five major classes of phytohormones, viz. auxins, cytokinins, gibberellins, ethylene and abscisic acid. Of these, cytokinins, 6N substituted adenine derivatives, are of special importance owing to their possible diverse roles in plant growth and development. They induce cell division, cell expansion in cotyledon, chloroplast and etioplast development, suppression of apical dominance and senescence, and differentiation of in vitro cultured cells. However, very little is known about the mechanism of cytokinin action at the molecular level. Cytokinins have been demonstrated to modulate the expression of genes coding for several enzymes including nitrate reductase, ribulose-l,5-bisphosphate carboxylase, RNA polymerase I, and pathogenesis-related (PR) enzymes, i.e. chitinases and β-1,3- glucanases. One of the important questions regarding cytokinin regulation of enzyme activities and/or the accumulation of their corresponding proteins and mRNAs is how the cytokinin signal is transduced. There is considerable evidence from earlier reports demonstrating that pathogens alter hormone physiology of the host plant and it has been proposed that the infection-associated enzyme changes might be mediated by phytohormones. In the present study, two PR enzymes, viz. cucumber chitinase and β-l,3-glucanase, have been chosen to examine the mode of regulation of their gene expression by cytokinins, including the identification of cytokinin signal transduction components. Plant chitinases and glucanases are important enzymes in plant defense mechanisms against fungal pathogens as they degrade the major fungal cell wall components, chitin and β-1,3-glucan, respectively. Besides their role in plant defense, they are known to be involved in diverse physiological and developmental processes, such as embryogenesis, seed germination and flower development, and are also developmentally and hormonally regulated. Initially, in order to study the effects of various cytokinins on chitinase and β-1,3-gIucanase enzyme activities and their gene expression, cotyledons excised from seven-day-old dark-grown cucumber seedlings were treated with water, and cytokinins, viz. benzyladenine, kinetin, zeatin and zeatin riboside. It was observed that chitinase and β-l,3-ghucanase enzyme activities and their transcripts were induced to varying extents following treatments of cotyledons with the cytokinins tested. However, a maximum increase in enzyme activities and their transcript levels was noticed in zeatin-treated cotyledons. Therefore, zeatin was used for further studies. The main objective of the present study was to investigate the cytokinin signal transduction mechanism involving the induction of expression of chitinase and β-1-3-glucanase. In order to obtain insights into the downstream components of the cytokinin-signaling pathway, effects of several agonists and antagonists of the signal transduction components on zeatin-induced chitinase and β-l,3-glucanase activities, and their protein and transcript levels were monitored by enzyme assay, by immunoblot analysis, and by northern analysis, respectively. Treatment of excised dark-grown cucumber cotyledons with staurosporine, a broad spectrum protein kinase inhibitor, reduced the zeatin-induced chitinase and β-l,3-glucanase enzyme activities and the accumulation of their proteins and transcripts. On the other hand, treatment with sodium fluoride, a general inhibitor of protein phosphatases, stimulated the basal chitinase and β-1,3-glucanase enzyme activities and their protein and transcript accumulation, whereas it had no effect on the zeatin-induced enzyme activities and their protein and transcript accumulation. These findings suggested that protein phosphorylation is critical in the cytokinin induction of expression of chitinase and β-l,3-glucanase. Since Ca2+ is known to be an important second messenger in several plant signal transduction pathways, the possible involvement of Ca2+ in the cytokinin-induced expression of chitinase andβ-l,3-glucanase was examined. The results of the present investigation showed that the chitinase and β-1,3-ghicanase activities and their proteins and transcripts were appreciably increased by exogenous CaCl2 treatment in control cotyledons. Treatment of cotyledons with zeatin plus CaCl2 did not result in a further increase in either these enzyme activities or their protein and transcript accumulation as compared to zeatin or CaCl2 treatment alone. The lack of additivity of zeatin plus CaCl2 treatment indicated a common mechanism of action of zeatin and Ca2+ in the induction of these enzyme activities and their gene expression. To test the occurrence of influx of extracellular Ca2+ by cytokinin, cotyledons were treated with the plasma membrane Ca2+ channel blocker, verapamil, and Ca2+ ionophore A23187. Verapamil treatment inhibited the zeatin-induced chitinase and β-1,3-ghicanase enzyme activities and their protein and transcript accumulation. An increase in the intracellular Ca2+ levels by means of Ca2+ ionophore treatment resulted in a significant increase in basal chitinase and β-l,3-glucanase activities and their protein and transcript accumulation. These results suggested that an influx of extracellular Ca2+ leading to increased levels of cytosolic Ca2+ is required for the cytokinin induction of expression of these enzymes. The correlation of chitinase and β-1,3-glucanase enzyme activities and their protein and transcript accumulation in the zeatin-treated cotyledons suggested that the cytokinin zeatin stimulates chitinase and β-l,3-glucanase accumulation at the mRNA level and that the increase in enzyme activities is due to an increase in the amount of the enzyme protein and not by the activation of the existing enzyme. Further, the effect of zeatin on both the enzyme activities and their transcript levels under conditions that inhibit protein synthesis was studied. Treatment of excised dark-grown cucumber cotyledons with cycloheximide, an inhibitor of protein synthesis, in the presence of zeatin, completely nullified the stimulatory effect of zeatin. These results indicated the requirement of cytokinin-induced enhanced concurrent protein synthesis in the observed stimulation of chitinase and β-l,3-glucanase enzyme activities as well as their transcript accumulation Ca2+ In an attempt to isolate the full length cucumber β-l,3-glucanase cDNA from a cucumber cDNA library, we isolated and sequenced one cDNA clone, which was 978 bp long and had a potential polyadenylation signal A ATA A starting 172 bases before the polyadenylation tail A deduced amino acid sequence of the cDNA, which was 242 amino acids in length, apparently encoded a partial β-amyrin synthase. Sequence comparison of the deduced partial amino acid sequence of cucumber β-amyrin synthase with other known plant β-amyrin synthase sequences available in databases revealed significant homologies to β-amyrin synthases from Panax, Pisum and Glycyrrhiza. Southern blot analysis indicated that there was only one β-amyrin synthase gene in the cucumber genome. RT-PCR analysis performed on total RNA isolated from zeatin- and salicylic acid-treated cotyledons using forward and reverse primers designed from the internal regions of the cDNA showed that the transcript levels of β-amyrin synthase were enhanced by both zeatin and salicylic acid. In conclusion, we have demonstrated that chitinase and β-l,3-glucanase accumulation is stimulated by exogenous cytokinin treatment of excised cucumber cotyledons, and this effect is correlated with the content of chitinase and β-1,3-glucanase transcripts as judged by northern analyses. Further, the findings reported in the thesis suggested that Ca2+ influx from extracellular space, protein phosphorylation by staurosporine-sensitive protein kinase(s) and concurrent protein synthesis are required for the signaling of cytokinin-induced expression of both these pathogenesis-related enzymes.
649

Die Physiologische Relevanz des G-Protein-gekoppelten Rezeptors GPR34

Liebscher, Ines 19 January 2011 (has links) (PDF)
Die Familie der G-Protein-gekoppelten Rezeptoren (GPCRs) bildet die größte Gruppe von Membranrezeptoren im menschlichen Organismus. Für viele GPCRs sind bisher die physiologischen Funktionen nicht bekannt. Das biologische Verständnis der Funktionen im menschlichen Organismus dieser sogenannten „orphan“ GPCRs (oGPCRs) hat, aufgrund möglicher kausaler Beteiligung an der Pathogenese von Erkrankungen sowie deren therapeutische Beeinflussbarkeit, hohe medizinische Relevanz. Die GPCRs der P2Y12-ähnliche Rezeptorgruppe besitzen eine große physiologische Bedeutung bei der Thrombozytenaggregation und der Induktion der Migration von immunokompetenten Zellen in Schädigungsgebiete. Der ADP-Rezeptor P2Y12 kann durch verschiedene pharmakologische Wirkstoffe beeinflusst werden, was bereits klinisch-therapeutisch genutzt wird. Diese Gruppe von GPCRs enthält jedoch auch Mitglieder, deren Funktionen völlig unbekannt sind. Einer dieser oGPCRs ist der GPR34. Ziel dieser Arbeit war es, mittels verschiedener in-vitro-Methoden und anhand eines GPR34-defizienten Mausstamms die physiologische Relevanz dieses P2Y12-ähnlichen Rezeptors zu analysieren. Dazu wurde ein GPR34-Knockout-Mausmodell etabliert. Die GPR34-Defizienz hatte keinen wesentlichen Einfluss auf die Entwicklung, Morphologie, das Wachstum oder die Fertilität bei Mäusen. Die Ergebnisse aus Immunisierungs– und Infektionsstudien zeigten jedoch, dass dieser evolutionär hoch konservierte Rezeptor eine wichtige Funktion in der Feinkontrolle der zellulären Immunabwehr ausübt. Neben einer verstärkten Antwort im Delayed-type Hypersensitivity (DTH)-Test war die Abwehr einer Cryptococcus-Infektion in diesem GPR34-defizienten Tiermodell beeinträchtigt. Signifikant erhöhte Zytokinspiegel nach Antigen- bzw. Pathogenexposition deuteten auf eine gestörte Immunregulation in GPR34-defizienten Mäusen hin. Weiterführende Untersuchungen sollten sich der Identifizierung des endogenen Agonisten und der Funktion des GPR34 bei der Koordinierung der zellulären Immunreaktion widmen.
650

The crosstalk between ITAM-associated receptors and Jak-STAT signaling pathways /

Park-Min, Kyung-Hyun. January 2007 (has links)
Thesis (Ph. D.)--Cornell University, January, 2007. / Vita. Includes bibliographical references (leaves 92-106).

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