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McKay, Jodi Ho-Jung. "HRas intracellular trafficking and signal transduction." [Ames, Iowa : Iowa State University], 2007.
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Legewie, Stefan. "Systems biological analyses of intracellular signal transduction." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2009. http://dx.doi.org/10.18452/16018.
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An der Interpretation extrazellulärer Signale beteiligte Regulationsnetzwerke sind von zentraler Bedeutung für alle Organismen. Extrazelluläre Signale werden gewöhnlich durch enzymatische Kaskaden innerhalb weniger Minuten in den Zellkern weitergeleitet, wo sie langsame Änderungen der Genexpression bewirken und so das Schicksal der Zelle beeinflussen. Im ersten Teil der Arbeit wird durch mathematische Modellierung untersucht, wie die MAPK Kaskade Signale von der Zellmembran in den Kern weiterleitet. Es wurden Netzwerkeigenschaften herausgearbeitet, die verhindern, dass die MAPK Kaskade fälschlicherweise durch genetische Mutationen aktiviert wird. Desweiteren wurde eine versteckte positive Rückkopplungsschleife identifiziert, welche die Aktivierung der MAPK Kaskade oberhalb eines gewissen Schwellwert-Stimulus verstärkt. Der zweite Teil der Arbeit konzentriert sich darauf, wie Änderungen der Genexpression auf langsamer Zeitskala in das Signalnetzwerk rückkoppeln. Eine systematische Genexpressionsdaten-Analyse ergab, dass transkriptionelle Rückkopplung in Eukaryoten generell über Induktion kurzlebiger Signalinhibitoren geschieht. Dynamische Modellierung und experimentelle Validierung von Modellvorhersagen ergab, dass das Inhibitorprotein SnoN als zentraler negativer Feedback Regulator im TGFbeta Signalweg fungiert. Der dritte Teil der Arbeit untersucht, wie die Genexpressionsmaschinerie intrazelluläre Signale interpretiert (“dekodiert“). Eine experimentelle und theoretische Analyse der cyanobakteriellen Eisenstress-Antwort ergab, dass IsrR, eine kleine regulatorische RNA, die Genexpression auf ausreichend starke und lange Stimulation beschränkt. Des Weiteren wurde ein “Reverse Engineering“-Algorithmus auf Hochdurchsatz-RNAi-Daten angewendet, um die Topologie eines krebsrelevanten Transkriptionsfaktornetzwerks abzuleiten. Zusammenfassend wurde in dieser Dissertation gezeigt, wie mathematische Modellierung die experimentelle Analyse biologischer Systeme unterstützen kann.<br>Intracellular regulatory networks involved in sensing extracellular cues are crucial to all living organisms. Extracellular signals are rapidly transmitted from the cell membrane to the nucleus by activation of enzymatic cascades which ultimately elicit slow changes in gene expression, and thereby affect the cell fate. In the first part of this thesis, the Ras-MAPK cascade transducing signals from the cell membrane to the nucleus is analyzed using mathematical modeling. Model analysis reveals network properties which prevent the MAPK cascade from being inappropriately activated by mutations. Moreover, the simulations unveil a hidden positive feedback loop which ensures strong amplification of MAPK signalling once extracellular stimulation exceeds a certain threshold. The second part of the thesis focuses on how slow gene expression responses feed back into the upstream signalling network. A systematic analysis of gene expression data gathered in mammalian cells demonstrates that such transcriptional feedback generally involves induction of highly unstable signalling inhibitors, thereby establishing negative feedback regulation. Dynamic data-based modelling identifies the SnoN oncoprotein as the central negative feedback regulator in the TGFbeta signalling pathway, and corresponding model predictions are verified experimentally in SnoN-depleted cells. The third part of the thesis focuses on how intracellular signals are decoded by the downstream gene expression machinery. A combined experimental and theoretical analysis of the cyanobacterial iron stress response reveals that small non-coding RNAs allow cells to selectively respond to sufficiently strong and sustained stimuli. Finally, a reverse engineering approach is applied to derive the topology of a complex mammalian transcription factor network from high-throughput knock-down data. In conclusion, this thesis demonstrates how mathematical modelling can support experimental analysis of biological systems.
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Bottomley, Matthew James. "Biophysical studies of intracellular signal transduction proteins : investigating the structure-function relationship." Thesis, University College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.286570.
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Hao, Baixia, and 郝佰侠. "Regulatory and functional studies of store-operated calcium entry." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/196486.
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Ca2+ signaling is essential for a wide variety of cellular activities, ranging from short term activities, such as synaptic and muscle contraction, to long term processes, such as proliferation and differentiation. Store-operated Ca2+ entry (SOCE), an important Ca2+ influx pathway in non-excitable cells, well coordinates Ca2+ release from ER and Ca2+ influx through plasma membrane. STIM1 and Orai1, serving as ER Ca2+ sensor and pore forming subunit, respectively, are the two essential components of SOCE machinery. In addition to activate Orai1 channel, studies have shown that STIM1 regulates other plasma membrane Ca2+ channels and senses a variety of cellular stresses to regulate SOCE. Therefore, it is of great interests to investigate the mechanisms and physiological functions of STIM1 and Orai1 mediated SOCE.
Here, we performed tandem affinity purification to identify STIM1 associated proteins in Hela cells stably expressing STIM1-His6-3×Flag. Four candidate proteins, including GRP78, HSP70, IQGAP1, and Actin, were identified by mass spectrometry analyses. Surprisingly, IQGAP1 failed to affect the activity of SOCE. Interestingly, GRP78 knockdown only affected the inactivation phase while exerted no effect on the activation phase of SOCE. In addition, GRP78 knockdown markedly induced cell apoptosis and dramatically increased the ER Ca2+ concentration. Moreover, GRP78 was involved in the regulation of SOCE by the ER stress. These data indicate that GRP78 is an important regulator of SOCE to prevent Ca2+ overload in cells. HSP70, however, significantly reduced the activity of SOCE by inhibiting STIM1 translocation to ER-PM junctions. Future studies will explore the mechanism of GRP78 and HSP70 in regulating SOCE by confocal and TIRF imaging. Embryonic stem (ES) cells proliferate unlimitedly and can differentiate into all fetal and adult cell types. This property endows ES cells to be the promising candidates in the therapy of neurodegenerative diseases. Thus, it is important to identify novel signaling molecules or events that could play a role in the neural commitment of ES cells. Accumulated evidences have documented the role of STIM1 and Orai1 mediated SOCE in neuronal activities. Yet, the role of SOCE in early neural development remains to be determined. Here we examined the role of STIM1 and Orai1 during neural differentiation of mouse ES cells. Both of STIM1 and Orai1 were expressed and functionally active in ES cells, and expressions of STIM1 and Orai1 were dynamically regulated during neural differentiation of mouse ES cells. STIM1 knockdown inhibited the differentiation of mouse ES cells into neural progenitors, neurons, and astrocytes. In addition, STIM1 knockdown caused severe cell death and markedly suppressed the proliferation of neural progenitors. Surprisingly, Orai1 knockdown had little effect on neural differentiation of mouse ES cells, but the neurons derived from Orai1 knockdown ES cells, like those from STIM1 knockdown cells, had defective SOCE. Taken together, our data indicate that STIM1 is required for neural differentiation of mouse ES cells independent of Orai1-mediated SOCE.<br>published_or_final_version<br>Physiology<br>Doctoral<br>Doctor of Philosophy
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Kemp, Daniel M. "Reporter gene analysis of regulatory mechanisms in cAMP signalling." Thesis, University of Kent, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.310202.
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Lau, See-yan. "A study of intracellular signals of K-opioids in non-neuronal cells /." Hong Kong : University of Hong Kong, 1997. http://sunzi.lib.hku.hk/hkuto/record.jsp?B19667139.
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Kawano, Yuichi. "Effect of hyperglycemia on glucose transport and intracellular signal transduction in skeletal muscle /." Stockholm, 1999. http://diss.kib.ki.se/1999/91-628-3593-9/.
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劉思恩 and See-yan Lau. "A study of intracellular signals of K-opioids in non-neuronal cells." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1997. http://hub.hku.hk/bib/B31214290.
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Sadreev, Ildar. "Mathematical modelling of inter- and intracellular signal transduction : the regulatory role of multisite interactions." Thesis, University of Exeter, 2015. http://hdl.handle.net/10871/21212.
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Signalling processes regulate various aspects of living cells via modulation of protein activity. The interactions between the signalling proteins can occur at single or multiple sites. Although single site protein interactions are relatively easy to understand, these rarely occur in living systems. It is therefore important to investigate multisite interactions. Despite the recent progress in experimental studies, the underlying molecular mechanisms and molecular functions of the multisite interactions are still not clear and therefore require systems approaches for deeper understanding, for example to understand how the system will react to perturbation of one of its components. The examples of the molecular functions that are studied in this thesis are: kinetics of multisite calcium binding to proteins such as calmodulin (CaM), multisite phosphorylation of interferon regulatory factor 5 (IRF-5) and signal transducers and activators of transcription (STATs). We also study the role of STATs in the overall immune response and in T cell phenotype switching as well as multisite phosphorylation of high osmolarity glycerol factor 1 (Hog1) in mitogen activated protein kinase (MAPK) cascade during the adaptation of Candida glabrata to osmotic stress. In this thesis, these examples are studied using the systems approach in the context of human diseases: cancer, candidiasis, immunity-related pathologies such as rheumatoid arthritis, inflammatory bowel disease and systemic lupus erythematosus. We discuss potential therapeutic implications of the proposed models in these diseases. The predictions of the models developed in this thesis are supported by the experimental data and propose possible mechanisms of the multisite interactions involved in the cellular regulation.
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Li, Sen, and 李森. "Intracellular alkalinization induces cytosolic Ca2+ increases by inhibiting sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B46940546.
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Hassan, Mohamed. "Interference of Hepatitis C virus (HCV) core protein with intracellular signal transduction processes in liver cells /." Aachen : Shaker, 2001. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=012995533&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.
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Chan, Ming Hang (Stanley), and stanley chan@baker edu au. "Investigation of The Intracellular Signalling Pathway for Interleukin-6 Gene Expression in Skeletal Muscle." RMIT University. Medical Sciences, 2007. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20080206.091026.
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Hui, Xin [Verfasser], and Peter [Akademischer Betreuer] Lipp. "Protein Kinase C, the Spatial and Temporal Modulator of Intracellular Signal Transduction / Xin Hui. Betreuer: Peter Lipp." Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2015. http://d-nb.info/1072409984/34.
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Winegar, Bruce D. (Bruce David). "Roles of Calcium Ions and Cyclic AMP in Olfactory Transduction." Thesis, North Texas State University, 1986. https://digital.library.unt.edu/ark:/67531/metadc331287/.
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The roles of Ca2 + and cAMP in olfactory transduction were explored using agents which affect calcium channels and second messenger systems. These agents were applied at certain calculated final concentrations onto olfactory epithelia of urethane-anesthetized frogs (Sana PiPlens) by two-sec aerosol spray. During extracellular recording, saturated vapors of isoamyl acetate were delivered every 100 sec in 0.3 sec pulses to produce an electroolfactogram (EOG). Inorganic cations that block inward calcium currents inhibit EOG responses with the following rank order: (La3+) > (Zn2+, Cd2+) > (Al3+, Ca2+, Sr2+) > (Co2+). Application of 7.5 mM La3+ eradicates £0G's, while Ba2+ (which can carry more current that Ca2+) initially produces significant enhancement (F=43.04, p<0.001, df=19). Magnesium ion has no effect on EOG's at 7.5 mM, while 1.5 X 10"4M Ca2+ is significantly inhibitory (F=5.74; p=0.0355; df=12). Control aerosol sprays of distilled water depress EOG's by an average of 5%. The organic calcium channel antagonists diltiazem and verapamil inhibit EOG's by 17% and 36X, respectively, at a concentration of 1.5 X 10~*M. Verapamil produces significant inhibition (F=17.17; p=0.002; df=ll) at 1.5 X 10" 5 M, while the 1,4-dihydropyridine calcium channel antagonists, nicardipine and nifedipine, do not inhibit beyond 1% DMSO controls. Several calmodulin antagonists decrease EOG's, but without correlation to their anti-calmodulin potency. Application of 1.5 X 10"*M chlorpromazine and N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide inhibit EOG's by 31% and 27%, respectively, while trifluoperazine inhibits by 23%. Dibutyryl cAMP, a lipophilic mimic of cAMP, produces 54% inhibition at 1.5 X 10" *M. Dibutyryl cGMP, cGMP, cAMP, and adenosine all decrease EOG's by less than 15% compared to distilled water controls. Forskolin, a reversible activator of adenylate cyclase, inhibits EOG's by 57% at 1.5 X 10"5M, which is significant beyond the 1% DMSO controls (F=17.17; p=0.002; df=ll). These data support the hypothesis that Ca2+ participates in olfactory transduction. Calcium ions could serve as charge carriers, second messengers, or both. Cyclic AMP could be involved with the primary excitatory process or sensory adaptation, or both.
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Tong, Chun-kit Benjamin, and 唐俊傑. "Molecular mechanism of disrupted capacitative calcium entry in familial Alzheimer's disease." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/205870.
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Presenilin (PS) is the catalytic subunit of the gamma-secretase which is responsible for the cleavage of amyloid precursor protein to form beta amyloid (Aβ). Mutations in PS cause familial Alzheimer’s disease (FAD) by increasing the Aβ plaques formation in the brain and thereby induce neurodegeneration. Apart from this, FAD-linked PS mutations have been demonstrated to disrupt cellular calcium (Ca2+) homeostasis. Ca2+is a vital secondary messenger that involved in various neurophysiological functions, including memory, learning, and neuroplasticity and mounting evidence suggesting that Ca2+dysregulation associated with PS mutations may play a proximal role in the AD pathogenesis. Yet, the molecular mechanism for Ca2+dysregulation in AD remains debatable. It has been reported that cellular Ca2+homeostatsis can be disrupted in various ways.
On the one hand, mutant PS has been demonstrated to exaggerate Ca2+release from the endoplasmic reticulum (ER) through different pathways. On the other hand, attenuatedCa2+influx from the extracellular medium through the capacitative Ca2+entry (CCE) pathway has also been reported to bring about cellular Ca2+disruption. However, the molecular mechanism for the PS mutation-mediated CCE deficits is largely unknown. For this reason, the objective of the current study is to elucidate the underlying molecular mechanism for attenuated CCE in AD.
In this study, human neuronal cell line SH-SY5Y is employed as a cellular model to investigate the effect of wild-type or FAD-linked PS1 mutation on CCE pathway. Using single cell Ca2+imaging technique, significant CCE deficits was observed in SH-SY5Y stably expressing FAD-linked PS1mutation, PS1M146L. Interestingly, this CCE attenuation in PS1 mutant expressing cells was not mediated by the down-regulation of STIM1 and Orai1 expression, the known essential molecular players in the CCE pathway. Instead, co-immunoprecipitation and proximity ligation assay have suggested a physical interaction between PS1 and STIM1 proteins. Moreover, a putative gamma-secretase mediated STIM1 cleavage was discovered by western blotting. In addition, confocal imaging showed that PS1M146L significantlyreduceSTIM1 puncta formation and ER translocation followed by the activation of CCE pathway by ER Ca2+store depletion with thapsigargin. This indicated that mutant PS1 attenuates CCE by affecting STIM1 oligomerization or its recruitment with Orai1. Taken together, our results suggested the negative regulatory role of PS on CCE pathway and hypothesized the molecular mechanism of CCE where FAD-linked PS mutation is perceived as a gain-of-function mutation and enhanced the negative impact on STIM1 to inhibit Ca2+entry.This hypothetic model provides new insights into the molecular regulation for CCE pathway and the identification of the interacting domains between PS1 and STIM1 may suggest novel targets for the development of therapeutic agents that help to treat the disease.<br>published_or_final_version<br>Physiology<br>Master<br>Master of Philosophy
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Zhong, Zhihui. "Proinsulin c-peptide : membrane interactions and intracellular signaling /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-886-6.
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Öberg, Camilla. "The life and death of the notch intracellular domain /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-464-X/.
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Al-Khalili, Lubna. "Gene regulation, intracellular signaling and membrane traffic : studies in primary human skeletal muscle cultures /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-866-1/.
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Ng, Dominic Chi Hiung. "Characterizing intracellular signaling mechanisms involved in the progression of cardiac hypertrophy and failure : involvement of JAK/STAT and MAPK pathways." University of Western Australia. Biochemistry and Molecular Biology Discipline Group, 2003. http://theses.library.uwa.edu.au/adt-WU2003.0032.
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[Truncated abstract] The innate ability of the heart to compensate for an increase in workload as a result of disease or injury, through an increase in size and mass is known as cardiac hypertrophy. The hypertrophy of the heart compensates for an increase in workload with an increase in cardiac output. However, excessive hypertrophy can result in cardiac dysfunction and substantially increases the risk of cardiac failure and mortality. The molecular mechanisms that regulate the development of cardiac hypertrophy and cardiac failure are not entirely understood. Traditionally, the G-protein Coupled Receptor (GPCR) and the downstream Mitogen-Activated Protein Kinase (MAPK) family of proteins have been implicated. However, elevated circulating and ventricular levels of several classes of cytokines also suggested that signaling by the downstream effectors of cytokine receptors, such as the Signal Transducers and Activators of Transcription (STATs), may be important. The aim of this thesis was, therefore, to characterize the involvement of MAPK and STAT pathways in regulating cardiac hypertrophy and cardiac failure. A function for MAPK and STAT signaling in regulating cardiac hypertrophy stimulated by the inflammatory cytokine IL-1Β was initially defined in primary cultures of neonatal rat cardiac myocytes. In this study, it was demonstrated that the chemical inhibition of ERK or p38MAPK was sufficient to inhibit IL-1Β-stimulated ANF expression. In contrast, simultaneous inhibition of both ERK and p38MAPK was required to ablate the hypertrophic morphology of cardiac myocytes treated with IL-1Β. These results demonstrated differential signaling from the MAPK isoforms in regulating the gene expression and morphological components of cardiac hypertrophy. In addition, it was revealed that IL-1Β treatment resulted in a delayed response (>60 min) in STAT3α tyrosine phosphorylation, which was subsequently shown to require the initial rapid activation of either ERK or p38MAPK. IL-1Β-stimulated STAT3 phosphorylation was also dependent on the de novo synthesis of secondary signaling molecules. The ablation of the STAT3 tyrosine phosphorylation by the inhibition of ERK or p38MAPK activity, correlated with the attenuation of IL-1Β-stimulated ANF expression, suggesting that signaling through STAT3α may be involved in regulating gene expression associated with IL-1Β cardiac hypertrophy
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Webb, Dominic-Luc. "Temporal monitoring of intracellular Ca²⁺ signaling and origins of Ca²⁺ oscillations /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-741-3/.
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Hung, Chun-hin, and 孔進軒. "Effect of novel Chinese specific presenilin-1 V97L mutation on intracellular calcium homeostasis in human neuroblastoma." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/193533.
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Presenilin-1 (PS1) mutations caused by the PSEN1 gene mutations are the major cause of early onset familial Alzheimer’s disease (EOFAD). Two Chinesespecific EOFAD related PS1 mutations, V97L and A136G, have been found. Studies suggested that V97L mutation lead to the overexpression of Aβ42 and tau hyperphosphorylation, which are the major hallmarks of Alzheimer’s disease (AD), while properties of A136G were unclear. Since calcium dysregulation was suggested to play an important role in AD, the research project investigated if V97L and A136G mutations also lead to altered endoplasmic reticulum (ER) 〖Ca〗^(2+) regulation. SH-SY5Y cells transduced with retrovirus carrying V97L mutant or A136 mutant PSEN1 were used as the experiment models.
In Western blotting, while the PS1 expression level was unaffected in V97L mutant, the expression level was significantly lower in A136G mutant. In carbachol (CCh) perfusion experiment, V97L mutant was found to exaggerate ER 〖Ca〗^(2+) release when stimulated by higher concentration (30, 100 and 300 μM) CCh, while A136G mutant exaggerated ER Ca2+ release when stimulated by 30 μM and 300 μM CCh, but not 100 μM CCh. In 5% fetal bovine serum (FBS) perfusion experiment, both V97L and A136G mutants were found to sensitize 〖Ca〗^(2+) oscillation, which the sensitization effect of V97L was 3 folds of A136G.
The results suggested that V97L mutation exaggerates ER 〖Ca〗^(2+) release, possibly via interaction with IP3R. However the results of A136G were inconclusive and contradicting, therefore further investigation is needed.<br>published_or_final_version<br>Physiology<br>Master<br>Master of Medical Sciences
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Ontiveros, Steven J. "Intracellular trafficking of the hantaviral nucleocapsid protein and its function in modulation of immune signaling." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2009. https://www.mhsl.uab.edu/dt/2010r/ontiveros.pdf.
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Kajikawa, Mariko. "An insulinotropic effect of vitamin D analog with increasing intracellular Ca[2+] concentration in pancreatic β-cells through non-genomic signal transduction". Kyoto University, 2001. http://hdl.handle.net/2433/150514.
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Kam, Wan-lung Kenneth. "Effect of ovariectomy and estrogen replacement on the [beta]-Adrenergic receptor signaling pathway and intracellular Ca2+ homeostasis in the rat heart." Click to view the E-thesis via HKUTO, 2005. http://sunzi.lib.hku.hk/hkuto/record/B31473143.
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Kinkl, Norbert. "Mechanisms of action of fibroblast growth factor 2 (FGF2) in rat retinal cells : photoreceptor survival and intracellular signaling." Université Louis Pasteur (Strasbourg) (1971-2008), 2001. http://www.theses.fr/2001STR13166.
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La dégénérescence des photorécepteurs est à l'origine de nombreuses pathologies rétiniennes aboutissant à une malvoyance voire à la cécité. Les facteurs neurotrophiques représentent une approche thérapeutique potentielle. Au cours de cette thèse nous nous sommes intéressés aux mécanismes d'actions in vitro d'un facteur neurotrophique exprimé dans la rétine, le FGF2. Afin d'étudier les effets directs du FGF2 sur la survie des photorécepteurs, nous avons mis au point un nouveau modèle de culture pure en photorécepteurs à partir de rétine jeune de rat. La pureté en photorécepteurs des cultures est > 99,5%, les cellules gliales de Müller (CGM) représentant < 0,5% de la population cellulaire. Grâce à ce modèle, nous avons montré que le FGF2 stimule la survie des photorécepteurs in vitro et qu'un autre facteur neurotrophique, l'EGF, accélère leur dégénérescence, en activant leurs récepteurs respectifs à activité tyrosine kinase. Les FGFR1-4 ainsi que différentes protéines impliquées dans la transduction du signal du FGF2 (ERK1/2, MEK1, MEK2, PLCg1, SHPTP-2, SOS1, SOS2, Grb2, Shc, Akt), sont exprimés de façon ubiquitaire, mais possèdent des niveau d'expression distinct, selon les différentes populations de cellules rétiniennes (photorécepteurs, rétine interne et CGM) in vivo et in vitro. Néanmoins, le FGF2 y induit des cascades de signalisation distinctes. Ceci indique l'existence de différentes voies de transduction du signal au FGF2 dans la rétine aboutissant toutes à l'activation de ERK1/2. L'inhibition pharmacologique de MEK bloque l'activation de ERK1/2 dans les photorécepteurs et inhibe leur survie induit par le FGF2, alors qu'au niveau des cellules de la rétine interne et des CGM, le FGF2 stimule également des voies d'activation de ERK1/2 indépendantes de MEK. L'ensemble de ces résultats apporte des données originales concernant les effets du FGF2 sur la survie des photorécepteurs ainsi que sur sa signalisation dans la rétine de rat in vitro, et pourrait contribuer à une application potentielle du FGF2 dans une approche clinique.
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Arastoo, Mohammed. "Characterisation of phospholipase C-η enzymes and their relevance to disease". Thesis, University of St Andrews, 2016. http://hdl.handle.net/10023/15698.
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Phospholipase C enzymes are a class of enzymes that catalyse the cleavage of the membrane phospholipid, phosphatidylinositol bisphosphate (PtdIns(4,5)P₂) into the second messengers, inositol trisphosphate (Ins(4,5)P₃) and diacylglycerol (DAG). Six classes of PLC enzymes have been identified based on their structure and mechanism of activation. PLCηs are the most recently identified family and consist of two isozymes, PLCη1 and PLCη2. The aim of this thesis is to further understand the mechanisms of PLCη activation, the role of PLCη2 in relation to neuritogenesis and their roles in certain disease states. Both isoforms were found to be activated by physiological concentrations of intracellular Ca²⁺. Activation of PLCη2 by Gß₁γ₂ was confirmed using a bacterial 2A co-expression system to allow expression of PLCη2, Gß₁ and Gγ₂ with a single plasmid. Localisation studies show a nuclear distribution for PLCη2, but a cytoplasmic distribution for PLCη1 in a neuroblastoma cells line (Neuro2A). PLCη2 has been implicated in brain development and neurite formation. Building on this, a neuronal differentiation model using RA-treated Neuro2A cells stably expressing mutant forms of PLCη2 was utilised, revealing that PLCη2 activity is essential for neuritogenesis but that this process is independent of the enzymes high sensitivity towards Ca²⁺. Furthermore, the direct interaction of PLCη2 and LIMK-1, a previously identified PLCη2 associated protein, is confirmed in the aforementioned neuronal model. Due to the high sensitivity of PLCη enzymes to Ca²⁺ and because of their presence within neurons, they may be involved in Ca²⁺ dysregulation that occurs in certain diseases such as Alzheimer's disease (AD). The role of PLCη2 was assessed in amyloid-ß (Aß) treated differentiated Neuro2A cells, a cellular model for AD pathogenesis. Also a developmental role for PLCη1 was investigated due to a recently identified PLCη1 polymorphism in patients with holoprosencephaly, an embryonic midline defect.
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Kral, Rosana Maria. "The versatility of Fas in death and survival signalling : role of a basic motif in the membrane-proximal intracellular region of the receptor." Nice, 2009. http://www.theses.fr/2009NICE4061.
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The receptor Fas/CD95 can trigger cell death if activated by its ligand, FasL. In addition, Fas can mediate proliferation and differentiation. An imbalance between these life-and-death decisions can cause diseases (e. G. Cancer), thus understanding how these different pathways are controlled is important for the development of therapeutic strategies. Our objective is to dissect the initial events of Fas-mediated signalling at the plasma membrane, focussing on the role of specialized nanodomains enriched in sphingolipids and cholesterol (SCNs or lipid rafts). Chakrabandhu et al. (2007) showed that palmitoylation of an intracellular cysteine constitutively targets a fraction of Fas to SCNs. FasL rapidly induces raft recruitment of partners to form the death-inducing signalling complex (DISC). Efficient DISC formation then occurs upon raft-dependent receptor internalization. Here we provide evidence for a second determinant of Fas SCN localization, a membrane-proximal intracellular group of basic residues. Despite being palmitoylated, a receptor mutated at these residues showed reduced SCN association and consequently, diminished ability to relay the death signal. This mutant also showed exacerbated promotion of non-death signals, as well as increased interaction with the actin cytoskeleton, that had previously been held to depend on SCN localization of Fas. This basic region is similar to motifs that target proteins to phosphoinositides (PIs), suggesting a potential link between Fas and PIs. These versatile regulatory lipids are rapidly convertible and can tightly control the presence of membrane-anchoring sites for proteins. We also studied this potential link<br>Le récepteur Fas/CD95 peut induire la mort cellulaire s’il est activé par son ligand, FasL. Fas peut aussi induire la prolifération et la différenciation. Une imbalance entre ces décisions de mort et survie peut entraîner des maladies (p. E. Cancer). Comprendre comment ces voies différentes sont contrôlées est important pour dévélopper des stratégies thérapeutiques. Notre objectif est de disséquer les evenements précoces de la signalisation de Fas à la membrane plasmique, en focalisant sur le rôle de domaines spécialisés, riches en sphingolipides et choléstérol (rafts). Chakrabandhu et al. (2007) ont identifié la palmitoylation d’un cystéine intracellulaire comme signal d’adressage constitutif de Fas aux rafts. FasL induit le recrutement rapide de partenaires aux rafts pour la formation du DISC (death-inducing signalling complex). Le DISC est formé plus efficacement après l’internalisation raft-dépendante de Fas. Ici, nous avons identifié un deuxième signal d’adressage aux rafts, un groupe intracellulaire de résidus basiques. Quoiqu’étant palmitoylé, un récepteur muté était moins associé aux rafts et, par conséquence, montrait une induction réduite du signal de mort. Ce mutant induisait des signaux de survie excessifs et montrait une association accrue avec le cytosquélette d’actine, qui a été considérée auparavant être dépendante de l’association aux rafts. Cette région basique est similaire aux motifs qui permettent l’interaction avec des phosphoinositides (PI), suggérant un lien potentiel entre Fas et les PI. Ces lipides versatiles sont rapidement convertibles et peuvent réguler la présence de sites d’ancrage pour des protéines à la membrane. Nous avons étudié ce lien
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Matos, Pinto Thiago. "Computational models of intracellular signalling and synaptic plasticity induction in the cerebellum." Thesis, University of Hertfordshire, 2013. http://hdl.handle.net/2299/11560.
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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.
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Magno, Aaron. "Proteins associated with the intracellular signalling tail of the calcium-sensing receptor and their impact on receptor function." University of Western Australia. School of Medicine and Pharmacology, 2009. http://theses.library.uwa.edu.au/adt-WU2010.0028.
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[Truncated abstract] The calcium-sensing receptor (CaR) is a G protein-coupled receptor that can respond to changes in extracellular calcium and plays an integral role in calcium homeostasis. Later studies revealed that the CaR was stimulated by not just calcium, but a diverse range of stimuli and that activation of the receptor regulated a host of different biological processes. The CaR is linked to these cellular responses via the various signalling pathways initiated by the receptor. Recent yeast two-hybrid studies have identified a number of accessory proteins that, through their interaction with the intracellular tail of the CaR, are able to regulate important functional aspects of the receptor, including its signalling and degradation. We hypothesised that many more proteins that bind to the CaR-tail await identification, especially since most of the previous studies used the yeast two-hybrid system to screen cDNA libraries generated from tissues that are important to whole body calcium homeostasis, such as the parathyroid gland and kidney. In order to identify novel binding partners of the CaR, which may affect its function, particularly in biological processes that might be unrelated to calcium homeostasis, our laboratory performed a yeast two-hybrid screen of an EMLC.1 mouse pluripotent haemopoietic cell line library using the intracellular tail of the human CaR as bait. This screen revealed a large number of
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CORRICELLI, Mariangela. "Two different points of view on signal transduction: defective autophagy as a key feature of Cerebral Cavernous Malformations and c-Src as modulator of intracellular Ca2+ homeostasis." Doctoral thesis, Università degli studi di Ferrara, 2018. http://hdl.handle.net/11392/2478764.
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Nel primo progetto abbiamo indagato il coinvolgimento dell’autofagia nella patogenesi delle Malformazioni Cavernose Cerebrali (CCM). CCM è una grave malattia cerebrovascolare che colpisce circa lo 0.3-0.5% della popolazione ed è caratterizzata da capillari dilatati e fragili che predispongono a convulsioni, deficit neurologici e fatali emorragie intracerebrali. CCM è una malattia genetica che può insorgere in modo sporadico o essere ereditata con modalità di trasmissione autosomica dominante. La causa è stata identificata in mutazioni che determinano la perdita di tre geni, KRIT1 (CCM1), CCM2 (MGC4607) e PDCD10 (CCM3), che si verificano nelle forme sia sporadiche sia familiari. L'autofagia è un processo di degradazione che preserva l'omeostasi intracellulare e svolge funzioni essenziali di controllo qualità. Infatti, diversi studi hanno identificato un’associazione tra disregolazione dell’autofagia e diverse patologie umane. Nel nostro lavoro mostriamo che la perdita del gene KRIT1 sopprime l'autofagia, portando ad un aberrante accumulo dell'adattatore autofagico p62/SQSTM1, difetti nei sistemi di controllo qualità e aumento dello stress intracellulare. La perdita di funzione della proteina KRIT1 attiva il pathway mTOR-ULK1, il principale regolatore dell'autofagia, e il trattamento con inibitori di mTOR reverte alcuni dei fenotipi molecolari e cellulari associati alle CCM. La riduzione nei livelli di autofagia è evidente anche in cellule endoteliali umane in cui è stato silenziato CCM2, in cellule e in tessuti di topo CCM3 knockout endotelio-specifico, e in lesioni CCM umane. Inoltre, la deregolazione dell'autofagia è altamente correlata alla transizione endoteliale-mesenchimale, evento cruciale che contribuisce alla progressione delle CCM. Complessivamente, i nostri dati indicano un ruolo chiave per la deregolazione dell’autofagia nella patogenesi delle CCM, fornendo così nuove possibilità per lo sviluppo di strategie farmacologiche per prevenire o contrastare gli esiti clinici avversi conseguenti alle lesioni. [da: Marchi et al. (2015) Defective autophagy is a key feature of cerebral cavernous malformations. EMBO Mol Med 7(11):1403-17].
Il secondo progetto è focalizzato sullo studio del ruolo del proto-oncogene c-Src nella modulazione del segnale Ca2+ intracellulare. c-Src è una tirosin chinasi di tipo non recettoriale e svolge un ruolo chiave in diverse vie di segnalazione coinvolte in eventi cellulari fondamentali, quali crescita cellulare, sopravvivenza, adesione, migrazione e invasione. È nota la correlazione diretta tra deregolazione o sovraespressione di c-Src e diversi tumori umani. Il calcio (Ca2+) è un secondo messaggero intracellulare altamente versatile che agisce in funzioni cellulari cruciali. Durante la carcinogenesi e la progressione del tumore, il segnale Ca2+ viene rimodellato in modo tale da supportare la maggior parte delle caratteristiche tipiche del cancro. In questo progetto abbiamo identificato una funzione chiave per c-Src nella modulazione dell'omeostasi del Ca2+ intracellulare. c-Src riduce il rilascio di Ca2+ dal reticolo endoplasmatico (ER) in seguito a stimolazione con agonista e altera i livelli intracellulari di Ca2+. Tale regolazione del segnale Ca2+ mediata da c-Src è strettamente dipendente dalla sua attività catalitica e dalla sua localizzazione. I nostri risultati hanno stabilito che c-Src controlla i livelli di Ca2+ intracellulare attraverso la fosforilazione di uno specifico target. Questa fosforilazione diretta media l'effetto regolatorio di c-Src sul rilascio di Ca2+ dal ER, influenzando quindi il segnale Ca2+ negli altri compartimenti. Nel complesso il nostro lavoro ha identificato un nuovo ruolo per c-Src nel controllo delle dinamiche del Ca2+ e contribuisce a chiarire in modo più dettagliato come c-Src agisca sia in un contesto fisiologico sia patologico.<br>In the first project we investigated the involvement of the autophagy in the pathogenesis of the Cerebral Cavernous Malformation (CCM). Cerebral cavernous malformation is a major cerebrovascular disease affecting approximately 0.3-0.5% of the population and is characterized by enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhages. Cerebral cavernous malformation is a genetic disease that may arise sporadically or be inherited as an autosomal dominant condition with incomplete penetrance and variable expressivity. Causative loss-of-function mutations have been identified in three genes, KRIT1 (CCM1), CCM2 (MGC4607), and PDCD10 (CCM3), which occur in both sporadic and familial forms. Autophagy is a bulk degradation process that maintains intracellular homeostasis and that plays essential quality control functions within the cell. Indeed, several studies have identified the association between dysregulated autophagy and different human diseases. Here, we show that the ablation of the KRIT1 gene strongly suppresses autophagy, leading to the aberrant accumulation of the autophagy adaptor p62/SQSTM1, defective quality control systems, and increased intracellular stress. KRIT1 loss-of-function activates the mTOR-ULK1 pathway, which is a master regulator of autophagy, and treatment with mTOR inhibitors rescues some of the molecular and cellular phenotypes associated with CCM. Insufficient autophagy is also evident in CCM2-silenced human endothelial cells and in both cells and tissues from an endothelial-specific CCM3-knockout mouse model, as well as in human CCM lesions. Furthermore, defective autophagy is highly correlated to endothelial-to-mesenchymal transition, a crucial event that contributes to CCM progression. Taken together, our data point to a key role for defective autophagy in CCM disease pathogenesis, thus providing a novel framework for the development of new pharmacological strategies to prevent or reverse adverse clinical outcomes of CCM lesions. [From: Marchi et al. (2015) Defective autophagy is a key feature of cerebral cavernous malformations. EMBO Mol Med 7(11):1403-17].
The second project was focused on the investigation of a potential role for the proto-oncogene c-Src in the modulation of intracellular Ca2+ signalling. c-Src is a non-receptor tyrosine kinase that plays a pivotal role in several signalling pathways involved in fundamental cellular events, including cell growth, survival, cell adhesion, migration and invasion. It is well established the link existing between c-Src deregulation or overexpression and several human cancers. Calcium (Ca2+) is a highly versatile intracellular second messenger that acts in crucial cellular functions. During carcinogenesis and tumour progression, Ca2+ signalling is significantly remodelled in a way that sustains most of typical cancer hallmarks, as uncontrolled proliferation and evasion of programmed cell death. We identified a key function for c-Src in the modulation of intracellular Ca2+ homeostasis. c-Src downregulates the Ca2+ release from the endoplasmic reticulum (ER) upon agonist stimulation and significantly alters the intracellular Ca2+ levels. c-Src regulation of Ca2+ signalling is strictly dependent on its catalytic activity and subcellular localization. Our results established that c-Src controls Ca2+ handling through phosphorylation of a specific molecular target. This direct phosphorylation mediates the c-Src effect on Ca2+ release from the intracellular store, thus affecting Ca2+ homeostasis at the other intracellular compartments. Overall, our work identified a new role for c-Src in the control of the intracellular Ca2+ dynamics and contribute to deeply understand how c-Src acts in the context of its physiological and pathological functions.
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Huang, Yun. "Integration of Extracellular and Intracellular Calcium Signals: Roles of Calcium-Sensing Receptor (CASR), Calmodulin and Stromal Interaction Molecule 1 (STIM1)." Atlanta, Ga. : Georgia State University, 2008. http://digitalarchive.gsu.edu/chemistry_diss/28/.
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Thesis (Ph. D.)--Georgia State University, 2008.<br>Title from title page (Digital Archive@GSU, viewed July 1, 2010) Jenny J. Yang, committee chair; Edward Brown, Giovanni Gadda, Zhi-ren Liu, committee members. Includes bibliographical references (p. 230-258).
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Mennour, Sabrina. "Activité de liaison à l’ARN des protéines de la voie de signalisation MAPK (Mitogen-Activated Protein Kinase) dans le mélanome LncRNA-Mediated Protein-Protein Scaffolding in Intracellular Signal Transduction Pathways." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASL062.
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Des études récentes ont souligné l'importance des ARN dans la régulation des interactions protéine-protéine. En permettant l'assemblage de complexes protéiques nucléaires, les ARN non codants agissent comme des échafaudages et favorisent ainsi les interactions protéine-protéine afin de réguler l'état de la chromatine. Les ARN sont également capables d’interagir avec des protéines pour en moduler leurs activités, leurs interactions ou encore leurs localisations. Cependant, le rôle potentiel des ARN dans la régulation des interactions protéine-protéine dans les principales voies de transduction du signal cytoplasmique reste largement inconnu. L’objectif de la thèse a consisté à rechercher et à mettre en évidence une activité de liaison directe à l’ARN de protéines impliquées dans les voies de transduction du signal cytoplasmique et d’évaluer le rôle des interactions ARN-protéines de transduction sur la signalisation intra-cellulaire. En utilisant des approches de CLIP (CrossLink et ImmunoPrécipitation) et de 2C (Complexe Capture : purification d’ARN basée sur leur affinité pour une matrice de silice), qui peuvent mettre en évidence des interactions directes entre les protéines et les ARN in vivo, nous avons démontré une interaction directe avec l’ARN de certaines protéines clés de la voie de signalisation MAPK. Des approches de microscopie telles que le PLA (Proximity Ligation Assay) nous ont permis de démontrer une modulation des interactions protéine-protéine dépendant de l'ARN dans la voie MAPK, suggérant qu'une composante ARN est impliquée dans la stabilisation de ces interactions protéine-protéine. Nous avons spécifiquement identifié un mutant de délétion BRAF, une protéine oncogène centrale et une cible thérapeutique dans le mélanome, qui montre une déficience dans son activité de liaison à l'ARN, ainsi qu’une activité de signalisation réduite. En mettant en évidence l'existence d'une modulation des interactions protéine-protéine médiée par l'ARN, cette étude démontre l'importance sans précédent de l'activité de liaison à l'ARN des principales protéines de transduction du signal qui devrait être prise en compte dans la compréhension et le ciblage des cellules tumorales<br>Recent studies have underscored the importance of RNAs in the regulation of protein-protein interactions. By allowing the assembly of protein complexes, non-coding RNAs act as scaffolds and thus promote protein-protein interactions in order to regulate the chromatin state. RNAs are also able to interact with proteins in order to modulate their activities, interactions or localisation. In the cytoplasm, signalling pathways are regulated through a cascade of protein-protein interactions. In the MAPK (Mitogen-Activated Protein Kinase) signalling pathway, the binding of a ligand to a membrane receptor triggers a cascade of phosphorylation and protein-protein interactions that allow the transduction of the signal. Abnormal activity of this pathway through increased ligand binding or activating mutations lead to cellular dysfunction associated with tumor initiation and progression.The potential role of RNAs in the direct regulation of protein-protein interactions of key cytoplasmic signal transduction pathways remains largely unknown. The aim of the thesis was to investigate and demonstrate the direct RNA binding activity of proteins involved in the MAPK pathway and to evaluate the role of RNA-protein interactions on intracellular signalling.Using a combination of CLIP (crosslinking and immunoprecipitation) and silica matrix-based affinity capture (2C complex capture) approaches that can uncover direct interactions between proteins and RNAs in vivo, we demonstrated a direct interaction between key MAPK signalling proteins and RNA in melanoma cells. Subsequent microscopy studies using proximity ligation assay (PLA) led us to demonstrate an RNA-dependent modulation of protein-protein interactions in the MAPK pathway, suggesting that an RNA component is involved in the stabilization of these protein-protein interactions. We specifically identified a deletion mutant in BRAF, a central oncogenic protein and therapeutic target in melanoma, that lacks RNA binding activity and harbors decreased signalling activity.By highlighting the existence of an RNA-mediated modulation of protein-protein interactions, this study shows the unprecedented importance of the RNA binding activity of key signal transduction proteins that should be considered in the understanding and targeting of tumor cells
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Rico, Bautista Elizabeth. "Negative regulation of growth hormone (GH) signaling /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-184-9/.
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Gatlin, Jesse C. "Eicosanoid-mediated repellent signaling in the nerve growth cone : a role for the PKC substrate MARCKS /." Connect to full text at ProQuest Digital Dissertations. IP filtered, 2005.
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Thesis (Ph.D. in Cell and Developmental Biology) -- University of Colorado at Denver and Health Sciences Center, 2005.<br>Typescript. Includes bibliographical references (leaves 123-141). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;
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Del, Río Iñiguez Iratxe. "Intracellular vesicle traffic, immunological synapse and T cell activation. Modulation by Human Immunodeficiency Virus type 1 Rac1-Rab11-FIP3 regulatory hub coordinates vesicle traffic with actin remodeling and T cell activation Rab11-FIP3 regulation of Lck endosomal traffic controls TCR signal transduction." Thesis, Sorbonne université, 2018. http://www.theses.fr/2018SORUS561.
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La formation et les fonctions de la synapse immunologique sont le résultat d’un processus de polarisation cellulaire du lymphocyte T. Ce processus dépend de l’action coordonnée du cytosquelette d’actine et de microtubules, et du trafic vésiculaire intracellulaire. Le récepteur T ainsi que des protéines impliquées dans sa signalisation intracellulaire sont associés à la membrane plasmique et à des compartiments vésiculaires endosomiaux, et circulent en continu entre ces deux localisations. Je montre dans cette thèse que la tyrosine kinase Lck et la GTPase Rac1 sont associés aux endosomes exprimant Rab11. Leur localisation intracellulaire et leurs fonctions dans la formation de la synapse immune, l’activation des lymphocytes T et les remaniements du cytosquelette d’actine dépendent de Rab11 et de son effecteur FIP3. Je me suis intéressée également à la protéine Nef du VIH-1, importante pour la réplication du virus et pour la pathogénèse associée au SIDA. Nef affecte le trafic endosomial, l’activation et le cytosquelette des lymphocytes T infectés. Nous avons mis en évidence que Nef concentre de façon spécifique des formes actives des protéines de signalisation dans le compartiment endosomial Rab11. Ceci conduit à une activation de certains gènes associés aux réponses précoces et tardives des lymphocytes T. Ce processus est contrecarré par la déplétion de FIP3, qui disperse le compartiment concentrant ces protéines. En conclusion, nos données révèlent de nouveaux mécanismes impliquant le trafic vésiculaire intracellulaire dans le contrôle de l’activation et du cytosquelette des lymphocytes T et leur détournement par le virus du SIDA<br>The immunological synapse is the result of a T cell polarization process that depends on the orchestrated action of the actin and microtubule cytoskeleton and of intracellular vesicle traffic. The T cell antigen receptor (TCR) and various components of its proximal signaling machinery are associated with the plasma membrane and vesicular endosomal compartments, continuously trafficking between the two locations. I show in this thesis that the subcellular localization and function of the tyrosine kinase Lck and the actin cytoskeleton regulator Rac1, depend on the Rab11 recycling endosomal compartment, and more in particular, on the Rab11 effector FIP3. Importantly, FIP3-dependent Lck and Rac1 localization controls early TCR signaling, intracellular calcium concentration, IL-2 gene expression and morphological events, like T cell spreading and synapse symmetry. Moreover, I investigated how the HIV-1 accessory protein Nef, which is crucial for virus replication in vivo and AIDS pathogenesis, specifically hijacks several active signaling molecules, concentrating them in the Rab11 endosomal compartment, and concomitantly inducing the upregulation of some early and late T cell activation genes. Interestingly, dispersion of this concentration by depleting Rab11-FIP3, counteracted Nef-induced gene expression upregulation. Therefore, by modifying their endosomal traffic, Nef hijacks signaling and actin cytoskeleton regulators to dually modulate their functional outputs. In conclusion, our data shed new light into the molecular mechanisms orchestrating endosomal traffic with T cell activation and cytoskeletal rearrangements, and their subversion during HIV-1 infection
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Sammons, Wendy L. "Generation and characterization of an attenuated mutant in a response-regulator gene of Francisella tularensis live vaccine strain (LVS)." [Tampa, Fla.] : University of South Florida, 2007. http://purl.fcla.edu/usf/dc/et/SFE0002268.
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Aggarwal, Kamna. "A View of the IMD Pathway from the RHIM." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/463.
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Innate immunity is the first line of defense against invading pathogens. It functions to eliminate pathogens and also to control infections. The innate immune response is also important for the development of pathogen-specific adaptive immune responses. As a result, the study of innate immune signaling pathways is crucial for understanding the interactions between host and pathogen. Unlike mammals, insects lack a classical adaptive immune response and rely mostly on innate immune responses.
Innate immune mechanisms have been widely studied in the fruit fly, Drosophila melanogaster. The genetic and molecular tools available in the Drosophila system make it an excellent model system for studying immunity. Furthermore, the innate immune signaling pathways used by Drosophila show strong homology to those of vertebrates making them ideal for studying these pathways. Drosophila immunity relies on cellular and humoral innate immune responses to fight pathogens. The hallmark of the Drosophilahumoral immune response is the rapid induction of antimicrobial peptide genes in the fat body. The production of these antimicrobial peptides is regulated by two immune signaling pathways-Toll and Immune Deficency (IMD) pathways.
The Toll pathway responds to many Gram-positive bacterial and fungal infections , while the IMD pathway is potently activated by DAP-type peptidoglycan (PGN) from Gram-negative bacteria and certain Gram-positive bacteria. Two receptors, PGRP-LC and PGRP-LE, are able to recognize DAP-type PGN at the cell surface or in the cytosol, respectively, and trigger the IMD pathway. Upon binding DAP-type PGN, both PGRP-LC and PGRP-LE dimerize/ multimerize and signal to the downstream components of IMD pathway. It is unclear how the receptor activates its downstream components.
My work has focused on understanding the molecular events that take place at the receptors following there activation. In these studies I have identified a common motif in the N-terminal domains of both the receptors, known as the RHIM-like domain. The RHIM-like domain is critical for signaling by either receptor, but the mechanism(s) involved remain unclear. IMD, a downstream component of the pathway, associates with both PGRP-LC and -LE but the interaction of PGRP-LC with IMD is not mediated through its RHIM-like domain. Also, mutations affecting the PGRP-LC RHIM-like motif are defective in all known downstream signaling events. However, the RHIM-like mutant receptors are capable of serving as a platform for the assembly of all known components of a receptor proximal signaling complex. These results suggest that another, unidentified component of the IMD signaling pathway may function to mediate interaction with the RHIM-like motif.
I performed a yeast two-hybrid screen to identify proteins that might interact with the receptor PGRP-LC through its RHIM- like domain. With this approach, two new components of the IMD pathway were identified. The first component I characterized is called Rudra and it is a critical feedback inhibitor of peptidoglycan receptor signaling. The other factor is known as RYBP, it includes a highly conserved ubiquitin binding motif (NZF), and RNAi studies suggest it is a critical component of the IMD pathway. The identification and characterization of these two new components of the IMD pathway has provided a new insight into the molecular events that take place proximal to the receptor.
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Girard, Emmanuelle. "Caractérisation de nouveaux régulateurs du transport intracellulaire du cholestérol : mise en évidence du rôle de la dynamine et des GTPases Rab7 et Rab9." Phd thesis, Université Paris Sud - Paris XI, 2013. http://tel.archives-ouvertes.fr/tel-00923162.
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Le transport intracellulaire du cholestérol et sa distribution correcte au niveau des différentes membranes sont essentiels pour assurer de nombreuses fonctions cellulaires. Malgré l'importance de ce transport les mécanismes de sa régulation restent encore mal connus. L'objectif de cette thèse était de mieux caractériser les acteurs du transport intracellulaire du cholestérol. Dans ce contexte, nous nous sommes intéressés à deux acteurs de ce transport : la dynamine et les Rab GTPases. Dans la première partie de la thèse nous avons utilisé le dynasore, un inhibiteur pharmacologique de la dynamine pour étudier le rôle de la dynamine dans le contrôle du transport endolysosomal dans les cellules HeLa et les macrophages humains. Nous avons ainsi confirmé le rôle de la dynamine dans la sortie du compartiment endolysosomal et la régulation de l'homéostasie du cholestérol. Dans la deuxième partie de la thèse, nous avons étudié le rôle de Rab7 et de Rab9 dans le transport du cholestérol en utilisant la technique d'ARN interférence ainsi que l'expression de mutants dominant négatifs. Nous avons montré qu'en plus de son rôle classique dans les étapes tardives du transport du cholestérol, Rab7 contrôle les étapes précoces du transport endosomal. Enfin, nous avons évalué le rôle de Rab7 dans notre modèle de macrophages humains surchargés. Nous avons mis en évidence un effet limité de l'inactivation de Rab7 sur le contrôle de l'homéostasie du cholestérol mais à l'inverse un effet majeur pour l'efflux du cholestérol vers l'apo AI. En conclusion, notre étude a permis de mieux caractériser le transport vésiculaire du cholestérol et de démontrer son importance dans la régulation de l'homéostasie intracellulaire en cholestérol. Nos résultats permettent également d'établir le rôle critique de Rab7 dans le trafic des LDL au niveau des endosomes précoces.
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Pelletier, Nadine. "Régulation des gènes C/EBPS par des voies de signalisation intracellulaire dans les cellules épithéliales intestinales." Mémoire, Université de Sherbrooke, 1996. http://savoirs.usherbrooke.ca/handle/11143/3110.
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Nous avons utilisé la lignée épithéliale intestinale de crypte de rat IEC-6 afin de mieux comprendre le rôle des C/EBPs dans la différenciation et la prolifération de l'intestin. Nous avons vérifié l'expression des ARNm de C/EBP$\alpha$, C/EBP$\beta$ et C/EBP$\delta$ suite à la stimulation des cellules IEC-6 par la forskoline et l'IBMX qui stimulent la PKA, le TPA qui stimule la PKC et la thapsigargine qui augmente la concentration de Ca$\sp{2+}$ cytoplasmique. La forskoline et l'IBMX induisent les niveaux d'ARNm des trois C/EBPs avec différentes cinétiques tandis que le TPA induit seulement les niveaux d'ARNm de C/EBP$\alpha$ et de C/EBP$\beta.$ La thapsigargine induit les niveaux d'ARNm des trois isoformes par un mécanisme de régulation post-transcriptionnel sans affecter les niveaux de protéines. Les variations des niveaux d'ARNm, induits par les trois voies de signalisation, étaient indépendantes d'une synthèse nouvelle de protéines. Les analyses des niveaux de protéine par Western nous ont permis de constater une induction des trois isoformes en accord avec celle des ARNm suite à une stimulation par la forskoline et l'IBMX. Nous avons observé que le patron d'expression des protéines C/EBP$\alpha$ et C/EBP$\beta$ concorde avec celui des ARNm après une stimulation par le TPA. La capacité de liaison à l'ADN des C/EBPs n'est pas affectée significativement par les voies de la PKA, de la PKC et du Ca$\sp{2+}.$ Les voies de la PKA et de la PKC peuvent moduler l'expression de certains gènes de la réponse inflammatoire comme l'haptoglobine et le thiostatin. La stimulation de ces différentes voies inhibe la prolifération cellulaire des cellules IEC-6 en absence et en présence de sérum. Finalement, nous avons observé que ces différentes voies pouvaient induire à des niveaux variables l'ARNm de p21, un gène très important dans l'arrêt de la prolifération cellulaire. [Résumé abrégé par UMI] [Symboles non conformes]
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Dinerstein-Cali, Hélène. "Etude des molecules impliquees dans la transduction du signal du recepteur de l'hormone de croissance (doctorat : endocrinologie et interactions cellulaires)." Paris 11, 2000. http://www.theses.fr/2000PA11T005.
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Nissen, Johan. "Fonction de coactivateur in vivo et in vitro : lien entre la signalisation intracellulaire et la régulation génique." Montpellier 2, 2001. http://www.theses.fr/2001MON20048.
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Ichas, François. "La mitochondrie, organelle excitable : participation active à la signalisation calcique intracellulaire." Bordeaux 2, 1997. http://www.theses.fr/1997BOR28475.
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Kiefer, Céline. "Mécanisme cinétique et moléculaire de CD38/NAD+glycohydrolase en relation avec la signalisation intracellulaire." Université Louis Pasteur (Strasbourg) (1971-2008), 2002. http://www.theses.fr/2002STR13048.
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Le CD38/NAD+glycohydrolase (EC 3. 2. 2. 5 et 3. 2. 2. 6), conservé dans l'évolution, est à la fois une glycoprotéine transmembranaire de type II qui possède des propriétés de signalisation notamment chez les lymphocytes et d'adhésion cellulaire et une ectoenzyme qui transforme le nicotinamide adénine dinucléotide (NAD+) en ADP-ribose cyclique (ADPRc), un second messager impliqué dans la mobilisation du calcium intracellulaire dans une variété de cellules aussi bien chez les mammifères que chez les plantes et les invertébrés. La question posée était de savoir si l'activité catalytique du CD38/NADase est directement responsable de la signalisation intracellulaire induite par son activation. Selon notre hypothèse, seul un changement conformationnel de l'enzyme, conduisant à une interaction avec des partenaires de surface cellulaire, serait responsable de son implication dans la signalisation intracellulaire et indépendamment des produits de la réaction enzymatique. Ce travail de thèse consistait donc à éclaircir cette problématique selon deux approches complémentaires : 1) après avoir établi un schéma cinétique unificateur pour les enzymes de mammifères et d'invertébrés impliqués dans la biosynthèse de l'ADPRc, il s'agissait d'étudier le mécanisme cinétique et moléculaire en modulant les différentes constantes cinétiques et vérifier l'implication de l'ADPRc complexé à l'enzyme dans la signalisation 2) dans une deuxième partie, il s'agissait d'étudier la relation entre la structure du CD38/NADase et ses fonctions catalytiques et de signalisation en effectuant différentes mutations à des endroits de l'enzyme susceptibles d'être impliqués dans la catalyse et/ou la liaison du substrat. Ce travail a permis de conclure que seule la fixation de ligands (substrat, anticorps) serait responsable d'un changement conformationnel du CD38/NADase, conduisant à un dialogue avec les partenaires de signalisation<br>CD38/NAD+glycohydrolase (EC 3. 2. 2. 5 et 3. 2. 2. 6) is at the same time an evolutionarily conserved type II transmembrane glycoprotein, which has signalling (e. G. In lymphocytes) and adhesion functions and also a multifunctional enzyme that catalyses the conversion of nicotinamide adenine dinucleotide (NAD+) into cyclic adenosine diphophoribose (cADPR), a second messenger involved in intracellular calcium mobilisation in a variety of cells from mammals to plants and invertebrates. The question raised was the following: is the catalytic activity of CD38/NADase directly responsible for the intracellular signalling triggered by its activation ? According to our hypothesis, only a conformational change of the enzyme that is followed by a cross talk with partner's proteins is responsible for its implication in intracellular signalling events and independently of its reaction products. This thesis aimed to elucidate this problem according to two complementary approaches : 1) after we established an unifying kinetic scheme for the mammalian and invertebrate enzymes involved in cADPR biosynthesis, we studied the kinetic and molecular mechanism by modulating the different kinetic constants, we thus want to verify the implication of cADPR complexed to the enzyme in the signalling events 2) in a second part, we studied the relationship between the structure of CD38/NADase and its catalytic and signalling functions by mutating different putative residues involved in catalysis and/or substrate binding. This work concludes that only ligand binding (substrate, antibody) could be responsible for conformational changes of CD38/NADase, which trigger a cross talk with signalling partners
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Descamps, Simon. "Mise en évidence du rôle du Nerve Growth Factor dans le cancer du sein : effets biologiques et signalisation intracellulaire." Lille 1, 2000. https://pepite-depot.univ-lille.fr/LIBRE/Th_Num/2000/50376-2000-129.pdf.
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Le Nerve Growth Factor (ngf) est le facteur de croissance neurotrophique par excellence. Nous avons démontré que le ngf est un puissant mitogène pour les cellules de cancer du sein mais qu'il n'a pas d'effet sur la croissance des cellules épithéliales normales. Nous avons également mis en évidence l'activité anti-apoptotique du ngf sur les cellules mammaires cancéreuses. Nous avons ensuite étudié les mécanismes de signalisations responsables de l'effet mitogène et de l'effet anti-apoptotique du ngf. Nos résultats démontrent que le ngf active les récepteurs trka et les map-kinases pour stimuler la prolifération et active p75 et le facteur de transcription nf-b pour inhiber l'apoptose. Nous avons donc démontré que le ngf emprunte deux voies de signalisation bien distinctes pour aboutir à ces deux effets biologiques. Nous avons enfin mesuré par rt-pcr quantitative en temps réel, l'expression des ARNm des récepteurs du ngf, trka et p75, sur une série de biopsies de cancers du sein. Nos résultats montrent que l'expression importante du récepteur trka est associée a une survie prolongée des patientes. L'ensemble de notre travail montre pour la première fois que le ngf est impliqué dans le développement du cancer du sein et ouvre de nouvelles perspectives pour le pronostic et le traitement de cette pathologie.
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Tesz, Gregory J. "Role of MAP4K4 Signaling in Adipocyte and Macrophage Derived Inflammation: A Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/380.
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Human obesity is increasing globally at an impressive rate. The rise in obesity has led to an increase in diseases associated with obesity, such as type 2 diabetes. A major prerequisite for this disease is the development of insulin resistance in the muscle and adipose tissues. Interestingly, experiments in rodent models suggest that adipocytes and macrophages can profoundly influence the development of insulin resistance. Accordingly, the number of adipose tissue macrophages increases substantially during the development of obesity. Numerous research models have demonstrated that macrophages promote insulin resistance by secreting cytokines, like TNFα, which impair whole body insulin sensitivity and adipose tissue function. Additionally, enhancements of murine adipose function, particularly glucose disposal, prevent the development of insulin resistance in mice on a high fat diet. Thus, mechanisms which enhance adipose function or attenuate macrophage inflammation are of interest.
Our lab previously identified mitogen activated protein kinase kinase kinase kinase 4 (MAP4K4) as a potent negative regulator of adipocyte function. In these studies, TNFα treatment increased the expression of adipocyte MAP4K4. Furthermore, the use of small interfering RNAs (siRNA) to block the increase in MAP4K4 expression protected adipocytes from some of the adverse effects of TNFα. Because MAP4K4 is a potent negative regulator of adipocyte function, an understanding of the mechanisms by which TNFα regulates MAP4K4 expression is of interest. Thus, for the first part of this thesis, I characterized the signaling pathways utilized by TNFα to regulate MAP4K4 expression in cultured adipocytes. Here I show that TNFα increases MAP4K4 expression through a pathway requiring the transcription factors activating transcription factor 2 (ATF2) and the JUN oncogene (cJUN). Through TNFα receptor 1 (TNFR1), but not TNFR2, TNFα increases MAP4K4 expression. This increase is highly specific to TNFα, as the inflammatory agents IL-1β, IL-6 and LPS did not affect MAP4K4 expression. In agreement, the activation of cJUN and ATF2 by TNFα is sustained over a longer period of time than by IL-1β in adipocytes. Finally, MAP4K4 is unique as the expression of other MAP kinases tested fails to change substantially with TNFα treatment.
For the second part of this thesis, I assessed the role of MAP4K4 in macrophage inflammation in vitro and in vivo. To accomplish this task, pure β1,3-D-glucan shells were used to encapsulate siRNA. Glucan shells were utilized because they are effectively taken up by macrophages which express the dectin-1 receptor and they survive oral delivery. I demonstrate that these β1,3-D-glucan encapsulated RNAi particles (GeRPs) are efficiently phagocytosed and capable of mediating the silencing of multiple macrophage genes in vitro and in vivo. Importantly, oral treatment of mice with GeRPs fails to increase plasma IFNγ and TNFα or alter serum AST and ALT levels. Orally administered GeRPs are found in macrophages isolated from the spleen, liver, lung and peritoneal cavity and mediate macrophage gene silencing in these tissues.
Utilizing this technology, I reveal that MAP4K4 augments the expression of TNFα in macrophages following LPS treatment. Oral delivery of MAP4K4 siRNA in GeRPs silences MAP4K4 expression by 70% and reduces basal TNFα and IL-1β expression significantly. The depletion of MAP4K4 in macrophages protects 40% of mice from death in the LPS/D- galactosamine (D-GalN) model of septicemia, compared to less than 10% in the control groups. This protection associates with significant decreases in serum TNFα concentrations following LPS/D-GalN challenge. Consistent with reduced macrophage inflammation, hepatocytes from mice treated orally with GeRPs targeting MAP4K4 present less apoptosis following LPS/D-GalN treatment. Thus, MAP4K4 is an important regulator of macrophage TNFα production in response to LPS.
The results presented here add to the knowledge of MAP4K4 action in adipocyte and macrophage inflammation substantially. Prior to these studies, the mechanism by which TNFα controlled MAP4K4 expression in adipocytes remained unknown. Considering that MAP4K4 is a negative regulator of adipocyte function, identifying the mechanisms that control MAP4K4 expression was of interest. Furthermore, the role of macrophage MAP4K4 in LPS stimulated TNFα production was also unknown. To address this question in vivo, new technology specifically targeting macrophages was needed. Thus, we developed a technology for non toxic and highly specific macrophage gene silencing in vivo. Considering that macrophages mediate numerous diseases, the application of GeRPs to these disease models is an exciting new possibility.
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Neyraud, Vincent. "L'ubiquitination des GTPases Ral : Un nouveau mécanisme de régulation diu trafic intracellulaire de Ral et des micro-domaines menmbranaires lipidiques." Paris 11, 2010. http://www.theses.fr/2010PA11T085.
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Kretz, Carole. "Induction du LTR de VIH-1 dans des conditions de stress cellulaire : rôles de NF-κB et de l'état redox intracellulaire". Lyon 1, 1997. http://www.theses.fr/1997LYO10076.
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L'infection par vih-1 est caractérisée par une phase de latence clinique particulièrement longue. La progression de la maladie jusqu'au stade sida nécessite une forte activation de la réplication virale. Parmi les stimuli induisant cette activation, on trouve des stress cellulaires tels que choc thermique, choc oxydant ou irradiation aux uv. L'action de ces agents s'exerce sur la transcription du provirus et, pour certains, (tnfalpha, peroxyde d'hydrogene) a été corrélée à l'activation du facteur de transcription cellulaire nf-kappa b, capable de se lier en deux sites, au ltr de vih-1. Ce travail de thèse s'attache à étudier l'influence d'un stress oxydant ou thermique sur le fonctionnement du ltr de vih-1 et de nf-kappa b. Il précise le rôle des radicaux libres oxygénés (rlo) dans l'activation de nf-kappa b par un choc oxydant. Ainsi, la diminution du taux de rlo intracellulaire par surexpression de la glutathion peroxydase ou de la protéine de stress hsp27 inhibe l'activation, par un stress oxydant, de nf-kappa b et les étapes de phosphorylation et de dégradation de sa sous-unité inhibitrice i kappa b-alpha. D'autre part, il décrit le mécanisme d'activation du ltr de vih-1 par le choc thermique ; ce mécanisme fait intervenir l'activation du facteur nf-kappa b. Cette stimulation de nf-kappa b est indépendante d'une dégradation de sa sous-unité inhibitrice i kappa b-alpha, mais dépend du potentiel redox cellulaire. Enfin, il met en évidence de nouveaux inducteurs du ltr de vih-1, que sont les analogues d'acides aminés. Ces composes exercent leur effet par l'intermédiaire d'une activation de nf-kappa b, concomitante d'une dégradation de i kappa b-alpha, mais sans étape de phosphorylation préalable de la sous-unité inhibitrice ; cette activation dépend, de plus, d'une variation de l'état redox intracellulaire. Ces résultats illustrent l'avantage sélectif que nf-kappa b confère au virus vih-1, dans des conditions de stress cellulaire.
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Launay, Sophie. "Etude des ATPases-Ca2+ de type SERCA lors de la différenciation hématopoiétique." Paris 5, 2000. http://www.theses.fr/2000PA05S001.
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L'activation cellulaire se traduit par un signal calcique, qui implique une libération puis une réincorporation de Ca²+ dans le réticulum endoplasmique (RE) par les récepteurs de l'inositol trisphosphate (R-IP3) et les SERCA (sarco/endoplasmic reticulum Ca²+-ATPase), respectivement. Afin d'avancer dans la compréhension de ce signal, nous avons focalisé notre attention sur le rôle des nouvelles formes de SERCA3 co-exprimées avec la forme SERCA2b dans les principales voies de l'hématopoïèse, modèle peu étudié jusqu'alors. Lors de l'activation lymphocytaire, nous avons observé que l'expression des SERCA est modulée de façon différentielle : l'expression des SERCA3 diminue alors que celle de SERCA2b augmente. De là, nous avons étudié à bipotentialité de la différenciation myelo-monocytaire. Des régulations d'expression des SERCA ont été retrouvées, divergeant ou convergeant selon la voie de différenciation empruntée. Lors de la différenciation granulocytaire, l'expression des SERCA3 augmente et celle de SERCA2b diminue, alors que lors de la maturation monocytaire, l'expression des deux types de SERCAs augmente. Nous avons montré que ces régulations in vitro étaient retrouvées in vivo lors d'une pathologie myéloïde, la leucémie promyelocytaire aiguë pour laquelle la thérapie utilisée différencie les cellules en granulocytes. Enfin, nous avons étudié la différenciation megacaryocytaire, en complétant l'étude des SERCA par celle des R-IP3. Dans ce cas, une surexpression des SERCA3 est observée, corrélée à celle des R-IP3, sans modulations de SERCA2b. L'hématopoïèse est donc associée à des régulations d'expression des protéines contrôlant la concentration de Ca²+, avec une constante, la surexpression de SERCA3. Notre travail a ainsi permis de découvrir la complexité de l'organisation du RE et de mettre l'accent sur la régulation de ce nouveau gène SERCA3, qui semble jouer un rôle fondamental dans la régulation de l'homéostasie calcique au cours de l'hématopoïèse.
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Pasquet, Jean-Max. "Etude des signaux intracellulaires et des mécanismes impliqués dans l'activation des plaquettes sanguines humaines conduisant à l'exposition des phospholipides procoagulants et à l'émission de microparticules." Bordeaux 2, 1996. http://www.theses.fr/1996BOR28447.
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L'asymétrie des phospholipides membranaires des cellules eucaryotes est maintenue par l'activité d'une Mg2+-ATPase, l'aminophospholipide translocase. Les plaquettes sanguines perdent cette asymétrie par activation physiologique (thrombine et collagène) ou non-physiologique (ionophore calcique, inhibiteurs de Ca2+-ATPases, de tyrosine-kinases ou de tyrosine-phosphatases) et deviendraient procoagulantes. L'exposition des aminophospholipides serait due à l'activation d'un transporteur protéique spécifique : la "scramblase". La formation de microparticules consécutivement à l'exposition des aminophospholipides est corrélée à l'activation d'une thiolprotéase cytosolique, la calpaine, qui participe à la réorganisation du cytosquelette plaquettaire et à la transduction des signaux intracellulaires lors de l'activation. L'activation de la calpaine et la formation de microparticules dépendent d'un influx massif de Ca2+, la concentration intracellulaire minimale à atteindre est de 3µM pour que ces réponses aient lieu. Cependant, l'exposition des aminophospholipides n'est pas nécessairement suivie de l'activation de la calpaine et de la formation de microparticules. Alors que l'activation plaquettaire s'accompagne généralement d'une forte augmentation des protéines tyrosine-phosphorylées, la formation des microparticules est corrélée à une déphosphorylation massive. Cette déphosphorylation est indépendante de l'influx de Ca2+ mais elle est étroitement corrélée à la mobilisation des réserves internes calciques. Les réponses plaquettaires tardives que sont l'exposition des aminophospholipides et la formation de microparticules sont sous le contrôle de tyrosine-phosphatases non identifiées à ce jour. Le ou les tyrosine-phosphatases mises en jeu contrôleraient négativement l'exposition des aminophospholipides et positivement la formation de microparticules. L'étude d'une pathologie présentant un défaut d'exposition des aminophospholipides procoagulants, le syndrome de Scott, pour laquelle une forte diminution des tyrosine-phosphorylations semble liée à une hyperactivité des tyrosine-phosphatases serait en faveur de cette hypothèse<br>Phospholipid asymmetry is maintained by a Mg2+-ATPase : the aminophospholipid translocase. Procoagulant phospholipid exposure in platelets could be induced by physiologic agonist such as thrombin and collagen or by chemical agents such as calcium ionophore. Ca2+-ATPase inhibitors and tyrosine kinase or tyrosine phosphatase inhibitors. Aminophospholipid exposure seems to be linked to a protein transporter : the "scramblase". Aminophospholipid exposure and microparticle formation are uncoupled events since it was possible to induce the former without the latter. When both responses occured, aminophospholipid exposure on the platelet surface proceedes microparticle formation. Microparticle formation requires calpain activation induced by a large Ca2+ influx and the proteolysis of cytoskeletal proteins. The intracellular Ca2+ minimal level measured for calpain activation and microparticle formation was 3 µM. In contrast to the strong increase in the tyrosine phosphorylated proteins which occured during activation, membrane vesiculation was correlated to dephosphorylation. This dephosphorylation was independent of Ca2+ influx but was strictly related to a sustained Ca2+ mobilization. Aminophospholipid exposure could be induced by inhibition of tyrosine kinase or tyrosine phosphatase while microparticle formation could be induced by tyrosine kinase inhibitor but was inhibited by tyrosine phosphatase inhibitor. A model could be proposed in which aminophospholipid exposure and microparticle formation are respectively negatively and positively regulated by tyrosine phosphatases. The study of a pathology, the Scott syndrome, characterized by a defect in aminophospholipid exposure, in membrane vesiculation and showing a lower tyrosine phosphorylated protein pattern upon thrombin plus collagen activation, seems to favour this model
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Bastin, Guillaume. "Les résidus cystéines en positions 2 et 12 de RGS4 influencent son trafic intracellulaire et ses fonctions." Thesis, Lille 1, 2013. http://www.theses.fr/2013LIL10003/document.
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Les protéines RGS (Regulator of G-protein Signaling) sont des inhibiteurs des voies de signalisation des protéines G. RGS4 atténue l’activité de protéines G dans plusieurs tissus tel que la diminution de son activité peut accroître la sévérité de la bradycardie, cardiomyopathies liées au diabète, l’invasion de cellule cancéreuse du sein, résistance à l’insuline et intolérance au glucose. RGS4 a été localisé à la membrane plasmique ainsi que dans des compartiments intracellulaires, cependant, son mode de trafique intracellulaire reste méconnu. En utilisant des outils de microscopie confocale sur cellules vivantes et méthode de détection d’activité des voies de signalisation conditionnée par les protéines G, nous avons caractérisé l’importance de deux sites de palmitoylation, ces deux sites : Cys2 et Cys12 montrent des intérêts complémentaires dans le trafic de RGS4 vers la membrane cellulaire. Dans un axe linéaire, nous avons identifié DHHC3 et 7, deux enzymes de palmitoylation participant au trafique intracellulaire de RGS4 et donc à la maximalisation de son activité inhibitrice des voies de signalisation contrôlées par Galphaq. Enfin des marqueurs de membranes endosomales, les protéines rab ont permis de caractériser les voies de trafic intracellulaire empruntée par RGS4, par exemple RGS4 est internalisé de la membrane plasmique par Rab5 et recyclé à la membrane cellulaire par Rab11. L’activation ou inhibition de Rab5 et 11 ont permis d’observer des changements d’activité de RGS4. Ces travaux confèrent une base de données pour des études ultérieures visant à développer des stratégies thérapeutiques à accroître les fonctionnalités de RGS4<br>RGS proteins (Regulator of G-protein Signaling) are potent inhibitors of heterotrimeric G-protein signaling. RGS4 attenuates G-protein activity in several tissues such that loss of its function may lead to bradycardia, diabetic cardiomyopathy, breast cancer cell invasion, insulin resistance and glucose intolerance. RGS4 has been localized to both plasma membrane and intracellular pools, however, the nature of its intracellular trafficking remains to be elucidated. G-protein inhibition requires the presence of RGS4 at the plasma membrane. In this work, we characterized the complementary roles of two putative palmitoylation sites on RGS4 to target intracellular compartments and plasma membrane. We identified palmitoylation on Cys2 and 12 respectively important for RGS4 endosomal targeting and plasma membrane localization, when mutations were introduced to the palmitoylation sites, RGS4 capability of inhibiting Gq-mediated signaling was impaired. As a continuum we identified two palmitoylating enzymes, DHHC3 and 7 as modulator of RGS4 localization and function. Knock downs of DHHC3 and 7 impaired RGS4 endosomal and plasma membrane targeting and capability of inhibiting M1-muscarinic receptor signaling. Finally we used live cell confocal microscopy to define RGS4 intracellular trafficking routes. Specifically Rab5 mediated RGS4 trafficking from the plasma membrane to intracellular compartments while Rab11 mediated RGS4 trafficking to the plasma membrane. Activation and inhibition of Rab5 and 11 routes impaired RGS4 capability of inhibiting M1-muscarinic receptor signaling pathway. These novel findings provide a strong rationale for future studies aimed at developing new strategies to increase the function of RGS4