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Wilson, Rona Kirstin. "Intracellular pathways in prion peptide trafficking." Thesis, University of Glasgow, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.433105.
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`Arnold, Claire. "Intracellular signalling pathways in myeloproliferative neoplasms." Thesis, Queen's University Belfast, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.680884.
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Vicario, Chiara. "Magnetogenetic Control of Intracellular Signaling Pathways." Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066581/document.
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Le contrôle de l'organisation spatiotemporelle des biomolécules à l'intérieur des cellules vivantes est fondamental pour déchiffrer les mécanismes qui règlent les voies de signalisation cellulaire et leur régulation. Dans ce projet de thèse nous présentons une nouvelle méthode pour induire une perturbation très spécifique et locale des voies de signalisation à l'intérieur des cellules vivantes: la magnétogénétique. Elle est basée sur l'utilisation de nanoparticules magnétiques biofonctionnalisée, pour induire et maintenir des gradients de protéines à l'intérieur des cellules vivantes.Nous avons modifié la taille et la surface de deux différentes types de particules, les nanoparticules superparamagnétiques synthétiques avec couche de silice et les nanoparticules basées sur la protéine GFP-ferritine, afin d'assurer une mobilité libre dans le cytosol. Ces nanoparticules peuvent être localisées rapidement dans les cellules vivantes grâce à leur diffusion biaisée par des forces magnétiques faibles, dans l'ordre du fN. En combinaison avec une fonctionnalisation de surface spécifique pour la capture des protéines d'intérêt ainsi que un chargement efficace des nanoparticules dans le cytoplasme, nous présentons une technologie capable de contrôler des gradients de protéines intracellulaires avec une résolution spatiale du micromètre et une résolution temporelle de quelques dizaines de secondes.Dans ce travail nous avons montré la possibilité de contrôler avec précision la perturbation des voies de signalisation associées aux petites protéines Rho GTPases et nous avons quantifié la propagation du signal en termes de recrutement des effecteurs et des changement morphologiquesControlling the spatio-temporal organization of biomolecules inside living cells is a major rerequisite for deciphering mechanisms governing cell signaling and its regulation. In this thesis project, a new method to induce a highly specific and local perturbation of signaling pathways inside living cells is presented: magnetogenetics. It is based on the use of biofunctionalized magnetic nanoparticles, to induce and maintain protein gradients inside living cells. We tailored the size and surface properties of both synthetic silica core shell nanoparticles and superparamagnetic GFP-ferritin-based nanoparticles in order to ensure unhindered mobility in the cytosol. These nanoparticles can be rapidly localized in living cells by exploiting biased diffusion at weak magnetic forces in the fN range. In combination with nanoparticles' surface functionalization for specific in situ capturing of target proteins as well as efficient delivery into the cytosplasm, this work presents a novel technology for controlling intracellular protein gradients with a spatial resolution of micrometers and a temporal resolution of a few tens of seconds. In this work we showed the possibility to precisely control the perturbation of the signaling pathways associated to the small Rho GTPases proteins with relative quantification on signal propagation in terms of effector recruitment and morphological changes
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Hutchinson, James Lawrence. "Salmonella interactions with host intracellular trafficking pathways." Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611631.
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Nesbeth, Darren Nicholas. "Biochemical studies of intracellular trafficking pathways in eukaryotes." Thesis, Imperial College London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.313830.
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Romero, Alirio Jose Melendez. "Intracellular signalling pathways activated by Fc#gamma#RI." Thesis, University of Glasgow, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.266462.
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Cullen, Peter J. "A study of the regulation of intracellular calcium." Thesis, University of East Anglia, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.277302.
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Behar, Marcelo S. Dohlman Henrik G. "Dynamic regulation and information transfer in intracellular-signaling pathways." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2008. http://dc.lib.unc.edu/u?/etd,1546.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2008.Title from electronic title page (viewed Sep. 16, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Physics and Astronomy." Discipline: Physics and Astronomy; Department/School: Physics and Astronomy.
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Pouliot, Philippe. "Implication of intracellular signalling pathways in allergic asthma pathogenesis." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=115896.
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The regulation of systemic immune responses is dependent on individual cell responses that will concur to induce a coherent response against a stimulus. In turn, cell response is dependent on the processing of intracellular signals generated at the cell membrane and transmitted through successive protein modifications to the nucleus in order to activate gene transcription. This is referred to as intracellular signalling. Tight control of these mechanisms is required to generate an appropriate cell response to environmental stimulations and globally to establish an appropriate immune response. Among protein modifications used to transmit a signal to the nucleus, protein tyrosine phosphorylation represents a pivotal method used by immune cells to rapidly induce signalling. While protein tyrosine kinases (PTKs) phosphorylate proteins, protein tyrosine phosphatases (PTPs) regulate the signalling by removing the phosphate group. The goal of this study was to better characterize intracellular signalling events involved in allergic asthma, a chronic inflammatory disease involving a Th2 immune response. In a first time, we investigated the role of PTPs in the development of asthma. We show that inhibition of global PTP activity in mice, during either the allergen sensitization or the allergen challenge phase, reduces asthma development and is linked to an increased Th1 response in the spleen and lung. Secondly, we revealed that TC-PTP inhibition reduces asthma development, while PTP-1B inhibition exacerbates inflammatory cells recruitment to the lung. Inhibition of either SHP-1 or PTP-PEST activity did not significantly modulate asthma development in our model. In a third set of experiments, we got interested in the signalling pathways triggered by the pro-inflammatory molecules myeloid-related proteins (MRPs) 8 and 14. MRPs are small cytosolic proteins recently described to have extracellular functions. MRP8 expression is resistant to corticosteroid treatment, and potentially promotes inflammation in corticosteroid-treated patients. We identified that MRPs induce signal through the action of TLR-4 and trigger the activation of MEK/ERK and JNK pathways that lead to NF-kappaB translocation. Collectively, our data provide a new characterization of signalling pathways engaged in allergic asthma. This should be helpful in the elaboration of new therapeutic approaches targeting precise pathways to inhibit mechanisms of inflammation.
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Fitzgerald, Jonathan Basil. "Chondrocyte gene expression and intracellular signaling pathways in cartilage mechanotransduction." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/33869.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2005.Includes bibliographical references (p. 152-167).
Chondrocytes respond to in vivo mechanical loads by regulating the composition of the cartilage extracellular matrix. This study utilized three loading protocols that span the range of forces and flows induced by in vivo loading. Constant (static) compression of cartilage explants induces a transient hydrostatic pressure buildup and fluid exudation from the compacted matrix until relaxation leads to a new equilibrium compressed state. Dynamic compression induces cyclic matrix deformation, hydrostatic pressures, fluid flows, and streaming currents. Dynamic tissue shear causes cyclic matrix deformation only. After applying these loading protocols to intact cartilage explants for 1 to 24 hours, we used real-time PCR to measure the temporal expression profiles of selected genes associated with cartilage homeostasis. In concurrent experiments, we assessed the involvement of intracellular signaling pathways using molecular inhibitors. In order to interpret the results we developed two techniques that reliably clustered intermediate-sized datasets using principal component analysis and k-means clustering. Mechanical loading regulated a variety of genes including matrix proteins, proteases, protease inhibitors, transcription factors, cytokines, and growth factors. Static compression transiently upregulated matrix proteins, however, mRNA levels were suppressed by 24 hours.
(cont.) Dynamic compression and dynamic shear increased matrix protein transcription particularly after 24 hours. In contrast, matrix proteases were upregulated by all 24 hour loading regimes, particularly static compression. Taken together these results demonstrate the functionally-coordinated regulation of chondrocyte gene transcription in response to mechanical forces, and support the hypothesis that dynamic loading is anabolic for cartilage and static loading is anti-anabolic. Intracellular calcium release, cAMP activation of protein-kinase-A, and the phosphorylation of MAP kinases (ERK1/2, p38), were all identified as signaling events necessary for mechanically-induced transcription. In addition, we measured the immediate, transient increase in mRNA levels of transcription factors downstream of the MAP kinase pathway (c-Fos and c-Jun), in response to all three loading types. The prevention of protein synthesis during static compression suppressed mechanically-induced transcription suggesting that signaling molecules are synthesized in response to mechanical forces. Comparison of this well characterized model of normal cartilage mechanotransduction to what occurs within diseased cartilage will hopefully provide insight into the mechanisms driving the progression of osteoarthritis.
by Jonathan Basil Fitzgerald.
Ph.D.
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Sumita, Takuya. "Studies on intracellular protein degradation pathways in plant fungal pathogens." Kyoto University, 2019. http://hdl.handle.net/2433/242706.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第21829号
農博第2342号
新制||農||1068(附属図書館)
学位論文||H31||N5201(農学部図書室)
京都大学大学院農学研究科地域環境科学専攻
(主査)教授 田中 千尋, 教授 本田 与一, 准教授 刑部 正博
学位規則第4条第1項該当
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BONFILI, LAURA. "Modulation of intracellular proteolytic pathways in neoplastic and neurodegenerative diseases." Doctoral thesis, Università degli Studi di Camerino, 2014. http://hdl.handle.net/11581/401774.
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The ubiquitinâ€proteasome system (UPS) and autophagy, the two major intracellular protein degradation systems, play a critical role in the regulation and maintenance of cellular homeostasis. The proteasome is known to degrade the majority of intracellular proteins, including cyclins, metabolic enzymes, antigen, transcription factors, and tumour suppressor proteins. Autophagy, or selfâ€eating, is a lysosomal degradation pathway in charge of recycling dysfunctional organelles and aggregated proteins. Impairments in the functionality of proteolytic pathways favour the accumulation of misfolded and abnormal proteins, resulting in the deposition of toxic aggregates that characterize diverse pathologic conditions such as cancer and neurodegenerations. For many years UPS and autophagy have been thought as separated pathways whereas an increasing number of data recently elucidated their intimate correlation. This PhD thesis is focused on understanding the role of UPS and autophagy in such diseases aimed at better clarifying their interplay. The potential of natural occurring compounds as proteasome modulators in the treatment and prevention of cancer is widely documented, with epigallocatechinâ€3â€gallate (EGCG) being the most studied polyphenol. In particular, owing to EGCG instability under physiological conditions, its degradation pattern was monitored and an equally active metabolite has been isolated and evaluated for its ability to modulate both proteasome functionality and apoptotic pathways. Furthermore, the marine sponge metabolite petrosaspongiolide M (PM), a natural proteasome inhibitor, has been considered, describing the molecular mechanism of interaction of PM with the immunoproteasome, a proteasome isoform with a prevalent role in immune response. This natural compound was also able to impair autophagy, with p62 serving as the link between the two proteolytic processes. Among polyphenolsâ€based anticancer approaches, curcumin represents a promising but poor bioavailable compound. Considering that metal complexes offer an opportunity for the design of bioactive compounds with anticancer properties, and that ruthenium is a valid non toxic alternative to Cisplatin, three ruthenium(II)curcumin complexes containing different arene moieties have been synthesized and analyzed for their ability to modulate proteasome functionality, comparing their efficacies with that of free curcumin in isolated proteasome complexes and in cultured colon cancer cells. A stable and effective curcumin derivative, in terms of proteasome inhibitory ability, antioxidant capacity, DNA binding ability has been identified, thus proving that the complexation of curcumin with ruthenium(II) is a good starting point for the development of curcuminâ€based anticancer drugs. Successively, a series of ruthenium(II) arene complexes with the 4â€(biphenylâ€4â€carbonyl)â€3â€methylâ€1â€phenylâ€5â€pyrazolonate ligand, and different ancillary ligands have been synthesized and characterized, identifying the hexamethylbenzene−ruthenium complexes as the most efficacious, in terms of antiproliferative activity in four human cancer cell lines, through the induction of apoptosis. Finally, regarding the interplay of UPS and autophagy in cancer, the effect of the hunger hormone ghrelin on both proteasome and autophagy has been analysed. Interestingly, this endogenous compound triggers apoptosis in colon cancer cells, via proteasome inhibition and autophagy induction, with p53 protein having an interactive role. Aging and neurodegenerative conditions, including Alzheimer disease (AD) are characterized by alterations in the normal cellular homeostasis with deregulation of the proteolysis. UPS and autophagy interplay in AD has been studied using human SHâ€SY5Y neuroblastoma cells stably transfected either with wildâ€type βâ€amyloid precursor protein (AβPP) gene or mutant Val717Gly AβPP gene as experimental model. The APPmut clone produces and releases significantly higher amounts of Aβ42, the amyloid peptide which is more prone to aggregation. The overâ€expression of the APP correlated with an increase in oxidative stress and with a reorganization of the cellular proteolytic machineries. Additionally, HDAC6â€increased expression has been identified as the cellular attempt to activate compensatory autophagy in such altered scenario. The existence of an amyloid (Aβ42) threshold level beyond which proteasomeâ€dependent proteolysis becomes definitely dysfunctional has been proved. Moreover, these cellular models have been used to demonstrate a role of the APP in affecting the downstream effects of proteolysis inhibition. The occurrence of APP wildâ€type form or the APP Val717Gly mutated form was observed to impair both proteasome or autophagy activities upon treatment with proteasome or autophagy inhibitors. Collectively, our results provide evidences on the key role of UPS and autophagy in both neoplastic and neurodegenerative diseases and gain insight into the interplay between the two pathways in proteinopathies, thus representing a real contribution in the development of new strategies to modulate the two pathways for therapeutic purposes.
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Birdsell, Dawn Nice. "REGULATION OF AQUAPORIN-1 ION CHANNEL FUNCTION BY INTRACELLULAR SIGNALING PATHWAYS." Diss., Tucson, Arizona : University of Arizona, 2005. http://etd.library.arizona.edu/etd/GetFileServlet?file=file:///data1/pdf/etd/azu%5Fetd%5F1143%5F1%5Fm.pdf&type=application/pdf.
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Roberts, Susan Read. "Mechanotransduction pathways associated with intracellular calcium in chondrocytes within 3D constructs." Thesis, Queen Mary, University of London, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.270626.
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Rapecki, Stephen Edward. "Regulation of leukocyte cytokine production by inhibitors of intracellular signalling pathways." Thesis, Open University, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368573.
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Heyward, Catherine Anne. "Investigation of potential phosphatidic acid target proteins in intracellular signalling pathways." Thesis, Birmingham City University, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.479113.
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Liu, Ke, University of Western Sydney, of Science Technology and Environment College, and of Science Food and Horticulture School. "Role of second messengers in controlling growth patterns of corneal epithelial cells." THESIS_CSTE_SFH_Liu_K.xml, 2002. http://handle.uws.edu.au:8081/1959.7/387.
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The purpose of this thesis was to investigate mechanisms contolling the growth of corneal epithelial cells, particularly the intracellular signals involved with stratification compared with cellular migration and maturation. Buttons of epithelium were cultured in different culture media. The explants were monitored microscopically for their growth patterns and finally fixed and examined for cytokeratin, vimentin and actin. Different growth patterns were observed in the different media, indicating that different signalling patterns must be operating in these cells depending upon the media in which they were grown. To investigate the intracellular pathways controlling the different growth patterns, the protein phosphorylation of different cultures was investigated. The two proteins, p57 and p30, are strongly suggested to be associated with stratification of the epithelial cells. The possible involvement of the common serine kinase, PKC, in controlling the growth pattern of corneal epithelial cells were also investigated. The results suggested that an intracellular pathway involving PKC promotes the maturation and spread of the cells but is not involved in their stratification. These experiments taken together indicate that the different aspects of corneal epithelia cell growth are tightly controlled and may occur quite independently. Specific protein expression appears to be important for stratification, and phosphorylation of proteins by PKC appears to be involved with the maturation of epithelial cells from basal cells. It also indicates that the mature cells are capable of producing the extracellular matrix protein fibronectin which appears to have an important role in causing the spread as distinct from the stratification of the corneal epithelial cells.Doctor of Philosophy (Ph.D.)
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Rosowski, Emily E. (Emily Elizabeth). "Modulation of innate immune signaling pathways by the intracellular pathogen Toxoplasma gondii." Thesis, Massachusetts Institute of Technology, 2013. http://hdl.handle.net/1721.1/83639.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2013.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references.
Toxoplasma gondii, an obligate intracellular protozoan parasite, is one of the most successful eukaryotic pathogens. It can infect virtually any warm-blooded animal, including humans, in whom it can cause serious disease. Its success is likely due to its ability to modulate host immune responses and host innate immune signaling pathways allowing it to establish a chronic infection with few symptoms in its hosts, which favors transmission to new hosts. Here, we report that Toxoplasma activates NF-[kappa]B and inhibits STAT1 signaling pathways to promote both its own survival and the survival of its host. We identified GRA15, a novel Toxoplasma secreted factor that activates the host cell NF-[kappa]B pathway. GRA15 is polymorphic between Toxoplasma strains and only active in the type II clonal lineage. GRA15 expression increases host pro-inflammatory cytokine production in vivo, thereby helping the host to control parasite growth. Conversely, Toxoplasma infection dampens the activation of other immune responses by inhibiting IFN-[gamma] and STAT1 signaling. All of the Toxoplasma strains that we have tested directly inhibit the activity of STAT1, the transcription factor through which IFN-[gamma] signals. We found that infection does not inhibit STAT1 phosphorylation, dimerization, nuclear translocation, or DNA binding. Instead, Toxoplasma must act even farther downstream, perhaps by inhibiting the recruitment of co-activators or RNA polymerase. Infection actually increased the association of STAT1 with DNA, which has been shown previously to be associated with decreased STAT1 transcriptional activity. The Toxoplasma effector that inhibits STAT1 remains unknown, but our results suggest that it is not secreted into the host cell upon invasion but must interface with its cellular target after the parasitophorous vacuole is formed. A deeper knowledge of how and why Toxoplasma modulates these processes will help us to understand more about the basic signaling pathways themselves and to discover clues on how to better treat Toxoplasma infections in humans.
by Emily E. Rosowski.
Ph.D.
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Koudelka, Juraj. "Determining TrkB intracellular signalling pathways required for specific aspects of gustatory development." Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/8830.
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Neurotrophins BDNF and NT4 influence the development of the rodent gustatory system. Despite binding to the same receptor, TrkB, they have different roles. BDNF is chemo-attractive for gustatory neurons and regulates gustatory neuron targeting and number during development. NT4 regulates gustatory neuron number earlier in development than BDNF, but it is not chemo-attractive and does not regulate gustatory neuron targeting. To elucidate the mechanisms that regulate these processes we have examined which TrkB intracellular signalling pathways are required for specific aspects of gustatory development by studying the effect of specific point mutations in TrkB docking sites. We found that the TrkB/Shc docking site is involved in regulating the survival of geniculate ganglion neurons as a point mutation in this adaptor site (TrkbS/S) caused large losses of these neurons as early as E12.5. These losses were exacerbated throughout development until after birth. A point mutation in the TrkB/PLCγ (TrkbP/P) docking site did not cause loss of geniculate ganglion neurons at any point during development. Animals with a point mutation in both docking sites (TrkbD/D) caused a further decrease in neuron numbers compared to animals with a mutation in only one of the docking sites, similarly to what has previously been shown in Trkb null animals. We concluded that the TrkB/Shc docking site is crucial for determining the survival of geniculate ganglion neurons during mouse gustatory development, while the TrkB/PLCγ docking site does not affect the neuronal survival directly and likely plays a role in maintenance of these neurons. Examining the targeting of geniculate ganglion afferents into the tongue revealed large deficits in innervated neural bud and taste bud numbers in TrkbS/S animals both before and after birth. This was concluded to be reflecting the lack of neuronal survival in this ganglion, a result that was mirrored in TrkbD/D animals. TrkbP/P animals, on the other hand, exhibited a developmental delay in innervation. This was indicated by a low amount of innervated neural buds following the initial innervation period, which was compensated for by a large increase in the number of innervated taste buds by birth. By adulthood, the numbers of taste buds present on the tongues of TrkbP/P animals reached normal numbers compared to control animals. This suggested that the TrkB/PLCγ docking site is involved primarily in innervation. Finally, we examined the morphology of taste buds in newly born and adult animals. We found that the low amount of geniculate ganglion afferents innervating the tongue in TrkbS/S and TrkbD/D animals caused a decrease in size of taste buds. This effect was seen to be partially rescued by adulthood in TrkbS/S animals but not in TrkbD/D animals due to lack of viability. The morphology of taste buds was unaffected in TrkbP/P animals until adulthood, at which point the size of the taste buds was increased. These results are in agreement with previous findings showing dependency of taste bud morphology on the amount of innervation. Overall, our findings show a differential role of TrkB adaptor sites in gustatory development. Despite activated by the same ligands, the docking sites on this receptor are able to exert different influence on signalling pathways downstream of TrkB affecting neuronal survival, targeting and morphology of taste buds.
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Vine, Sarah. "Modulation of intracellular signalling pathways in retinal microvascular cells by elevated glucose." Thesis, King's College London (University of London), 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.415918.
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Cerqueira, Vera. "Role of intracellular signalling pathways in conferring resistance to endocrine therapies in breast cancer." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/4511.
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Breast cancer is the most prevalent form of cancer in women and accounts for 519,000 annual deaths (WHO Statistics). It has long been established that oestrogen (E2) stimulates tumour growth of oestrogen receptor (ER) positive breast cancer and is involved in the pathogenesis of the disease. Consequently, therapeutic approaches targeting the ER were developed. The use of endocrine therapy is an integral component in treating breast cancer however resistance to such drugs is a major limitation. Unfortunately, even initially responding tumours eventually develop resistance - acquired resistance. The aim of this study was to determine which intracellular pathways may be important in conferring acquired endocrine resistance. In order to do so, a three-stage MCF-7 cell model emulating the clinical development of acquired endocrine was used. MCF-7/LCC1 (LCC1) and MCF-7/LCC9 (LCC9) cells lines were derived from the oestrogen dependent and antioestrogen sensitive MCF-7 cell line. LCC1 cells remain responsive to endocrine therapies but their growth is not dependent on oestrogenic stimulus. LCC9 cells, on the other hand are fully resistant to endocrine therapies and completely oestrogen independent. A number of different cell membrane receptors and intracellular pathways have been implicated in endocrine resistance including HER receptor family, PI3K/Akt & MEK/ERK pathways. These pathways are of particular interest since they are able to activate ER in the absence of oestrogenic stimulus. It is likely that several pathways may be important in conferring resistance to endocrine therapies therefore the experiments in this study focussed on the transcriptional regulation of HER receptors, the activation of the Akt pathway and its implication to basic cellular processes. Following E2 treatment (48h), HER2/3/4 mRNA and protein levels were reduced in MCF- 7 and LCC1 but not in the endocrine-resistant LCC9 cell line as measured by QRT-PCR and Western blotting. The anti-estrogen fulvestrant (ICI 182,780) reversed the E2 modulation. A previous study has shown that ER and the HER2 promoter compete for limiting amounts of SRC-1 in oestrogen-responsive ZR-75-1 cells, causing HER2 repression after E2 stimulation (Newman et al.,Oncogene, 19, 490-7, 2000). ER RNAi abolished E2 repression of HER2 in MCF-7 and LCC1 cells. Furthermore, LCC9 cells have reduced SRC-1 recruitment to ER (assessed by ChIP) allowing SRC-1 to bind to the HER2 promoter. SRC-1 RNAi reduced HER2 transcription in MCF7 cells in a manner similar to E2 whilst it did not restore E2 repression in LCC9 suggesting that the latter cells have alternative mechanisms regulating HER2 transcription. RNAis against the other two p160 co-activators TIF2 and AIB1 did not restore E2 mediated HER2 repression in LCC9 cells. The importance of redundancy between p160 co-activators was also determined by performing double knockouts. SRC-1/TIF2 and TIF2/AIB1 double siRNAs had little effect on HER2 mRNA levels however SRC-1/AIB1 siRNA restored oestrogen mediated downregulation of HER2 transcription in LCC9 cells. This data indicates that SRC-1 and AIB1 co-activators play a role in the transcriptional regulation of HER receptor particularly in MCF-7 and LCC1 cells. The regulation of this transcriptional mechanism is altered in resistant LCC9 cells but, as evidenced by the double knockouts, p160 coactivators are still able to affect HER expression in these cells. This mechanism was further studied in primary breast cancer tumour material. The importance of the Akt pathway in this cell line model was also investigated as phospho-Akt levels are elevated in LCC1 and LCC9 cells. This in turn was shown to activate mTOR and ER (Ser167 residue phosphorylation) thereby contributing to increased growth and ligand independent activation of the oestrogen receptor respectively. Activation of PI3K and PTEN is unchanged in LCC1 and LCC9 cells suggesting that these proteins are not responsible for elevated Akt phosphorylation. In contrast, these cells do express higher levels of phospho-IGFR due to the high availability of receptor ligands (IGFI & IGFII). This is likely to be, at least partially, responsible for the elevated Akt activation. Moreover, the role of Akt isoforms was also determined as they are known to have different functions. The levels of Akt 2 phosphorylation are higher in endocrine resistant cell lines in comparison to parental MCF-7 cells. Interestingly, the Akt 3 phosphorylation is present in all cell lines whilst Akt 1 phosphorylation is minimal. Nevertheless, Akt RNAi studies reveal that Akt 1 and 2 siRNA dramatically reduce growth in MCF-7, LCC1 and LCC9 cells. These results suggest that Akt 2 phosphorylation may play a part in conferring endocrine resistance but the other isoforms are also important for normal cellular growth. The cell cycle profiles of LCC1 and LCC9 are very similar to MCF-7. Similarly, migration levels are unchanged in endocrine resistant cell lines. However, in the presence of antioestrogenic drugs, apoptosis in LCC1 and LCC9 cells in reduced in comparison to the parental MCF-7 cell line. Furthermore, LCC1 and LCC9 cells have higher invasion rates. The deregulation of HER receptor expression and elevated Akt activation may together confer survival advantage in LCC1 and LCC9 cells whilst also increasing their invading potential.
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Pollock, Jamie. "Calcium mobilisation from intracellular stores in cultured DRG neurones : modulation by metabotropic glutamate receptors, TNF and sphingolipids." Thesis, University of Aberdeen, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325230.
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Jones, Sarah Elizabeth. "The effect of cAMP elevation on intracellular signalling pathways in prostate epithelial cells." Thesis, University of Glasgow, 2010. http://theses.gla.ac.uk/1668/.
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Chronic IL-6 signalling contributes to the pathophysiology of many diseases including prostate cancer. Relevant to prostate cancer is the ability of the pro-inflammatory cytokine IL-6 to activate the oncogenic signalling protein STAT3, thus inhibition of STAT3 activation is a popular avenue of research to augment prostate cancer therapies. In this study, the endogenous anti-inflammatory molecule cAMP was investigated as a mechanism by which to inhibit IL-6-induced STAT3 activation in the DU145, LNCaP and PZ-HPV-7 prostate epithelial cells. Elevation of cAMP attenuated IL-6-mediated activation of STAT3 which was mimicked via selective activation of the exchange protein activated by cAMP. Inhibition of protein kinase A (PKA) alone also attenuated IL-6-induced STAT3 activation, suggesting a role for PKA activity in sustained IL-6 signalling in these cells. In DU145 and PZ-HPV-7 cells, the inhibitory effect of cAMP elevation was correlated with an increase in protein levels of suppressor of cytokine signalling 3. However, this was not the case in LNCaP cells in which cAMP elevation was instead associated with morphological changes consistent with neuroendocrine-like differentiation associated with terminal disease. PKA activation was required for cAMP-mediated changes in LNCaP cell morphology and could be recapitulated by reagents which inhibited RhoA/ROCK signalling, suggesting that cAMP elevation is able to inhibit RhoA activation via a PKA-dependent pathway. Additionally, cAMP elevation activated ERK1/2 and selective blockade of ERK signalling attenuated the effects of cAMP elevation on cell morphology. Selective activation of ERK1/2 did not induce the early changes in cell morphology associated with increased intracellular cAMP concentrations, suggesting that another, related pathway was responsible for this phenomenon. Genetic or pharmacological inhibition of the MEK5/ERK5 signalling pathway significantly attenuated the rapid cAMP-mediated changes in LNCaP cell morphology, suggesting this pathway may be a possible target by which to inhibit the onset of neuroendocrine differentiation. To summarise, this study demonstrates that whilst the ability of intracellular cAMP elevation to inhibit STAT3 activation is common to the prostate epithelial cell lines used, the downstream effects of cAMP elevation can vary dramatically. Thus, whilst modulation of cAMP signalling may represent a suitable therapeutic strategy when considering some aspects of prostate cancer, the impact on other signalling events must be considered.
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Jury, Elizabeth Carole. "Dysregulated intracellular signalling pathways in T lymphocytes from patients with systemic lupus erythematosus." Thesis, University College London (University of London), 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.411032.
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FUMAGALLI, SILVIA. "Characterization of the intracellular pathways involved in IL-22 production by dendritic cells." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2012. http://hdl.handle.net/10281/29857.
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Dendritic cells (DCs) play a key role in the inflammatory process. They participate to the activation of the immune response by a direct cellular contact through receptor-ligand mediated interactions or by the production of specific cytokines able to modulate different cell type activity. In this study, we show that DCs produce the novel cytokine IL-22 in response to TLRs ligands and we studied IL-22 gene transcript modulation both in vitro (Bone Marrow-derived DCs) and in vivo (CD11c+) derived DCs. Several non hematopoietic cells, in particular epithelial cells, express the IL-22 receptor that once induced, stimulates the production of different molecules such as antimicrobial peptides that are active both locally and systemically. Although it is known that innate lymphoid cells are able to produce IL-22 under several conditions and through the activation of different transcription factors, very little it is known about DCs.
The ability of DCs to produce IL-22 in response to the activation of TLRs and c-Type lectins receptors has been characterized. DCs derived IL-22 has been shown to induce MYD88-dependent and TRIF-independent pathways. Moreover, the intracellular signalling involved in IL-22 production in DCs has been monitored by using different inhibitors. We found that JNK and ERK MAP kinases play a major role in IL-22 production whereas p38 is not implicated. By blocking the NFkB canonical pathway (p65) with the inhibitor Bay11-7082, we confirmed that it takes part together with AP-1 in IL-22 production.
Finally, we tested the role of the aryl hydrocarbon receptor (AhR), as it has been reported to be essential in CD4+cells (Th17 subtype) derived IL-22. The IL-22 gene transcription dependence on AhR has been measured by using agonist and antagonist molecules that confirmed a crucial role for AhR in IL-22 production also in DCs. In conclusion, our study provides the first evidence that DCs are able to produce IL-22 upon TLRs and C-type lectins ligation. IL-22 is known to be expressed in many chronic inflammatory conditions, including psoriasis and rheumatoid arthritis, and its up-regulation often correlates with disease activity. Gaining a better understanding of the IL-22 expression and regulation is important for the development of IL-22 as a potential drug target.
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Raman, Malavika. "Identification of intracellular signaling pathways regulated by the TAO family of mammalian STE20p kinases." Access to abstract only; dissertation is embargoed until after 5/15/2007, 2006. http://www4.utsouthwestern.edu/library/ETD/etdDetails.cfm?etdID=163.
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Didangelos, Athanasios. "Intracellular signalling pathways activated in cartilage by injury and their modulation by synovial fluid." Thesis, Imperial College London, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.445330.
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Perkins, Jonathan Edward. "A study of EGF-induced intracellular signalling pathways and effects in first trimester trophoblast." Thesis, University of Birmingham, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.398444.
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Rohwer, Forest. "Interleukin-2 activation of intracellular signaling pathways : effects on gene expression, proliferation, and apoptosis /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 1997. http://wwwlib.umi.com/cr/ucsd/fullcit?p9806513.
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Whitfield, Robert. "The role of intracellular signalling pathways in CHO cell growth in a synthetic environment." Thesis, University of Sheffield, 2014. http://etheses.whiterose.ac.uk/6454/.
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Industrial batch and fed-batch culture of Chinese hamster ovary (CHO) cell lines for the production of recombinant proteins, utilises the ability of engineered CHO cells to grow and proliferate in suspension in a synthetic environment that is free of many of the extracellular signals (e.g. exogenous growth factors, cell to matrix and cell to cell contacts) that control growth in vivo. This functional capability derives from extensive cell culture experimentation and lengthy adaptation processes to generate production cell lines. Many different approaches have been used in this study to develop an understanding of the underlying signalling pathway changes that facilitate CHO cell adaptation to suspension growth in a synthetic environment. Initial characterisation experiments compared the growth of a parental adherent CHO cell line (CCL-61) and a directly adapted suspension CHO cell line (CHO-SA) in different growth environments. Results from this characterisation showed that in this model system, unadapted CCL-61 cells are unable to grow and proliferate in suspension conditions, while suspension CHO-SA cells are able to grow and proliferate in suspension conditions but only when an exogenous growth factor, in this case Insulin, is present in the culture media. Differences in the PI3K/Akt and MAPK signalling pathway activation status between the unadapted CCL-61 and suspension adapted CHO-SA cell lines growing in adherent and suspension conditions were comparatively mapped by western blotting analysis and immunoprecipitation/immunoblot analysis. Results indicate that a shift in signalling flux occurs when cells are taken from an adherent growth environment to a suspension growth environment. In adherent cells, integrin-mediated attachment to the extracellular matrix stimulates an up-regulation in signalling flux via the MAPK pathway. When integrin-mediated attachment is abolished, as in suspension growth, a shift in signalling flux is seen towards the PI3K/Akt pathway. Further experimental data using chemical inhibitors against specific signalling intermediates such as PI3K, Akt and MEK 1/2 suggests that activation of these signalling intermediates is vital for cell survival and proliferation in differing synthetic environments and therefore they V are viable targets for genetic engineering strategies used to functionally substitute for extracellular signals. Confocal microscopy and flow cytometry were used to deduce how the PI3K/Akt and MAPK signalling pathways interact with specific cell surface membrane transducers such as integrins and specific receptor tyrosine kinases (RTKs) such as the insulin receptor. Analysis of integrin expression levels between adherent and suspension cell lines shows that despite the lack of integrin-mediated adhesion utilized by suspension cells, the expression levels of various integrins, inclucing β1, 5 and 1, are not down-regulated in suspension adapted CHO cells. Further analysis of the activation status of key adaptor proteins such as Focal Adhesion Kinase (FAK) demonstrates how changes at the cell surface are transmitted to downstream signaling networks.
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SALVATORE, DANIELA. "ASSESSMENT OF INTRACELLULAR REDOX-BALANCE ROLE IN TMZ-RESISTANCE-RELATED CYTOPROTECTIVE PATHWAYS IN GBM." Doctoral thesis, Università degli Studi di Milano, 2020. http://hdl.handle.net/2434/702299.
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Background: Glioblastoma (GBM) represents the most aggressive astrocytic brain tumour in adults and exhibits a dismal prognosis related to resistance to therapy, which is principally based on Temozolomide plus RT (Stupp protocol). Among crucial factors which are involved in resistance to TMZ treatment, HIF-1α emerges since it activates several pathways, among which angiogenesis and EMT. Beyond its primary alkylating power, TMZ can exerts other secondary effects within cells, as ER stress-mediated ROS release from mitochondria and activation of autophagy. Several works have described HIF-1α degradation in lysosomes through the chaperone mediated autophagy (CMA) pathway. Importance of this indirect therapeutic potential has to be yet fully elucidated but the comprehension of the main mechanisms is shedding new light on tumour resistance strategies. At the same time, new procedures for the improvement of radiotherapy treatments could help the development of more precise approaches. Aims: The main purposes of this research project have been: to understand the molecular mechanisms underlying GBM resistance to TMZ, to investigate HIF-1α role in resistance and to assess a new strategy able to restore sensitivity to this treatment. Moreover, since the need for a murine model of resistant GBM to test the efficacy of the new therapeutic strategy, a secondary aim of this project has been the setting up of an orthotopic model of GBM, resistant to TMZ, and to characterize it as regards its radio-responsiveness. Final aim of the study has been to use the same orthotopic model to set up a non-invasive procedure to assess GBM by means of PET using 18F-Fluciclovine, whose cellular uptake is dependent upon the rate of activity of amino acid transporters (ASCT2). Materials and methods: HIF-1α activity crucial role in responsiveness to TMZ has been evaluated characterising two human GBM cell lines, U251-responsive cells to TMZ and T98-resistant ones, through molecular, biochemical ang gene expression analyses, by means of gene silencing and PX-478-mediated pharmacological inhibition. Moreover, CMA engagement in responsiveness to TMZ has been evaluated, following the experimental approach used for HIF-1α and through biochemical and protein studies of CMA pathway. Mitochondrial ROS contribution and the detox machinery role in TMZ treatment have been investigated, respectively, treating cells with MitoT utilising all the aforementioned techniques (including scratch test) and by means of gene expression profile and protein analyses. H2O2 treatments, which retrace the experimental approach used for MitoT, have been used for testing the potential ROS role in reverting resistance to TMZ. Murine glioma cell line CT-2A, after having been characterised as U251 and T98 for evaluating its responsiveness to TMZ, have been stereotaxically injected (i.c.) in C57BL/6J mice to set up orthotopic glioma models. MRI scans have been carried out at days 9 and 15 after i.c. injection for monitoring tumour growth. MRI has been exploited also for monitoring tumour growth after RT (15Gy-hemibrain). 18F-Fluciclovine-PET has been performed 16 days after i.c. injection. Untreated and RT-treated mice brains have been collected for IHC analyses regarding ASCT2 expression levels. Results: This study allowed the identification of response biomarkers and mechanisms involved in GBM resistance to TMZ treatment. HIF-1α has been identified as a crucial factor in resistance to TMZ: hypoxic cells are characterized by a lower sensitivity to the drug, while a significant decrease both in viability and HIF-1α activity has been detected after treatment in sensitive cells while no modulation in HIF-1α activity and viability was observed in T98 resistant cells. These results were confirmed by assessing also apoptosis-, CMA- and EMT-related gene expression. Further results showed the involvement of CMA in HIF-1α degradation and the consequent cytotoxicity due to the drug: in fact, LAMP-2A silencing induced resistance in previously sensitive cells, while HIF-1α gene silencing reverted T98 phenotype from a previously resistant to a sensitive one. Also, the PX-478 mediated HIF-1α activity abrogation confirmed the previous result. CMA activation following TMZ treatment can be induced by ROS release from the mitochondria as demonstrated by using the MitoT. The study of detox machinery showed a down-regulation in sensitive cells after TMZ treatment, consistently with the temporary ROS fluctuations. At the same time, an external-mediated increase in intracellular ROS level by H2O2 treatment resulted to be able to induce the same mechanisms activated by TMZ in sensitive cells after ROS release, determining a responsive profile both in sensitive and resistant cells. Of note, here we report that, only the concurrent treatment with H2O2 and TMZ induced a completely significant responsive profile in resistant cells, confirming the crucial role of ROS, CMA, HIF-1α modulation and the drug. The final result of this work is the development of a TMZ-resistant murine orthotopic model by i.c. injection of CT-2A cells. This model resulted to be responsive to RT, as reported by MRI studies. 18F-Fluciclovine tracer was used to monitor tumour extent in view of its implementation on the evaluation of response to treatment by PET. Finally, preliminary IHC data of untreated and RT-treated mice brains have confirmed a down-regulation of ASCT2 expression after RT compared to the untreated ones. Conclusions: In this thesis work, it has been demonstrated that HIF-1α activity is a key player in resistance to TMZ, that CMA has a crucial role in mediating TMZ-induced cytotoxicity and that the induction of this pathway was due to a transitory increase in intracellular ROS level. Moreover, an exogenous increase in ROS levels has been able to restore a completely responsive profile in resistant cells opening the way for the evaluation of new therapeutic approaches. CT-2A orthotopic murine models represent a good opportunity for the in vivo assessment of these new treatments, especially using the non-invasive multimodal imaging, strategy described herein.
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Hofer, Matthias Philipp. "Identification and characterisation of regulators of oligodendrocyte precursor cell differentiation and associated intracellular signalling pathways." Thesis, University of Cambridge, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.648833.
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Nguyen, Bao-Anh Vu. "Characterisation and modulation of the intracellular inflammatory signalling pathways activated during surgery with cardiopulmonary bypass." Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/33259.
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Surgery with cardiopulmonary bypass (CPB) is associated with post-operative complications due to systemic inflammation. However, the intracellular signalling pathways that promote inflammation in cardiac surgery with CPB are uncertain. The studies presented in this thesis were designed to illuminate these molecular mechanisms, thereby informing the development of novel anti-inflammatory strategies. This was addressed through a clinical trial to determine the effects of CPB on inflammatory signalling in leukocytes (Chapter 4). In this study, the induction of reactive oxygen species (ROS) and the activation of NF-κB and p38 MAP kinase within leukocytes was compared in patients exposed to miniaturised CPB (mCPB; an optimised form of CPB designed to attenuate systemic inflammatory activation) or conventional CPB (cCPB). Twenty-six patients undergoing surgical revascularisation for advanced coronary artery disease were randomised to undergo surgery with either cCPB or mCPB. Blood samples were collected pre-operatively and at various times after the initiation of CPB and analysed by intracellular staining and flow cytometry for intracellular markers of activation. p38 MAP kinase phosphorylation in granulocytes was enhanced in patients receiving cCPB compared to mCPB (p < 0.05). Levels of ROS in lymphocytes were elevated in cCPB compared to mCPB (p < 0.01) whereas ROS levels in granulocytes and monocytes were similar between groups. NF-κB phosphorylation in leukocyte sub-sets, leukocyte tissue migration as well as conventional markers of inflammation were comparable between the investigative groups. A porcine model was also established to study the signalling pathways that promote systemic inflammation in response to cardiac surgery with CPB under well-controlled experimental conditions. The influence of sulforaphane, an anti-inflammatory compound derived from green vegetables, on inflammation and injury in response to CPB was also studied. It was observed that pre-treatment of animals with sulforaphane reduced p38 MAP kinase (p < 0.05) and NF-κB (p < 0.05) phosphorylation in leukocytes exposed to CPB and protected porcine kidneys from exhibiting histological features of early injury. A small clinical study demonstrated biologically significant levels of sulforaphane could be determined in plasma, with lower levels of p38 MAP kinase (p < 0.01) and attenuated ROS (p < 0.01) in the early stages following consumption. In conclusion, systemic inflammatory responses following CPB were associated with activation of p38 MAP kinase and NF-κB pathways in circulating leukocytes in both porcine and clinical studies. Inflammatory responses to CPB can be reduced by miniaturisation of the CPB circuit and pharmacologically using sulforaphane.
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Thakker, Parva. "T Cell Intrinsic and Extrinsic Role of XIAP, During CD8 T Cell Response Against Intracellular Pathogens." Thesis, Université d'Ottawa / University of Ottawa, 2021. http://hdl.handle.net/10393/42419.
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The magnitude and effectiveness of CD8 response against intracellular pathogens is
directed by survival and apoptotic signals that govern the fate of T cells. XIAP is a bona fide endogenous inhibitor of apoptotic signals. In this thesis, I have investigated the role of XIAP at various stages of CD8 T cell response. I used both in vivo and in vitro models to show that XIAP acts in a CD8 T cell extrinsic and intrinsic manner to regulate the expansion and contraction phases of the CD8 T cell response, respectively. During the expansion phase, XIAP prevents the cell death of APCs to promote APC-T cell interaction and cytokine release, which facilitates the proliferation and survival of activated T cells. During the contraction phase, XIAP functions in a cell-intrinsic fashion to inhibit the proapoptotic signals in the activated CD8 T cells to prolong the immune response. Finally, I also demonstrate that the expression of XIAP in T cells is critical for their differentiation in to memory subsets. Overall, I present that XIAP plays a critical role in generating an effective CD8 T cell immune response.
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Marshall, Kirsty Mary. "Investigation of the intracellular pathways required for 5HT-induced mitogenesis and their role in pulmonary hypertension." Connect to e-thesis, 2007. http://theses.gla.ac.uk/103/.
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Thesis (Ph.D.) - University of Glasgow, 2007.Ph.D. thesis submitted to the Division of Neuroscience and Biomedical Systems, Institute of Biomedical and Life Sciences, University of Glasgow, 2007. Includes bibliographical references. Print version also available.
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Marshall, Kirsty M. "Investigation of the intracellular pathways required for 5HT-induced mitogenesis and their role in pulmonary hypertension." Thesis, University of Glasgow, 2008. http://theses.gla.ac.uk/103/.
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ABSTRACT Pulmonary arterial hypertension (PAH) is a rare and progressive disease characterised by increased pulmonary vascular resistance and elevated pulmonary artery pressure, leading to right ventricular failure and eventually death. The monoamine 5-hydroxytryptamine (5HT) has been implicated in the processes of pulmonary vasoconstriction and pulmonary artery remodelling that contribute to the development of the PAH. However, the signalling mechanisms utilised by 5HT that contribute to pulmonary vascular remodelling are still unclear and appear to be cell-type specific, with much of the work having been carried out in pulmonary artery smooth muscle cells (PASMCs). Fibroblasts also contribute significantly to the pulmonary vascular remodelling that occurs during PAH, however little is known of the role 5HT plays in this cell type. Using Chinese Hamster Lung Fibroblast (CCL-39) cells as a model system to investigate the mitogenic effects of 5HT, this study has characterised potential 5HT-mediated signalling pathways in fibroblasts that may contribute to pulmonary vascular remodelling. 5HT was found to induce a rapid and transient activation of extracellular regulated mitogen-activated protein kinase (ERK), a process central to the mitogenic effects of 5HT in CCL-39 cells. Furthermore, the 5HT transporter (5HTT), 5HT1B and 5HT2A receptors were all required for optimal ERK-dependent proliferation. Pharmacological inhibition of the Rho/ROCK (Rho-associated kinase) pathway significantly inhibited 5HT-stimulated ERK activation, cyclin D1 accumulation and proliferation. Inhibition of ROCK had no effect on the translocation of active ERK to the nucleus, but did however selectively inhibit 5HT-induced activation of a cytoplasmic pool of ERK. Additionally, ROCK inhibition had no effect on the ability of 5HT to activate mitogen-activated protein kinase kinase (MEK), suggesting ROCK is required for maintaining functional interactions between MEK and ERK. Sensitivity to ROCK inhibition is restricted to 5HT1B receptor activation of the ERK pathway. Moreover, the role of ROCK in maintaining cytoskeletal integrity is important in mediating 5HT-induced ERK activation, as disruption of the actin cytoskeleton markedly and specifically reduces 5HT- stimulated ERK activation. Using a model of PAH, arising from overexpression of 5HTT (5HTT+), the effects of ROCK inhibition in vivo were investigated. ROCK 1 and ROCK 2 transcripts were upregulated in response to chronic hypoxia, with the upregulation of ROCK 1 potentiated in 5HTT+ mice. Administration of the ROCK inhibitor Y27632 had significantly greater effects in 5HTT+ mice compared to WT, highlighting the functional importance of the increase in ROCK 1 transcript. Hypoxia-induced pulmonary vascular remodelling and elevated right ventricular pressure were attenuated more significantly by ROCK inhibition in 5HTT+ mice than in WT. Furthermore, ROCK inhibition only reduced hypoxia-derived right ventricular hypertrophy significantly in 5HTT+ animals and not WT. In conclusion, this study highlights a role for ROCK in the pulmonary vascular changes that occur during PAH and proposes a new mechanism by which cross-talk between ROCK and 5HT signalling systems occurs.
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Hutchinson, J. L. "Intracellular targeting mechanisms of Salmonella virulence effector proteins, and bacterial interactions with host antigen presentation pathways." Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604849.
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SPI2 effectors are responsible for an observed Salmonella-specified downregulation of MHC Class II at the surface of infected cells. The cooperative contribution of SPI2 effector interactions to the maturation of the Salmonella-containing vacuole (SCV) leads to the hypothesis that correct localisation of effectors following their secretion into the host cell is important to their function. Work described herein characterises the localisation of a key virulence factor, SifA, which contains a cysteine-rich C-terminal motif previously proposed to be a site for host lipid attachment. A role was demonstrated for lipid modification in retention of SifA at the SCV following secretion. However, membrane association and initial localisation of SifA to the SCV was shown to be independent of C-terminal lipid attachment, and instead likely dependent on the protein N terminus which shares homology with a subset of SPI2 effector N termini previously implicated in intracellular targeting. This thesis also characterises the distribution of host antigen presenting molecules within infected cells, drawing comparison between MHC Class II and the four surface isoforms of the non-classical lipid antigen-presenting CD1 molecules. Salmonella was shown to traffic through CD1-positive compartments yet in contrast to MHC Class II none of the CD1 isoforms were downregulated at the surface of infected MelJuso cells.
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Rohl, Joan. "Intracellular trafficking and secretion of matrix metalloproteinases during macrophage migration." Thesis, Queensland University of Technology, 2016. https://eprints.qut.edu.au/102374/1/Joan_Rohl_Thesis.pdf.
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This thesis advances the knowledge of intracellular trafficking pathways for the cell surface delivery of Matrix metalloproteinases 9 and 14 in macrophages. As elevated and persistent levels of these proteolytic enzymes contribute to excessive inflammation and poor wound healing outcomes, the findings from this thesis could lead to the development of improved therapeutics for the treatment of chronic wounds. Trafficking machinery proteins responsible for cell surface delivery of Matrix metalloproteinase 14, matrix degradation and macrophage invasion was identified and could be used as novel therapeutic targets.
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Hurst, Samantha. "n-3 Polyunsaturated fatty acid effects on inflammatory mediator activity and intracellular signalling pathways in chondrocyte metabolism." Thesis, Cardiff University, 2005. http://orca.cf.ac.uk/55511/.
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Previous studies have shown that supplementation of n-3 polyunsaturated fatty acids (PUFAs) has a beneficial effect on reducing the expression and activity of degradative enzymes and inflammatory factors known to cause damage and destruction of cartilage in arthritic diseases. The aims of this thesis was to use a well-established in vitro model of cartilage degradation to further these studies and to investigate how n-3 PUFAs effect the expression of inflammatory factors at a proteomic level and to use specific inhibitors to identify possible signalling pathways involved in cartilage metabolism. The results of this thesis research indicate that n-3 PUFAs abrogate IL-1-induced cyclooxygenase-2 (COX-2) mRNA expression, protein levels and activity, measured as PGE2 production, in both normal bovine and human osteoarthritis articular cartilage chondrocytes. These studies were followed by the use of a simple array system to analyse the expression of several marker genes from different signalling pathways after IL-1 exposure, plus or minus n-3 PUFA supplementation. This led us to identify three possible pathways involved in IL-1-induced cartilage catabolism and inflammation. These were analysed further with the use of specific inhibitors to ascertain whether the inhibition profiles were similar to those seen by n-3 PUFAs. Two main pathways, the extracellular signal-regulated kinase (ERK) pathway and NFkappaB pathway were identified. Further analysis using the ERK pathway inhibitor, U0126, showed that it decreased IL-1-induced glycosaminoglycan release from the tissue, endogenous aggrecanase activity, ADAMTS-4 (but not ADAMTS-5) mRNA levels, MMP-3 and MMP-13 mRNA levels, COX-2 message, protein levels and PGE2 production in a manner similar to that seen with n-3 PUFA supplementation. Collectively, these results suggest that n-3 PUFAs may be directing their effects through the ERK pathway.
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Pirkl, Martin Franz-Xaver [Verfasser], and Rainer [Akademischer Betreuer] Spang. "Indirect inference of synergistic and alternative signalling of intracellular pathways / Martin Franz-Xaver Pirkl. Betreuer: Rainer Spang." Regensburg : Universitätsbibliothek Regensburg, 2016. http://d-nb.info/1110148615/34.
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Xiong, Qingming. "Anaplasma phagocytophilum and Ehrlichia ewingii Exploit Host Signaling Pathways for Their Infection." The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1245167091.
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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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Biever, Anne. "Regulation of the protein synthesis machinery in the striatum." Thesis, Montpellier, 2016. http://www.theses.fr/2016MONTT002.
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Le striatum dorsal et le noyau accumbens (NAc) jouent un rôle crucial dans la sélection et l’exécution de mouvements résultant de l’intégration de signaux dopaminergiques et d’informations glutamatergiques sensorielles. A ce jour, les mécanismes moléculaires à travers lesquels la dopamine (DA) régule la plasticité des neurones épineux moyens du striatum (MSNs) sont peu connus. La synthèse des protéines est un événement essentiel requis pour la plasticité synaptique et la mémoire à long terme. Dans de nombreuses régions cérébrales, l’initiation, étant l'étape limitante de la synthèse protéique, est contrôlée par la phosphorylation de facteurs d’initiation de la traduction (eIFs). Notre hypothèse est que la DA pourrait réguler la traduction d’ARNm dans le striatum à travers des mécanismes moléculaires similaires. La première partie de cette thèse visait à étudier le rôle de la DA dans la régulation de la machinerie de traduction dans les MSNs. Pour ce faire, nous avons analysé au niveau du striatum, la phosphorylation de différents eIFs en réponse à l’administration aigue ou répétée de d-amphetamine (d-amph), entraînant une augmentation transitoire ou de longue durée de la transmission dopaminergique, respectivement. Bien que l’administration de la d-amph est associée à une légère augmentation de pS209-eIF4E, l’état de phosphorylation de S1108-eIF4G reste inchangé. En revanche, une forte augmentation de p51-eIF2α a été observée après administration répétée d-amph. Nous démontrons que la phosphorylation de 51-eIF2α est corrélée à une diminution transitoire de la synthèse protéique globale dans le striatum. En outre, la d-amph induit également une importante augmentation de la phosphorylation de la protéine ribosomale S6 (rpS6). Cet effet se produit spécifiquement dans MSNs exprimant le récepteur D1 à la DA et implique la cascade de signalisation AMPc/PKA/DARPP-32, tout en étant indépendant des voies mTORC1/S6K et ERK. La phosphorylation de rpS6 est couramment utilisée pour marquer de l'activité neuronale bien que son rôle biologique dans le cerveau reste énigmatique. Compte tenu sa régulation significative par la DA, la deuxième partie de cette thèse a eu pour but d’acquérir de nouvelles connaissances sur la fonction de la phosphorylation de rpS6 en utilisant un modèle de souris rpS6 déficient de ses sites de phosphorylations, rpS6P-/-. Dans ces souris transgéniques la synthèse protéique globale est normale dans diverses régions du cerveau. Néanmoins, les souris rpS6P -/- présentent une altération de la traduction d'un sous-ensemble de ARNm, ceci sélectivement dans le NAc, suggérant le rôle potentiel de la phosphorylation de rpS6 dans la régulation de la traduction de transcrits bien spécifiques au sein de cette sous-région du striatum. Dans l'ensemble, les résultats présentés dans cette thèse permettent une meilleure compréhension des mécanismes engagés par DA pour contrôler la traduction d’ ARNm dans les MSNs du striatumThe dorsal striatum and the nucleus accumbens (NAc) process dopamine (DA) signals in order to generate appropriate behavior in response to given glutamatergic sensory cues. The molecular mechanisms through which DA promotes long-lasting changes in striatal GABAergic medium-sized spiny neurons (MSNs) are still not fully understood. It is widely accepted that protein synthesis is an essential event required for several forms of synaptic plasticity and long-term memory. In various brain areas, initiation is the rate-limiting step of translation and is regulated through phosphorylation of translation initiation factors (eIFs). Whether DA could regulate mRNA translation in the striatum through similar mechanisms is yet poorly investigated. A first part of this thesis aimed to shed light on the role of DA in the regulation of the translational machinery in MSNs. Here, we measured the phosphorylation state of eIFs following single and repeated in vivo d-amphetamine (d-amph) administration, resulting in a transient or long-lasting increase of the dopaminergic transmission, respectively. Although d-amph exposure slightly enhances the striatal pS209-eIF4E, pS1108-eIF4G remains unchanged. In contrast, a strong increase in p51-eIF2α is observed after repeated d-amph administration. We demonstrate that d-amph-induced p51-eIF2α is associated to a transient decrease in generall striatal protein synthesis. In addition, d-amph markedly increases the striatal phosphorylation of the 40S ribosomal protein S6 (rpS6). This effect occurs selectively in D1 DA receptor (D1R)-expressing MSNs and requires the cAMP/PKA/DARPP-32 cascade but is independent of mTORC1/S6K and ERK signaling. rpS6 phosphorylation is commonly used as a marker for neuronal activity even though its biological role in the brain remains puzzling. Given the significant regulation of striatal rpS6 phosphorylation by DA, the second part of this thesis sought to gain new insights into the function of this post-translational event by using a phosphodeficient rpS6P-/- mouse model. We showed that rpS6P-/- mice display unaltered global protein synthesis in different brain regions. Nonetheless, rpS6P-/- mice exhibit impaired translation of a subset of mRNA selectively in the NAc, pointing to the potential role of rpS6 phosphorylation in the regulation of transcript-specific translation within this striatal sub-region. Overall, the results presented in this thesis provide a better understanding of the mechanisms engaged by DA to control mRNA translation in striatal MSNs
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Al-Rasheed, Nawal Mohammed. "Proinsulin C-peptide : activation of intracellular signalling pathways and modulation of transcription factors in opossum kidney proximal tubular cells." Thesis, University of Leicester, 2006. http://hdl.handle.net/2381/29949.
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In recent years an increasingly substantial body of data, supports a role for C-peptide in several biological activities. However, the precise molecular mechanisms of C-peptide action are not fully understood. The aim of this thesis was to study the intracellular signalling pathways and the transcription factors that C-peptide activates in proximal tubular cells using opossum kidney cells (OK) as a model. Using specific inhibitors and phospho-specific antibodies, intracellular signalling pathways activated by C-peptide were examined by kinase assay and Western blotting. The results show that C-peptide is able to activate extracellular signal regulated kinase (ERK), phosphatidylinositol 3-kinase (PI 3-kinase) and PKC-a. ERK activation was attenuated by PKC inhibitor pre-treatment and activation of ERK and PKC-a were abolished in the absence of extracellular Ca2+. Elevations of [Ca2+]i were examined using confocal microscopy. C-peptide induced transient increase in [Ca2+]i but the response of cells was variable. Thymidine incorporation assay was used to assess proliferation. C-peptide was found to be a functional mitogen in this cell type stimulating significantly increased cell proliferation. Proliferator-activated receptor (PPAR) transcriptional activity was measured using a luciferase reporter assay in OK cells. C-peptide induced concentration-dependent stimulation of PPARy activity. C-peptide also substantially augmented ciglitazone-stimulated PPARy activity. GW9662, an irreversible PPARy antagonist, blocked PPARy activation by ciglitazone, but had no effect on C-peptide-stimulated PPARy activity. C-peptide stimulation of PPARy was attenuated by wortmannin pre-treatment, and by expression of a dominant negative PI 3-kinase p85 regulatory subunit (Ap85). C-peptide had no effect on protein expression levels of PPARy. PPARy phosphorylation was examined by [32P]-orthophosphate labelling of OK cells and immunoprecipitation of phospho-PPARy. C-peptide-induced PI 3-kinase dependent phosphorylation of PPARy. C-peptide is able to protect against tumor necrosis factor-alpha- (TNF-a) induced proximal tubular cells toxicity. Stimulation with 300ng/ml TNF-a for 24 hours resulted in significant reduction of cell viability which was reversed by pretreatment with C-peptide. TNF-a induced apoptosis was detected by measuring histone associated DNA fragments and DNA nick end-labelling of OK cells. Incubation of cells with 300ng/ml TNF-a for 24 hours induced apoptosis, but C-peptide pr-etreatment protected against TNF-a induced apoptosis. The protective effects of C-peptide were associated with activation of nuclear factor kB (NFkB) and increased expression of TNF receptor-associated factor 2, the product of an NFkB-dependent survival gene. This was dependent upon activation of PI 3-kinase, but not ERK. All C-peptide effects were abolished by pretreatment with PTX implicating a G-protein coupled receptor (GPCR), to either Goii or GOo, in the transduction of these events. C-peptide increased [35S]-GTPyS binding to Ga* in OK cell membranes. This study has now for the first time demonstrated specifically that Ga* proteins are activated by C-peptide binding to a GPCR. Despite being ignored for many years it is now clear that C-peptide possesses important biological properties and may potentially protect against diabetic complications.
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Watt, Fiona Elizabeth. "Investigation of the effects of injury upon intracellular signalling pathways and expression of inflammatory response genes in articular cartilage." Thesis, Imperial College London, 2009. http://hdl.handle.net/10044/1/4701.
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Damage to joints predisposes to osteoarthritis. The mechanism by which injury to cartilage might lead to net matrix loss and cartilage degeneration remains unknown. Following experimental sharp injury to porcine articular cartilage (dissection from, or scoring of the articular surface), our group has previously shown rapid activation of the 3 mitogen activated protein kinase (MAPK) pathways in cartilage. Activation of ERK, and probably also p38 MAPK, is due to release of fibroblast growth factor (FGF) from the matrix after injury. However, despite a long search, the cause of JNK activation following sharp injury remains unknown. I investigated the extent and regulation of inflammatory signalling after cartilage injury and whether it was sufficient to cause expression of inflammatory response genes. In this work, I show that a number of intracellular signalling pathways including PI3 kinase and IκB kinase (IKK) (which leads to activation of NFκB) are activated by sharp injury to cartilage. The signalling following injury was sufficient to induce a wide range of inflammatory response mRNAs, including pro-inflammatory cytokines such as IL-6, COX-2 and proteinases such as MMP-1 and ADAMTS-4 in a pattern which was not entirely IL-1-like. Pharmacological inhibition experiments suggested that the production by injured cartilage of an inflammatory response gene which could be assayed at the protein level, activin A, was regulated by FGF-mediated pathways (ERK) as well as by NFκB and tyrosine kinases (src family kinases). Given these findings, the role of tyrosine kinases in the early response of cartilage to injury was explored. By phosphotyrosine immunoprecipitation and purification from injured cartilage lysates, FAK and its substrate paxillin were identified from silverstained gels by mass spectrometry. The phosphorylation of these src substrates accounted for rapidly inducible bands seen on phosphotyrosine western blotting of injured cartilage lysates. However, no evidence of a role for src kinases in the regulation of MAPK/IKK signalling upon injury was found. In contrast, blockade of another tyrosine kinase, the FGF receptor, led to partial inhibition not only of the ERK pathway following sharp injury, but also the other MAPKs and IKK. Whilst activation of the same pathways was also seen following injury to synovium, FGF receptor inhibition had no effect on this signalling. This suggested that FGF may have a pro-inflammatory action following injury in vivo which is a particular feature of cartilage.
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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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Velthuis, Jarig Herman Lambertus. "Natural killer cell-induced apoptosis in rat colon carcinoma cells : a study on effector mechanisms and their intracellular signaling pathways /." Leiden : [Universiteit Leiden], 2003. http://catalogue.bnf.fr/ark:/12148/cb399324262.
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Tjong, Yung-wui, and 鍾勇會. "Mechanisms of endogenous nitric oxide production and intracellular pathways in rat hippocampal CA1 calcium response to hypoxia and in-vitro ischemia." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B30073005.
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Liu, Ke. "Role of second messengers in controlling growth patterns of corneal epithelial cells." Thesis, View thesis, 2002. http://handle.uws.edu.au:8081/1959.7/387.
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The purpose of this thesis was to investigate mechanisms contolling the growth of corneal epithelial cells, particularly the intracellular signals involved with stratification compared with cellular migration and maturation. Buttons of epithelium were cultured in different culture media. The explants were monitored microscopically for their growth patterns and finally fixed and examined for cytokeratin, vimentin and actin. Different growth patterns were observed in the different media, indicating that different signalling patterns must be operating in these cells depending upon the media in which they were grown. To investigate the intracellular pathways controlling the different growth patterns, the protein phosphorylation of different cultures was investigated. The two proteins, p57 and p30, are strongly suggested to be associated with stratification of the epithelial cells. The possible involvement of the common serine kinase, PKC, in controlling the growth pattern of corneal epithelial cells were also investigated. The results suggested that an intracellular pathway involving PKC promotes the maturation and spread of the cells but is not involved in their stratification. These experiments taken together indicate that the different aspects of corneal epithelia cell growth are tightly controlled and may occur quite independently. Specific protein expression appears to be important for stratification, and phosphorylation of proteins by PKC appears to be involved with the maturation of epithelial cells from basal cells. It also indicates that the mature cells are capable of producing the extracellular matrix protein fibronectin which appears to have an important role in causing the spread as distinct from the stratification of the corneal epithelial cells.
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Guo, Daorong. "Dissecting Signaling Pathways that Regulate Axonal Guidance Effects of Sonic Hedgehog: A Dissertation." eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/531.
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During development, axons respond to a variety of guidance cues in the environment to navigate to the proper targets. Sonic hedgehog (Shh), a classical morphogen, has been shown to function as a guidance factor that directly acts on the growth cones of various types of axons. We previously found that Shh affects retinal ganglion cell (RGC) axonal growth and navigation in a concentration-dependent manner. However, the signaling pathways that mediate such events are still unclear.
In this thesis, we show that high concentrations of Shh induce growth cone collapse and repulsive turning of the chick RGC through rapid increase of Ca2+ in the growth cone, and specific activation of PKCα and Rho signaling pathways. We further found that integrin linked kinase (ILK) acts as an immediate downstream effector of PKCα. PKCα directly phosphorylates ILK in vitro at two previously unidentified sites threonine-173 and -181. Inhibition of PKCα, Rho, and ILK by pharmacological inhibitors and/or dominant-negative approaches abolished the negative effects of high-concentration of Shh. We provide evidence that Rho likely functions downstream of PKC and suggest that PKC, Rho and ILK may cooperatively mediate the negative effects of high concentrations of Shh. Furthermore, retroviral expression of dominant-negative constructs of PKCα (DN-PKCα) and ILK-double mutants (ILK-DM) resulted in misguidance of RGC axons at the optic chiasm in vivo. These results demonstrate that new signaling pathways composed of PKCα, Rho, and ILK play an important role in Shh-induced axonal chemorepulsion.
In contrast, we show that attractive axonal turning in response to low concentrations of Shh is independent of PKCα, but requires the activity of cyclic nucleotides cAMP. Taken together, our results suggest that the opposing effects of Shh on axon guidance are mediated by different signaling pathways.