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1
Mahnel, Petr. "Optimalizace výroby firmy AKT." Master's thesis, Vysoká škola ekonomická v Praze, 2009. http://www.nusl.cz/ntk/nusl-72128.
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This thesis aims to provide a reader with practical example of optimization achieved using the Lingo software. The main focus is improvement of efficiency and shortening of a production process in AKT, a company engaged in production of car parts. It is a software, which accommodates needs of the company as well as production requirements. The theoretical part is focused on detailed description of methods and procedures used in a practical part. These methods should help the reader to understand a nature of formulas relevant for the subject. Practical part focuses on the optimization using data obtained from the company AKT, evaluation of the data and subsequent consultations with a manager of production, followed by the assessment of practical relevance for the production.
2
Aiken, Andrew. "AKT-R4 a diagnosis tool." Thesis, Available from the University of Aberdeen Library and Historic Collections Digital Resources, 2008. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?application=DIGITOOL-3&owner=resourcediscovery&custom_att_2=simple_viewer&pid=25223.
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Meadows, Kafi, Seema Iyer, Mark Stevens, Duanning Wang, Sharon Shechter, Carole Perruzzi, Todd Camenisch, and Laura Benjamin. "Akt promotes Endocardial-Mesenchyme Transition." BioMed Central, 2009. http://hdl.handle.net/10150/610167.
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Endothelial to mesenchyme transition (EndMT) can be observed during the formation of endocardial cushions from the endocardium, the endothelial lining of the atrioventricular canal (AVC), of the developing heart at embryonic day 9.5 (E9.5). Many regulators of the process have been identifiedhowever, the mechanisms driving the initial commitment decision of endothelial cells to EndMT have been difficult to separate from processes required for mesenchymal proliferation and migration. We have several lines of evidence that suggest a central role for Akt signaling in committing endothelial cells to enter EndMT. Akt1 mRNA was restricted to the endocardium of endocardial cushions while they were forming. The PI3K/Akt signaling pathway is necessary for mesenchyme outgrowth, as sprouting was inhibited in AVC explant cultures treated with the PI3K inhibitor LY294002. Furthermore, endothelial marker, VE-cadherin, was downregulated and mesenchyme markers, N-cadherin and Snail, were induced in response to expression of a constitutively active form of Akt1 (myrAkt1) in endothelial cells. Finally, we isolated the function of Akt1 signaling in the commitment to the transition using a transgenic model where myrAkt1 was pulsed only in endocardial cells and turned off after EndMT initiation. In this way, we determined that increased Akt signaling in the endocardium drives EndMT and discounted its other functions in cushion mesenchymal cells.
4
Park, Sungman. "AKT function and human oncogenesis." [Tampa, Fla.] : University of South Florida, 2007. http://purl.fcla.edu/usf/dc/et/SFE0001885.
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Palková, Anežka. "Spolupráce EU - AKT na příkladu Haiti." Master's thesis, Vysoká škola ekonomická v Praze, 2011. http://www.nusl.cz/ntk/nusl-113492.
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This thesis focuses on cooperation of the European Union with the African, Carribean and Pacific Group of States. Its aim is to describe the relations in a complex way and to record the changes that are connected with the evolution of cooperation. Attention is also paid to conditionality of cooperation. Introductory part describes EU development cooperation and humanitarian aid. Historical evolution of EU-ACP cooperation follows together with the details on the Cotonou Agreement. Last part is the case study on Haiti.
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Middel, Ines Kristin. "Quantitative Untersuchung der Proteinkinase AKT am Ovarialkarzinom." Köln Deutsche Zentralbibliothek für Medizin, 2010. http://d-nb.info/1000727416/34.
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Ng, Foong Loo Yvonne Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "Insulin action: unravelling AKT signalling in Adipocytes." Awarded by:University of New South Wales. Biotechnology & Biomolecular Sciences, 2009. http://handle.unsw.edu.au/1959.4/44628.
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The Ser/Thr kinase Akt plays an important role in many of insulin's actions including GLUT4 translocation to the plasma membrane (PM). However, there are several features of Akt's regulation of GLUT4 translocation that remain unclear. The goal of my thesis was to resolve some of the following questions: Is activation of Akt sufficient to stimulate GLUT4 translocation? What is the quantitative relationship in signal transmission between individual components within the Akt cascade? What is the role of Akt in insulin resistance? To determine if activation of Akt is sufficient to mediate GLUT4 translocation, I developed a drug-inducible heterodimerisation strategy to activate Akt2 independently of other potential insulin signalling pathways. These studies revealed that activation of Akt2 resulted in rapid stimulation of GLUT4 translocation to a similar extent with maximum insulin, indicating that Akt2 is sufficient for this event. It was previously observed that maximum effect of insulin on GLUT4 translocation was obtained with minimum activation of Akt. To resolve this discrepancy, the relationship between Akt signalling components was examined using a quantitative kinetic and dose response approach combined with hierarchical cluster analysis. Most notably I observed a strong relationship between Akt at the PM, but not Akt in the whole cell lysate, with its substrate phosphorylation. Active pools of phospho-Akt and -AS160, a major substrate involved in GLUT4 translocation, were found in the lipid raft, highlighting the importance of subcellular partitioning of key signalling components for achieving biological specificity. The involvement of Akt in insulin resistance was investigated using the heterodimerisation strategy. These studies revealed that insulin itself initiates a pathway that causes insulin resistance by converging on target(s) downstream of Akt. This inhibitory pathway emanates from PI3-kinase and is likely induced by a range of insults including chronic insulin and dexamethasone. In conclusion, Akt is a crucial element in the insulin action pathway that exhibits precise spatial regulation. While the role of this nanoregulation of Akt in disease remains to be evaluated, my studies suggest that the major defect contributing to insulin resistance occurs downstream of Akt. The elucidation of this target will have major implications for metabolic diseases.
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Rickle, Annika. "PTEN and Akt signalling in Alzheimer's disease /." Stockholm : Karolinska institutet, 2005. http://diss.kib.ki.se/2005/91-7140-514-3/.
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Hiester, Andreas [Verfasser]. "Proteininteraktion der Proteinkinase AKT 1 / Andreas Hiester." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2014. http://d-nb.info/1046174215/34.
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Fedrigo, Carlos Alexandre. "Inibição da via PI3K-Akt em gliomas." Pontifícia Universidade Católica do Rio Grande do Sul, 2012. http://hdl.handle.net/10923/4518.
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Previous issue date: 2012Glioblastoma multiforme (GMB) is the most malignant and common type of all astrocytic tumours. Current standard treatment for GBM patients involves maximum surgical resection of the tumour, followed by radiotherapy and chemotherapy, usually containing the alkylating agent Temozolomide (TMZ). Despite this aggressive combination therapy, the survival rate of GBM patients is still low. This work consisted in investigating the cytotoxic effects of Akt-inhibition by MK-2206 with irradiation (RT) and TMZ on in vitro human malignant glioma. Seven malignant glioma cell lines were cultured and tested for clonogenic survival, invasion inhibition, tumour spheroid growth and proliferation. The Akt-inhibitor MK-2206 and TMZ were added at different time treatments and in varying doses. Cultures were irradiated with single dose and with fractionated γ-irradiation. Cellular modulation of Akt and p-Akt were assessed by Western blot analysis. MK-2206 reduced the levels of phospho- Akt key protein in the PI3Kinase-Akt pathway, decreased cell survival, and inhibited invasion, proliferation and cell growth. The combination of MK-2206 and RT lead to enhanced inhibition of cell proliferation and invasion, which is not observed with RT alone. The radioenhancing effect of MK-2206 was most striking in inhibition of spheroid volume growth by fractionated RT; the radiosensitizing effect of MK-2206 was stronger than that of TMZ. MK-2206 enhanced the in vitro effects of RT and TMZ in terms of decreased cell survival, invasion, proliferation and growth in malignant glioma. Effects could be ascribed to inhibition of PI3K-Akt pathway.
O Glioblastoma multiforme (GBM) é o tipo mais maligno e mais comum de todos tumores astrocíticos. O tratamento atual para pacientes de GBM envolve máxima remoção cirúrgica, seguida de radio e quimioterapia, normalmente com o agente alquilante Temozolamida (TMZ). Apesar da agressividade da terapia combinada, o tempo de sobrevivência dos pacientes ainda é baixo. Este trabalho procurou investigar os efeitos citotóxicos do inibidor de Akt MK-2206 em combinação com irradiação (RT) e TMZ em um painel de células de gliomas humanos. Sete linhagens de glioma foram cultivadas e testadas em ensaio de sobrevivência clonogênica, inibição de invasão, e modelos de proliferação e crescimento de volume em esferóides. O inibidor MK-2206 e TMZ foram adicionados em diferentes tempos de tratamento e diferentes doses. As culturas foram irradiadas com doses únicas ou em terapias fracionadas com irradiação γ. A modulação celular de Akt e fosfo-Akt foi checada via Western Blot. O composto MK-2206 reduziu a fosforilação da proteína chave Akt na via PI3K, diminuindo a sobrevivência celular e inibindo invasão, proliferação e crescimento celular. A combinação de MK-2206 com RT levou a uma maior inibição de invasão e proliferação, o que não é observado somente com a RT. O efeito radiosensível de MK-2206 foi ainda maior na inibição do volume dos esferóides em terapia combinada com RT fracionada, sendo ainda maior do que o efeito combinado com TMZ. MK-2206 aumentou os efeitos in vitro de RT e TMZ em termos de redução de sobrevivência celular, invasão, proliferação e crescimento celular em gliomas malignos. Os efeitos podem ser atribuídos a inibição da via PI3KAkt.
11
Song, Jingwen. "Subcellular compartmentalization of Akt contributes to signaling intensity." Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=114537.
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Background: In the Current Model of Akt activation, a key event is its recruitment to the plasma membrane, where it is fully activated subsequent to sequential phosphorylation at positions Ser473 and Thr308. The distribution of Akt into various subcellular compartments may contribute to the different signaling pathways which it influences. There is evidence of compartment-specific regulators in plasma membrane (PM) and endosomes. Previous experiment in our laboratory showed that Akt achieved greater specific activity in PM compared to cytosol after insulin treatment. This raised the question whether Akt hyperactivity in PM is exclusively due to the high concentration in PM of activated Akt (theCurrent Model), or due to other factor (s) besides pSer473 and pThr308. Aim of study: The objective of our study was to assess the sequence and extent of the Akt activation state as a consequence of its subcellular localization. Therefore we compared the Akt activation state (Akt kinase activity / phosphorylation level, or Akt kinase activity/content) in two compartments (PM & cytosol) after insulin and EGF (Epidermal Growth Factor) administration. Results and conclusions: We found that Akt pThr308 correlates more closely to Akt kinase activity than pSer473; and concluded that Akt pThr308 is the important phosphorylation site determining Akt activity as stipulated in the Current Model. Second, Akt activity differed in PM vs cytosol with the activity in PM not fully explained by the levels of pSer473 and pThr308. We suggest that other regulator(s) exist in the plasma membrane which contributes to the up-regulation of Akt activity. Third, we found that the potential regulator(s) plays a comparable role to influence Akt activity in PM after both insulin and EGF administration.Mise en contexte: Dans le modèle actuel d'activation de l'Akt, un événement clé est le recrutement de cette protéine à la membrane plasmique, où elle est complètement activée à la suite de la phosphorylation séquentielle des acides aminés aux positions Ser473 et Thr308. La distribution de l'Akt dans les divers compartiments subcellulaires pourrait contribuer aux différentes voies de signalisations. Il est rapporté que des régulateurs de compartiments spécifique dans la membrane plasmique (PM) et les endosomes contribuent à son recrutement. Des expériences précédentes dans notre laboratoire ont démontré que l'Akt a atteint un niveau d'hyperactivité dans la PM en comparaison avec le cytosol après avoir ajouté l'insuline. Ceci amène à la question de l'hyperactivité de l'Akt dans la PM est exclusivement dûe à la haute concentration d'Akt activés dans la PM (le modèle actuel) ou d'autre(s) facteur(s) additionnel (les) contribue (nt) à son activation. But de l'étude : L'objectif de cette étude est de déterminer la conséquence et l'ampleur du niveau d'activation de l'Akt en fonction de sa localisation subcellulaire. Ainsi, nous avons comparé le niveau d'activation de l'Akt (l'activité Akt kinase / niveau de phosphorylation, ou l'activité Akt kinase / contenu) dans deux compartiments (PM & cytosol) après l'administration d'insuline et d'EGF (Epidermal Growth Factor). Résultats et conclusions : Nous avons observé que l'Akt pThr308 est corrélée plus étroitement à l'activité de l'Akt kinase qu'à pSer473 et avons conclu que l'Akt pThr308 est le site significatif pour déterminer l'activité de l'Akt tel que défini dans le modèle actuel. De plus, l'activité de l'Akt est différente dans la PM versus cytosol. Également, l'activité de l'Akt dans la PM n'est pas complètementexpliquée par les niveaux de pSer473 et pThr308. Nous spéculons que d'autres facteurs régulateurs pourraient jouer un rôle comparable dans l'activité de l'Akt dans la PM après l'administration d'EGF et d'insuline.
12
Peng, Zhengang, Jennifer Weber, Zhaosheng Han, Rulong Shen, Wenchao Zhou, James Scott, Michael Chan, and Huey-Jen Lin. "Dichotomy effects of Akt signaling in breast cancer." BioMed Central, 2012. http://hdl.handle.net/10150/610205.
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BACKGROUND:The oncogenic roles contributed by the Akt/PKB kinase family remain controversial and presumably depend on cell context, but are perceived to be modulated by an interplay and net balance between various isoforms. This study is intended to decipher whether distinct Akt kinase isoforms exert either redundant or unique functions in regulating neoplastic features of breast cancer cells, including epithelial-mesenchymal transition (EMT), cell motility, and stem/progenitor cell expansion.RESULTS:We demonstrate that overactivation of Akt signaling in nonmalignant MCF10A cells and in primary cultures of normal human mammary epithelial tissue results in previously unreported inhibitory effects on EMT, cell motility and stem/progenitor cell expansion. Importantly, this effect is largely redundant and independent of Akt isoform types. However, using a series of isogenic cell lines derived from MCF-10A cells but exhibiting varying stages of progressive tumorigenesis, we observe that this inhibition of neoplastic behavior can be reversed in epithelial cells that have advanced to a highly malignant state. In contrast to the tumor suppressive properties of Akt, activated Akt signaling in MCF10A cells can rescue cell viability upon treatment with cytotoxic agents. This feature is regarded as tumor-promoting.CONCLUSION:We demonstrate that Akt signaling conveys novel dichotomy effects in which its oncogenic properties contributes mainly to sustaining cell viability, as opposed to the its tumor suppressing effects, which are mediated by repressing EMT, cell motility, and stem/progenitor cell expansion. While the former exerts a tumor-enhancing effect, the latter merely acts as a safeguard by restraining epithelial cells at the primary sites until metastatic spread can be moved forward, a process that is presumably dictated by the permissive tumor microenvironment or additional oncogenic insults.
13
Fedrigo, Carlos Alexandre. "Inibi??o da via PI3K-Akt em gliomas." Pontif?cia Universidade Cat?lica do Rio Grande do Sul, 2012. http://tede2.pucrs.br/tede2/handle/tede/1696.
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Previous issue date: 2012-05-29Glioblastoma multiforme (GMB) is the most malignant and common type of all astrocytic tumours. Current standard treatment for GBM patients involves maximum surgical resection of the tumour, followed by radiotherapy and chemotherapy, usually containing the alkylating agent Temozolomide (TMZ). Despite this aggressive combination therapy, the survival rate of GBM patients is still low. This work consisted in investigating the cytotoxic effects of Akt-inhibition by MK-2206 with irradiation (RT) and TMZ on in vitro human malignant glioma. Seven malignant glioma cell lines were cultured and tested for clonogenic survival, invasion inhibition, tumour spheroid growth and proliferation. The Akt-inhibitor MK-2206 and TMZ were added at different time treatments and in varying doses. Cultures were irradiated with single dose and with fractionated γ-irradiation. Cellular modulation of Akt and p-Akt were assessed by Western blot analysis. MK-2206 reduced the levels of phospho- Akt key protein in the PI3Kinase-Akt pathway, decreased cell survival, and inhibited invasion, proliferation and cell growth. The combination of MK-2206 and RT lead to enhanced inhibition of cell proliferation and invasion, which is not observed with RT alone. The radioenhancing effect of MK-2206 was most striking in inhibition of spheroid volume growth by fractionated RT; the radiosensitizing effect of MK-2206 was stronger than that of TMZ. MK-2206 enhanced the in vitro effects of RT and TMZ in terms of decreased cell survival, invasion, proliferation and growth in malignant glioma. Effects could be ascribed to inhibition of PI3K-Akt pathway
O Glioblastoma multiforme (GBM) ? o tipo mais maligno e mais comum de todos tumores astroc?ticos. O tratamento atual para pacientes de GBM envolve m?xima remo??o cir?rgica, seguida de radio e quimioterapia, normalmente com o agente alquilante Temozolamida (TMZ). Apesar da agressividade da terapia combinada, o tempo de sobreviv?ncia dos pacientes ainda ? baixo. Este trabalho procurou investigar os efeitos citot?xicos do inibidor de Akt MK-2206 em combina??o com irradia??o (RT) e TMZ em um painel de c?lulas de gliomas humanos. Sete linhagens de glioma foram cultivadas e testadas em ensaio de sobreviv?ncia clonog?nica, inibi??o de invas?o, e modelos de prolifera??o e crescimento de volume em esfer?ides. O inibidor MK-2206 e TMZ foram adicionados em diferentes tempos de tratamento e diferentes doses. As culturas foram irradiadas com doses ?nicas ou em terapias fracionadas com irradia??o γ. A modula??o celular de Akt e fosfo-Akt foi checada via Western Blot. O composto MK-2206 reduziu a fosforila??o da prote?na chave Akt na via PI3K, diminuindo a sobreviv?ncia celular e inibindo invas?o, prolifera??o e crescimento celular. A combina??o de MK-2206 com RT levou a uma maior inibi??o de invas?o e prolifera??o, o que n?o ? observado somente com a RT. O efeito radiosens?vel de MK-2206 foi ainda maior na inibi??o do volume dos esfer?ides em terapia combinada com RT fracionada, sendo ainda maior do que o efeito combinado com TMZ. MK-2206 aumentou os efeitos in vitro de RT e TMZ em termos de redu??o de sobreviv?ncia celular, invas?o, prolifera??o e crescimento celular em gliomas malignos. Os efeitos podem ser atribu?dos a inibi??o da via PI3KAkt
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McKenzie, Maxine. "Akt signalling in the human parasite 'Schistosoma mansoni'." Thesis, Kingston University, 2017. http://eprints.kingston.ac.uk/41116/.
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The study of cell signalling in schistosomes is crucial in deepening our knowledge of the biology of these blood flukes, which affect hundreds of millions of people worldwide. Here, Akt/protein kinase B (PKB) signalling has been functionally characterised and mapped in Schistosoma mansoni; an Akt variant of approximately 52 kDa has been characterised and RNA interference of the S. mansoni Akt gene, resulted in an 84% reduction in Akt expression. The phosphorylation (activation) status of the characterised Akt protein was increased by host molecules, including insulin and L-arginine in somules and adult worms, and L-arginine and linoleic acid in cercariae. Akt phosphorylation (activation) was also attenuated by Akt Inhibitor X and herbimycin A treatment. Immunohistochemistry/confocal laser scanning microscopy revealed phosphorylated Akt in all S. mansoni human infective/resident life stages. Somules and adult worms displayed activated Akt primarily in the tegument, particularly the tubercles and gynaecophoric canal of adult males. Cercariae exhibited activated Akt in the nervous system and punctate regions along the length of the tail prompting investigation into the role of Akt in cercarial motility. Behavioural studies demonstrated a significant increase in cercarial swimming in response to host factors, which was attenuated following exposure to Akt inhibitor X. The striking activation of Akt observed in the tegument of adult worms stimulated research into its possible role in glucose uptake in this host-interactive layer. RNAi of Akt resulted in a 59% and 47% reduction in SGTP4 glucose transporter expression in male and female adult worms respectively with a concomitant reduction in glucose uptake by the parasite. In somules, the expression of SGTP4 and its evolution at the apical tegument membrane during transformation were significantly attenuated by Akt Inhibitor X; a 74% reduction in glucose uptake was also demonstrated following Akt inhibition. Bioinformatic analysis of S. mansoni Akt interacting proteins uncovered a putative connection between Akt and Rab vesicle trafficking proteins and a mechanistic model illuminating the possible role of Akt in the translocation of SGTP4 to the parasite surface was proposed. Collectively, this research highlights the significance of Akt in schistosome homeostasis and host-parasite interactions and thus demonstrates that Akt may be a suitable target for anti-schistosome drug development strategies.
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Watton, Sandra Jayne. "Cell adhesion and growth factor regulation of Akt." Thesis, University College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.394505.
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Crompton, Joseph. "Targeting Akt in cell transfer immunotherapy for cancer." Thesis, University of Cambridge, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.709380.
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Brustolon, Francesca. "Proteinchinasi di sopravvivenza: CK2, Akt, PRPK. Connessioni regolatorie." Doctoral thesis, Università degli studi di Padova, 2008. http://hdl.handle.net/11577/3426386.
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In this work I focused on functional correlations between some important protein kinases, tightly involved in cell viability. The project was divided in the following major parts:
a) CK2 indirectly modulates phosphorylation state of Akt pThr308.
Protein kinase CK2 is a Ser/Thr protein kinase composed of two catalytic (? and/or ?') and two regulatory (?) subunits. It is ubiquitous, constitutively active, and pleiotropic, with more than 300 protein substrates known so far. CK2 plays an antiapoptotic role, coordinating a network of signaling pathways essential for cell survival. One of these is rapresented by Akt, a kinase whose mechanism of activation is based on the phosphorylation of two key residues: Thr308 (by the PDK1 kinase) and Ser473 (by the mTOR/Rictor complex). On the other side, the dephosphorylation of Thr308 and Ser473 is accounted for by PP2A phosphatase and PHLPP, respectively.
CK2 acts directly on Akt, phosphorylating its Ser129 residue, both in vitro and in vivo, thus enhancing its catalytic activity.
Here we also show that downregulation of CK2 activity by inhibitors, or mutation of the CK2-dependent site in Akt (Ser129Ala), induce a lower phosphorylation of Thr308, which is not a direct target for CK2. This finding can be explained assuming that phosphorylation of Akt at Ser129 by CK2 facilitates the recognition of Thr308 by the kinase PDK1 or, alternatively, makes Akt less accessible to the phosphatase PP2A. We found that PDK1 does not discriminate between the two different forms of Akt, phosphorylated or not at the CK2 site; on the contrary, our data show that, in case of Ser129 phosphorylation, a lower degree of T308 dephosphorylation occurs. In fact, when we treat cells with the PP2A inhibitor okadaic acid, any difference at Thr308 phosphorylation is abrogated between wt Akt (phosphorylated at Ser129 by CK2) and Ser129Ala Akt mutant. We also found that Hsp90, involved in preserving the Thr308 phosphorylation state, is less tightly associated to Akt in case of the Ser129Ala mutation. This indicates that the regulation operated by CK2 on Akt can be ascribed, at least in part, to a protection from the pThr308-dephosphorylation process, possibly involving other protein partners, such as the Cdc37/Hsp90 complex.
b) Drug resistance and pro-survival protein kinases.
Given the pro-survival and anti-apoptotic function of CK2 and Akt, we also investigated their possible involvement in the multidrug resistance phenotype, a condition where a high proliferation rate and a reduced cell death degree result in the failure of cancer therapy.
We analysed the expression level of these two kinases and their role in some cell lines, available in the two variants normally sensitive (S) or resistant (R) to chemical apoptosis. Most of the work was focused on T-lymphoblastoid cells (CEM-S and CEM-R), but we also extended our analyses to other cell models, particullary to an osteosarcoma cell line (U2OS-S and U2OS-R) and to ovarian carcinoma cells (2008-S and 2008-R). We found that CK2 and Akt are differently expressed throughout our cellular models, causing different panels of endogenous substrates phosphorylation, whose identification can be useful to understand the drug resistance phenomenon.
We also compared CK2 activity of sensitive and resistant cells, and we evaluated the effect of CK2 specific inhibitors on cell viability, showing that CK2 blockade is effective in inducing cell death of resistant lines tested so far. We therefore conclude that inhibition of CK2, also considering the connections of this kinase with other survival pathways, such as Akt, can be considered as a promising tool to sensitize resistant cells to drug-induced apoptosis.
c) Regulation of PRPK by Akt.
Finally we analysed the functional correlation between Akt and PRPK (p53-related protein kinase), the human homologue of yeast Bud32. PRPK and Bud32 belong to a small subfamily of atypical protein kinases, called piD261. Despite Bud32, which is a kinase essential for yeast survival and morphology, the role of PRPK in the cell is still unclear. It is inactive unless it is previously incubated with cell lysates. We have seen that such an activation of PRPK is mediated by Akt, which phosphorylates PRPK at Ser250. Recombinant PRPK was shown to be phosphorylated in vitro by Akt and its phospho-form is recognized by a Ser250-phospho-specific antibody. Here we demonstrate that this phosphorylation takes place also in vivo: in fact cell co-transfection with Akt along with wild-type PRPK, but not with its Ser250Ala mutant, results in increased PRPK phosphorylation; moreover, the phosphorylation of p53 at Ser15, the only known substrate of PRPK, is markedly increased by co-transfection of Akt with wild-type PRPK and is abrogated by cell treatment with the Akt pathway inhibitor LY294002. Our data disclose an unanticipated mechanism by which PRPK can be activated and provide a functional link between this enigmatic kinase and the Akt signalling pathway.
The general conclusion from this work is that different survival kinases are connected and cooperate to the final purpose of ensuring a high degree of cell survival. It is therefore conceivable that even a small dis-regulation of one of them can in turn produce dramatic and pathological efforts. Thus, whenever therapeutic strategies are based on targeting one of these enzymes, this complex network of cross-talk should be always taken into account.
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Qin, Qizhi. "Evaluation of the therapeutic potential of Akt inhibition in a translational model of histiocytic sarcoma." Diss., Virginia Tech, 2018. http://hdl.handle.net/10919/97520.
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Histiocytic sarcoma (HS) is an exceptionally rare malignant neoplasm derived from dendritic cells and histiocytes, with no available effective treatment options. Akt signaling and proteasome dysfunction have been implicated in the pathogenesis of the disease, both in humans and dogs. Our work aims to investigate the importance of the Akt signaling pathway and evaluate the potential of Akt-targeted therapy in a canine model of histiocytic sarcoma.
We demonstrated Akt signaling to be active in 9 out of 10 canine HS tumor samples, regardless the presence of PTEN. Moreover, the Akt signaling pathway appears to be constitutively active in DH82 cells — a cell line model of canine HS, when compared to control canine dendritic cells. Pharmacologic Akt inhibition resulted in significant decrease in Akt S473 phosphorylation, GSK-3β S9 phosphorylation, Akt activity, cell viability, increased apoptosis, and resulted in sensitization to proteasome inhibition-depended cell death in a synergistic manner. Proteasome inhibition using carfilzomib, an irreversible proteasome inhibitor, induced dose-depended/caspase-3 independent cell death, at clinically relevant drug concentrations. The therapeutic effect of Akt inhibition was validated in vivo using a DH82 xenograft murine model. Akt inhibition lead to reduced tumor growth, prolonged overall survival, and ameliorated splenomegaly, but not affected the lung metastasis. Moreover, the therapeutic effect of Akt inhibition was potentiated in combination with carfilzomib.
In conclusion, targeting Akt signaling may represent an attractive potential therapeutic target for the HS. Future studies are required to examine the clinical efficacy of Akt-targeted therapy in dogs with HS using novel selective Akt inhibitors.Ph. D.
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Zhou, Xiangyu. "The contribution of single Akt isoforms to neuronal survival: characterization of a new mechanim of Akt activation in the PDK1 K465E mice." Doctoral thesis, Universitat Autònoma de Barcelona, 2014. http://hdl.handle.net/10803/285558.
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La vía de la fosfoinositol 3 quinasa (PI3K) desempeña un papel crucial en el control de la supervivencia y diferenciación neuronales. PDK1 es una quinasa master esencial que, en respuesta a la activación de PI3K, activa un elevado número que quinasas de la família Agc, incluidas las isoformas de PKB/Akt. La activación de Akt por PDK1 depende de la interacción de los dominios PH presentes en ambas quinasas con PtdIns(3,4,5)P3, el producto de la PI3K. La resolución de la estructura cristalográifca del dominio PH de PDK1 permitió el diseño de una mutación puntual dirigida, Lys 465 a Glu, que impide la interacción del dominio PH de PDK1 con PtdIns(3,4,5)P3. Los ratones PDK1K465E/K456E eran viables aunque pequeños y presentaban una modesta reducción de la activación de Akt comparados con los ratones control. Por el contrario, el resto de quinasas reguladas por PDK1, como RSK, S6K o SGK de activaban con normalidad.
Nuestros estudios previos demostraron que la supervivencia neuronal no estaba afectada en el ratón PDK1K465E/K465E, a pesar de que la activación de Akt estaba claramente reducida. Incluso tras el tratamiento con el compuesto inhibidor de Akt1 y Akt2 Akti-1/2, las neuronas corticales eran sorprendentemente capaces de sobrevivir. Es por ello que se propusoq eu Akt3 podria tener un papel central en el control de la supervivencia neuronal. Los resultados de este estudio demuestran que la activación de PKB/Akt en el córtex estaba menos comprometida que en tejidos de respuesta a insulina en el ratón PDK1K465E/K465E. se analizó la actividad de las tres isoformas de Akt, y se demostró que la actividad de PKBγ/Akt3 estaba preservada en comparación con PKBα/Akt1 or PKBβ/Akt2 el ratón PDKK465E/K465E, y que la proporción de actividad Akt3 es más elevada en neuronas que en otros tipos celulares, en el ratón PDK1K465E/k465E. PKBβ/Akt2 representa tan solo una pequeña fracción de la actividad PKB/Akt total en neuronas corticales lo que no hace pensar que tenga un papel esencial en el control de la supervivencia neuronal. Además, experimentos de interferencia de RNA demuestran que la inhibición de Akt1 y Akt3 compromete la supervivencia neuronal en el ratón PDK1K465E/K465E, mientras que la inhibición de Akt2 no tiene consecuencias. Se propone así un modelo cuantitativo de regulación de la supervivencia neuronal por Akt por el que Akt1 y Akt3 juegan un papel más importante en supervivencia porque representan un elevado porcentaje de la actividad Akt total. Además, se demuestra en esta tesis un nuevo mecanismo de activación de PKB/Akt en el ratón PDK1K465E/K465E por el que en ausencia de unión a PtdIns(3,4,5)P3 PDK1 puede aún interaccionar directamente con Akt y activarla.The phosphoinositide 3-kinase (PI3K) signaling pathway plays a central role in promoting neuronal survival and differentiation. PDK1 is a crucial master kinase that, in response to PI 3-kinase activation, switches on a number of AGC-kinase family members including PKB/Akt. Activation of Akt by PDK1 relies on the interaction of the PH domains present on both kinases with PtdIns(3,4,5)P3, the PI 3-kinase product. Crystal structure high-resolution of the PDK1 PH-domain allowed the design of a specific point mutation, Lys 465 to Glu, impairing the interaction of the PH-domain with PtdIns(3,4,5)P3. The PDK1K465E/K456E mice were shown to be viable but smaller, with a modest reduction in Akt activity compared with the wild type mice. By contrast, other PDK1 target such as RSK, S6K or SGK isoforms remained unaffected. Our former studies have indicated that neuronal survival was not compromised in the PDK1K465E/K465E mice, although activation of Akt was obviously reduced. Even after treated with the Akti-1/2 inhibitor, cortical neurons were still surprisingly capable to survive. Regarding this, I proposed that Akt3 plays key role in regulating neuronal survival. The results of my study indicate that the PKB/Akt activation in cortex was less reduced than in insulin responsive tissues in the PDK1K465E/K465E mice. I further analyze the activity of different Akt isoforms, and I found that PKBγ/Akt3 activity was preserved compared with PKBα/Akt1 or PKBβ/Akt2 in the PDKK465E/K465E mice, and also that the proportion of Akt3 phosphorylation at the PDK1 site is much higher in neurons than in other tissues in the PDK1K465E/k465E mice. The PKBβ/Akt2 only accounted for a small fraction of the total PKB/Akt activity in cortical neurons, which implies unimportant role in regulating the neuronal survival. Furthermore, The data from Akt shRNA interference and Hoechst assays demonstrate that the percentage of apoptotic cells was increased by Akt1 and Akt3 shRNAs infection in cortical neurons In the PDK1K465E/K465E mice, whereas Akt2 down-regulation had no significant effects on neuronal apoptosis. Here I proposed a model that the neuronal survival was regulated by PKB/Akt isoforms in a quantitative manner. Because of the high proportion of Akt phosphorylation at Thr 8/9 sites, Akt1 and Akt3 might play more important roles than Akt2 in neuronal survival. Besides this, I also uncovered a novel mechanism in the activation of PKB/Akt in the PDK1K465E/K465E mice, In which PDK1 can take advantage of the docking site mechanism to activate Akt, in spite of the absence of PtdIns(3,4,5)P3 binding.
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Sears, Daniel. "Identification of PI3K/Akt targets in chronic myeloid leukaemia." Thesis, Imperial College London, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.505451.
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Prescott, Gerald Robert. "The role of the kinase, Akt/PKBin regulated exocytosis." Thesis, University of Liverpool, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.479062.
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Amin, Elyas Oliver Muhammad. "Investigating how enteropathogenic Escherichia coli (EPEC) subverts AKT signalling." Thesis, University of Newcastle upon Tyne, 2017. http://hdl.handle.net/10443/3653.
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The phosphoinositide 3-kinase (PI3K) signalling pathway is activated in macrophages in response to many bacterial pathogens, triggering phagocytic uptake mechanisms and phosphorylation-associated activation of the serine/threonine kinase AKT. Enteropathogenic E. coli (EPEC) inhibits both PI3K mediated phagocytosis and AKT phosphorylation; dependent on a type 3-secretion system (T3SS) critical for delivering up to 24 known effector proteins into target cells. The efficient translocation of most EPEC effectors is dependent on the T3SS effector chaperone CesT. Although the effectors and mechanisms for inhibiting phagocytosis are well described, little is known how EPEC inhibits AKT phosphorylation. AKT activation is a multi-step process involving its recruitment to the cell membrane and phosphorylation of Thr308 by PDK1 and Ser473 by mTORC2. This activation process is supressed by inositol (such as PTEN) and protein (such as PP1 & PP2A) phosphatases. Altered AKT signalling is associated with many cancers, diabetes, cardiovascular and infectious disease, thus identifying how EPEC inhibits AKT activity could provide insight into its complex regulatory process and/or new therapeutic strategies. Screening of bacterial strains, lacking or expressing subsets of EPEC effectors, by western blot analysis suggests that the inhibitory mechanism depends on the CesT chaperone but not the function of the 21 most studied effectors. The EPEC inhibitory mechanism was investigated through the development of a two-wave infection model, examining for T3SS dependent changes in AKT phosphorylation (Thr308 & Ser473), membrane localisation and activity of AKT associated signalling proteins (PDK1 & PTEN). This strategy revealed inhibition of AKT phosphorylation to be stable (up to 3 h) and linked to increased activity of serine/threonine protein phosphatase(s). This finding was supported by phosphatase inhibitor studies, suggesting the involvement of a host activated or bacterial delivered protein phosphatase. Thus, this study provides new insights into the requirement of the EPEC effector repertoire and suggests a novel mechanism by which EPEC inhibits AKT signalling.
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Nunes, Tinoco Nunes. "Estudo funcional da via PI3K/AKT em Aedes aegypti." Botucatu, 2018. http://hdl.handle.net/11449/157112.
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Orientador: Jayme Augusto de Souza NetoResumo: Aedes aegypti é a espécie de mosquito emergente em áreas urbanas com maior impacto na saúde pública, sendo o principal vetor dos arbovírus dengue, Zika e chikungunya. Por esse motivo, se faz indispensável a compreensão de mecanismos moleculares associados a processos fisiológicos em A. aegypti, como resposta imune, comportamento e homeostase intestinal. Conforme observado em outros organismos, a via PI3K/AKT tem papéis importantes no metabolismo, na reprodução, na tolerância ao estresse e na imunidade. A quinase AKT atua como um regulador negativo da via PI3K/AKT, fosforilando o fator de transcrição FOXO e impedindo sua translocação nuclear. Nosso objetivo foi avaliar o perfil transcricional em A. aegypti com o gene akt silenciado e avaliar as consequências deste silenciamento sobre a microbiota bacteriana. Além disso, investigamos uma provável ativação mitocondrial quando do silenciamento de akt. Mostramos que o silenciamento de akt resultou na ativação de genes essenciais para a manutenção da homeostase intestinal do mosquito, como peptídeos antimicrobianos (AMPs) e genes codificadores de enzimas associadas à produção de espécies reativas de oxigênio (ROS). Notavelmente, observou-se uma forte repressão de 11 profenoloxidases (PPOs), além de uma potente indução de genes codificadores de subunidades da enzima NADH desidrogenase, em comparação com o respectivo grupo controle, sugerindo que tal indução esteja associada a um provável aumento da atividade mitocondrial. No context... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Aedes aegypti is the emerging mosquito species in urban areas with the greatest impact on public health, being the main vector of arboviruses dengue, Zika and chikungunya. For this reason, it is essential to understand the molecular mechanisms associated with physiological processes in A. aegypti, such as immune response, behavior and intestinal homeostasis. As observed in other organisms, the PI3K / AKT pathway has important roles in metabolism, reproduction, stress tolerance and immunity. AKT kinase acts as a negative regulator of the PI3K / AKT pathway, phosphorylating the FOXO transcription factor and preventing its nuclear translocation. Our objective was to evaluate the transcriptional profile in A. aegypti with the silenced akt gene and to evaluate the consequences of this silencing on the bacterial microbiota. In addition, we investigated a possible mitochondrial activation during the akt silencing. We have shown that akt silencing resulted in the activation of essential genes for the maintenance of mosquito intestinal homeostasis, such as antimicrobial peptides (AMPs) and genes encoding enzymes associated with the production of reactive oxygen species (ROS). Notably, a strong repression of 11 profenoloxidases (PPOs) was observed, as well as a potent induction of genes encoding subunits of the NADH dehydrogenase enzyme, in comparison with the respective control group, suggesting that such induction is associated with a possible increase in mitochondrial activity. In t... (Complete abstract click electronic access below)
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Tseng, Ping-Hui. "PDK-1/AKT pathway as targets for chemosensitizing effects." The Ohio State University, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=osu1131466383.
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Ramamoorthy, Mahesh. "Crosstalk Between MDM2 and Akt Signaling Pathway in Oncogenesis." VCU Scholars Compass, 2008. http://scholarscompass.vcu.edu/etd/1636.
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MDM2, the human homologue of the Mouse Double Minute 2 gene product, has been shown to be over-expressed in many cancers and to induce tumorigenesis. The role of MDM2 in oncogenesis was thought to be p53 dependent. However recent years have shown MDM2 to be a key player in a complex network of interactions that affect cell cycle, apoptosis, and tumorigenesis in a p53 independent manner. Here we report a novel p53 independent role for the multidimensional protein MDM2; its ability to induce phosphorylation of Akt at serine 473 residue. Transient and stable over-expression of MDM2 in cultured cell lines induces Akt phosphorylation. Silencing of MDM2 expression in cancer cells that over express MDM2 inhibits Akt phosphorylation suggesting endogenous MDM2 participates in Akt phosphorylation. Stable up-regulation of MDM2 expression reduced sensitivity of cells to chemotherapeutic drugs such as Etoposide, Carboplatin or Paclitaxel. The domain of MDM2 responsible for drug resistance overlaps with the Akt phosphorylation domain. An inhibitor of Akt phosphorylation abrogated MDM2-mediated Akt phosphorylation and reduction of Etoposide sensitivity indicating that MDM2 reduces Etoposide sensitivity of cancer cells by activating the Akt phosphorylation. A PI-3 kinase inhibitor Wortmannin inhibits the ability of MDM2 to induce Akt phosphorylation and silencing of Rictor, a known kinase of Akt, does not hamper the ability of MDM2 to induce phosphorylation of Akt. MDM2-mediated Akt phosphorylation does not require p53, and the p53-interaction domain of MDM2 is dispensable for Akt phosphorylation. The presence of MDM2 enhances the Insulin like Growth Factor 1 mediated activation of Akt. Further cells harboring MDM2 show enhanced Interleukin 8( IL8) activation, which could be a possible mechanism of Akt activation. Downstream of Akt activation we show increased events that have been correlated with Akt activation like increased Bcl-2 levels increased processing of NF-κB2, and GSK3α/β phosphorylation among others. Our observation reveals a novel signaling function of MDM2 important for regulation of cell growth and chemotherapeutic sensitivity through Akt phosphorylation.
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Erbele-Küster, Dorothea. "Lesen als Akt des Betens : eine Rezeptionsästhetik der Psalmen /." Neukirchen-Vluyn : Neukirchener Verl, 2001. http://catalogue.bnf.fr/ark:/12148/cb38818991m.
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Houssaini, Amal. "La voie de signalisation Akt/mTOR : rôle physiopathologique etcible thérapeutique dans l’hypertension artérielle pulmonaire expérimentale." Thesis, Paris Est, 2012. http://www.theses.fr/2012PEST0069.
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Les travaux de la thèse portent sur l'implication de la voie de signalisation Akt (sérine/thréonine kinase Akt) et mTOR (mammalian target of rapamycin) dans la physiopathologie de l'hypertension artérielle pulmonaire (HTAP) expérimentale. L'HTAP résulte d'une prolifération exagérée des cellules constitutives des vaisseaux pulmonaires, principalement les cellules musculaires lisses artérielles pulmonaires (CML-AP). De nombreux effecteurs biologiques et physiques préalablement identifiés agissent sur les CML-AP et participent à l'hyperplasie de celles-ci. Nous montrons que ces nombreux effecteurs convergent vers une voie de signalisation intracellulaire commune, la voie Akt/mTOR, qui de fait représente une cible thérapeutique pour le traitement de l'HTAP, et pourrait conditionner l'hyperplasie des CML-AP. mTOR est présent dans la cellule sous forme de deux complexes, mTORC1 et mTORC2, qui phosphorylent des substrats variés contrôlant la prolifération cellulaire. Les effecteurs de mTORC1 incluent les S6 kinases (S6K1 and S6K2) et les "eIF4E-binding proteins" (4EBP) alors que mTORC2 active la sérine/thréonine kinase Akt et parmi les kinases sous-jacentes, la kinase GSK3. La première étude est consacrée à l'évaluation des effets des inhibiteurs de protéases du VIH (ritonavir, amprenavir, nelfinavir) sur la progression de HTAP expérimentale, induite par la monocrotaline ou l'hypoxie. Nous montrons que ces deux formes d'HTAP sont associées à une activation de la voie Akt/mTOR dans les artères pulmonaires. Les traitements respectifs par les trois inhibiteurs des protéases du VIH durant 3 semaines induisent une réversibilité de l'HTAP, de l'hypertrophie ventriculaire droite et du remodelage des vaisseaux pulmonaires, de même qu'une inhibition de la phosphorylation d'Akt, de S6K et de GSK3. La prolifération des CML-AP induite par le PDGF ou le SVF 5%, associée à une augmentation de p-Akt et p-GSK3, est également bloquée par les inhibiteurs des protéases, de façon similaire et non additive à celle d'inhibiteurs spécifiques de la PI3 kinase et de GSK3. La conclusion est que ces traitements antirétroviraux inihibent la progression de l'HTAP en inhibant la voir Akt/mTOR dans les CML-AP. Cette proposition permettrait d'expliquer l'effet suspecté en clinique des traitements antirétroviraux sur l'HTAP compliquant l'infection par le VIH.Dans la seconde étude, nous montrons que les CML-AP en culture prélevées à partir de rats ayant développé une HTAP induite par la monocrotaline proliférent de façon exagérée en comparaison avec les cellules de rats témoins. Ce phénotype prolifératif est observé en présence de nombreux facteurs mitogènes parmi lesquels le SVF 5%, le PDGF, la sérotonine ou 5-HT, l'IGF1 ou l'IL1-beta, et est associé à une activation des substrats de mTORC1 et mTORC2. Le traitement in vitro par la rapamycine des CML-AP de rats avec HTAP établie permet d'inhiber la prolifération de ces cellules et de bloquer à la fois mTORC1 et mTORC2. De même, le traitement par la rapamycine de rats porteurs de l'HTAP préétablie pendant une semaine permet de normaliser la prolifération des CML-AP in vitro et in vivo et d'inhiber mTORC1 et mTORC2, effets non observés par l'Imatinib ou la Fluoxetine. De plus, le traitement des rats par la rapamycine prévient ou corrige l'HTAP induite par la monocrotaline de façon plus importante que l'Imatinib ou la Fluoxétine.Ces résultats indiquent donc que l'activation de la voie Akt/mTOR, très étroitement associée au développement de l'HTAP expérimentale, pourrait expliquer le phénotype prolifératif anormal des CML-AP inhérent à la pathologie, et ainsi représenter une cible thérapeutique de choix pour le traitement de l'HTAP humaineThe major objectives of research described in this thesis is focused on the cell signaling pathway of Akt (serine/threonine kinase Akt) and mTOR (mammalian target of rapamycin) in the patho-physiology of experimental pulmonary arterial hypertension (PAH). PAH occurs as a result ofhyperplasia of the components of pulmonary vessels, principally the pulmonary arterial smooth muscle cells (PA-SMCs). Numerous previously identified biological and physical effectors act on the PA-SMCs and participate in PA-SMC hyperplasia. Here we show studied that these different effectors converge into a common intracellular signaling pathway, Akt/mTOR signaling pathway, which represents actually a therapeutic target for PAH treatment, and could be involved in the hyperplasia of PA-SMCs. In cells mTOR, is presented in the form of two complexes, mTORC1 and mTORC2, which phosphorylate various substrates controlling the cellular proliferation. The effectors of mTORC1 include the S6 kinases (S6K1 and S6K2) and eIF4E-binding proteins (4EBP), meanwhile mTORC2 activates the serine/threonine kinase Akt and the underlying kinases, e.g. GSK3 kinase.The first study is devoted to evaluate the effects of the protease inhibitors of HIV (ritonavir, amprenavir, nelfinavir) on experimental PAH development induced by monocrotaline or hypoxia. We studied that the two forms of PAH are associated with an activation of Akt/mTOR signaling pathway in pulmonary arteries. The treatment by the three protease inhibitors of HIV during 3 weeks causes reversibility in experimental PAH with decreased right ventricular hypertrophy and pulmonary vascular remodeling as well as inhibition of phosphorylation of Akt, S6K and GSK3. The proliferation of PA-SMCs stimulated by PDGF or FCS 5%, which is associated with an increased p-Akt and p-GSK3, is also blocked by the proteases inhibitors, in a similar and non additive way like the specific inhibitors of PI3 kinase and GSK3. We conclude that the antiretroviral treatments significantly inhibits PAH development by inhibiting Akt/mTOR signaling pathway in PA-SMCs. This proposition allows explaining the effect of antiretroviral treatments of PAH accompanied with HIV in patients.In the second study, we studied that the cultured PA-SMCs extracted from the rats with monocrotaline induced-PAH(MCT-PAH) proliferates faster as compared to control. This proliferative phenotype is observed in the presence of different mitogenic factors including FCS 5%, PDGF, 5-HT, IGF1 or IL-1β, and is associated with an activation of the substrates of mTORC1 and mTORC2. Treatment with rapamycin in the PA-SMCs extracted from the rats with PAH in vitro inhibits the proliferation and also blocks the activation of mTORC1 and mTORC2. The treatment by rapamycin in the rats with PAH during one week allows normalizing the proliferation of PA-SMCs in vitro and inhibiting the activation of mTORC1 and mTORC2 in vivo. These effects were not observed when treated with imatinib or fluoxetine. Moreover, treatment with rapamycin prevents or reverse MCT induced PAH more significantly than that by imatinib or fluoxetine.These results indicate that the activation of Akt/mTOR signaling pathway isclosely related to experimental PAH development, which can explain the abnormal proliferative phenotype of PA-SMCs involved in the patho-physiology of PAH, and represent a therapeutic target for the treatment of PAH in human
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Schwarz, Angela. "Die Vaterstädtische Stiftung in Hamburg in den Jahren von 1849 bis 1945 : "... einen Akt der Gerechtigkeit durch einen Akt der Wohlthätigkeit zu verewigen..."." Hamburg Kovač, 2007. http://www.verlagdrkovac.de/978-3-8300-2797-3.htm.
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Schwarz, Angela. "Die Vaterstädtische Stiftung in Hamburg in den Jahren von 1849 bis 1945 "... einen Akt der Gerechtigkeit durch einen Akt der Wohlthätigkeit zu verewigen ..."." Hamburg Kovač, 2003. http://www.verlagdrkovac.de/978-3-8300-2797-3.htm.
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Higa, Thaís Tiemi. "Imunolocalização de supressores (FOXO3a e PTEN) e ativadores (Akt e phospho-Akt) da transição de folículos primordiais e primários em tecido ovariano humano." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/17/17145/tde-26042018-152648/.
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Mulheres com risco de falência ovariana prematura, assim como aquelas diagnosticadas com câncer que desejam preservar sua fertilidade, têm como opção a criopreservação do tecido ovariano. Esse tecido seria destinado, dependendo do caso, ao reimplante posterior ou para o cultivo in vitro de folículos ovarianos isolados do tecido criopreservado. Nesse contexto, os folículos primordiais são uma população importante tanto por serem mais resistentes ao processo de criopreservação, como por representarem cerca de 90% da população total folicular. Porém o uso destes folículos para procedimentos de Reprodução Assistida ainda é bastante limitado, pois os mecanismos responsáveis pelo seu processo de ativação ainda não são completamente conhecidos. A via de sinalização fosfatidilinositol 3- quinase (PI3K) foi recentemente identificada como determinante para o controle da ativação dos folículos primordiais. Portanto o objetivo deste estudo foi identificar e localizar os fatores componentes da via: supressores (FOXO3a e PTEN) e ativadores (Akt e Phospho-Akt). O que ofereceria uma valiosa ferramenta para elucidar os mecanismos envolvidos na ativação do pool de reserva folicular e permitiria o desenvolvimento de sistemas de cultivo folicular que atuassem diretamente nestes mecanismos. Sendo assim, foi realizado um estudo transversal com amostras de tecido ovariano humano, que foram submetidas à reação imunohistoquímica dos fatores previamente citados. Foram incluídas na casuística 40 pacientes, com idade média de 27,7 anos ± 7,26. Foi realizada uma análise comparativa da expressão dessas proteínas entre os folículos primordiais e primários. Foi encontrada diferença significativa para a proteína Akt, (p<0,05) em que os folículos primordiais (oócito e células da granulosa) manifestaram mais expressão da proteína Akt em comparação aos folículos primários. Também foi encontrada diferença significativa para a proteína phospho-Akt, porém apenas nas células da granulosa, em que houve maior expressão em folículos primordiais comparados aos primários. Enquanto ambos os estágios tiveram marcação negativa para o PTEN e FOXO3a na maioria dos folículos analisados. Sendo assim, neste estudo não foi possível identificar dentre as proteínas escolhidas uma que tivesse expressão claramente característica de uma ou de outra fase folicular, não sendo possível inferir que a atividade de qualquer uma das proteínas fosse estritamente ligada à ativação dos folículo primordiais.Women at risk of premature ovarian failure, as well as those diagnosed with cancer who wish to preserve their fertility, have, as option, the ovarian tissue cryopreservation. This tissue would be destined, depending on the case, to posterior reimplantation, or for the in vitro culture of ovarian follicles isolated from the cryopreserved tissue. In this context, primordial follicles are an important population of cells. As they are more resistant to the cryopreservation process and they represent about 90% of the whole follicular population. However, the use of these follicles for Assisted Reproduction procedures is still quite limited, since the mechanisms responsible for its activation process are not fully understood. The phosphatidylinositol 3-kinase (PI3K) signaling pathway has recently been identified as determinant for the control of primordial follicle activation. Therefore, the aim of this study was to identify and localize the components of this pathway: suppressors (FOXO3a and PTEN) and activators (Akt and phospho-Akt). This would offer a valuable tool to elucidate the mechanisms involved in the activation of the follicular reserve pool and would allow the development of in vitro culture protocols that would act directly in these mechanisms. Thus, a cross-sectional study with samples of human ovarian tissue was performed. These samples were submitted to the immunohistochemical reaction of the previously mentioned factors. Forty patients were included in the study, with a mean age of 27.7 ± 7.26. A comparative analysis of the expression of these proteins was performed between primordial and primary follicles. A significant difference was found for the Akt protein (p<0.05) in which the primordial follicles (oocyte and granulosa cells) showed more Akt expression than primary follicles. Another significant difference was found for the phosphor-Akt protein, but only for the granulosa cells, where there was a greater expression in primordial follicles compared to the primary ones. While both stages were negatively stained for PTEN and FOXO3a in most of the follicles analyzed. Thus, in this study it was not possible to identify among the selected proteins one that had clearly characteristic expression of one or the other follicular phase, and it was not possible to infer that the activity of any of the proteins was strictly linked to the activation of the primordial follicles.
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Favaro, Patricia Maria Bergamo. "FMNL1 : caracterização de um novo gene humano relacionado com a familia das forminas." [s.n.], 2006. http://repositorio.unicamp.br/jspui/handle/REPOSIP/311973.
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Orientador: Sara Teresinha Ollala SaadTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: Forminas são proteínas relacionadas com o controle de morfogênese, diferenciação embrionária, citocinese, polaridade e sobrevivência celular. São conservadas entre várias espécies, incluindo fungos, plantas, moscas de fruta, camundongos e humanos, apresentando múltiplos domínios. Com o objetivo de descrever novas proteínas, através das informações geradas pelo projeto Genoma do Câncer Humano, uma parceria entre a Fapesp e o Instituto Ludwig, duas EST humanas (expression sequence tags) foram identificadas com homologia aos genes da família das forminas. O objetivo do presente estudo foi obter a seqüência completa do RNAm deste novo gene e a estrutura primária da proteína para estudos de localização, interações com outras proteínas e estudos funcionais da mesma. Para obter a seqüência completa do RNAm, foram realizadas buscas no banco de dados genômico do NCBI (National Center for Biotechnology lnformation) e usadas técnicas de biologia molecular. Assim, após a obtenção da seqüência completa do novo RNAm foi possível o depósito do mesmo no banco de dados do portal NCBI, com o nome de Formina Leucocitária Humana (número de acesso AY278319). Posteriormente, baseando-se na nomenclatura de novos genes e proteínas, aprovada pelo Gene Nomenclature Committee RUGO, o novo gene foi renomeado formin-like 1, doravante FMNL 1. A produção de um anticorpo policlonal específico para a proteína codificada pelo FMNL1, permitiu o estudo de sua expressão em diversos tecidos humanos. Através da técnica de Western Blotting, foi observada expressão preferencial em células neoplásicas, especialmente linfóides. Dentre os tecidos normais, foi detectada expressão apenas em células mononucleares de sangue periférico, em linfonodo e preferencialmente em células CD19- quando comparadas com células CD19+ de tonsilas normais. Dentre as linhagens celulares neoplásicas, a expressão foi maior em MOLT-4 e Jurkat, quando comparadas com outras linhagens mielóides, indicando expressão preferencial da proteína em neoplasias linfóides. Além disso, a alta expressão da proteína foi observada em células mononucleares de sangue periférico de 20 pacientes com. LLC (leucemia linfóide crônica), quando comparadas com células mononucleares de sangue periférico de indivíduos normais. Quando a expressão da proteína FMNL1 foi estudada em diferentes tipos histológicos de Linfomas não-Hodgkin (LNH) e em linfonodos reativos, novamente foi observada a expressão preferencial no tecido neoplásico em relação ao tecido normal, sendo os LNH- T aqueles com expressão mais significativa. Esta nova proteína FMNL1 localiza-se no citoplasma, conforme verificado pela microscopia confocal e confirmado através da técnica de separação de frações. Das interações desta proteína, foi detectada a associação de FMNL 1 com AKT, sendo que essa associação é independente da ativação de AKT. Também foi observado que a ativação da via das caspases em duas linhagens celulares neoplásicas levava à diminuição da expressão da proteína FMNL 1. Em síntese, a expressão preferencial da proteína FMNL1 em tecidos neoplásicos línfóides e a sua associação com AKT, sugere a sua participação na sobrevivência dessas células e, possivelmente, na fisiopatologia das neoplasias hematológicas
Abstract: Formins are proteins that are involved in processes such as morphogenesis, embryonic differentiation, cytokinesis, cell polarity and survival. The formins are multidomain proteins that are conserved from plants to fungi and vertebrates. A search of the ORESTES database, generated from the Human Cancer Genome Project, identified 2 ESTs that were similar to the Formin-related proteins. The aim of this study was to obtain the full-Iength sequence ofthis new gene, the primary structure ofthe protein coded by this gene, its subcellular localization and to characterize its possible interaction with other proteins in addition to functional studies. In an attempt to obtain the full-Iength sequence of this rnRNA, searches in the human genome database at NCBI (National Center for Biotechnology Information) and molecular biology techniques were performed. The whole coding sequence ofthis gene was named Human Leukocyte Formin and was deposited at the human genome database at NCBI (GenBank Accession No. AY278319). Later, based on the HUGO Gene Nomenc1ature Committee, the new gene was renamed formin-like 1 and the symbol FMNLl will herein be used. The production of a polyc1onal antibody specific for the protein FMNLl allowed the study of the protein's expression in different human tissues. Using the Westem Blotting technique, a preferential expression of this protein in lymphoid malignancies was observed. Among normal tissues, a low expression in lymph node, peripheral blood mononuClear cells and in CD19- cells (when compared with CD19+ cells from normal tonsils) was observed. Among the malignant celllines, a high expression of the protein was detected in MOLT-4 and the Jurkat cell line, when compared with myeloid cancer cell lines, suggesting a preferential expression of FMNLl in lymphoid malignancies. Moreover, a high expression of the protein in peripheral blood mononuc1ear cells from 20 chronic lymphocytic leukemia patients was observed when compared with peripheral blood mononuc1ear cells of normal donors. When the expression of FMNLl protein was compared among different histological types of non-Hodgkin's lymphoma (NHL) and reactive lymph nodes, again, a preferential expression of the protein in the malignant tissue was detected and the T cell NHL presented the highest expression. This new protein is located in the cytoplasm of the cells, as observed by confocal microscopy and confirmed by separation of the cellular ftaction. With regard to the interactions ofthis new protein, an association ofFMNLI with AKT was detected, and this association was not dependent on AKT activation. A decrease in FMNLI expression in 2 cancer celllines after caspase activation was also detected. ln conclusion, the preferential expression of FMNLI in lymphoid malignancies and its association with AKT, suggests the role ofFMNLI in cell survival and a possible role in the pathophysiology ofhematological malignancies
Doutorado
Biologia Estrutural, Celular, Molecular e do Desenvolvimento
Doutor em Fisiopatologia Medica
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Lammert, Angela [Verfasser]. "Charakterisierung der molekularen Mechanismen der K-Ras/Akt-regulierten Motilität sowie der Funktion von Akt-Effektoren in Karzinomzelllinien mit onkogenem K-Ras / Angela Lammert." Gießen : Universitätsbibliothek, 2019. http://d-nb.info/1176965751/34.
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Na, Shin-Young. "PKB-Akt a critical regulator of lymphocyte development and function /." Doctoral thesis, [S.l.] : [s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=976116464.
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Ferber, Ines. "Radio-Chemoresistenz beim kleinzelligen Bronchialkarzinom möglicher Einfluss AKT-abhängiger Signaltransduktionswege /." [S.l.] : [s.n.], 2004. http://archiv.ub.uni-marburg.de/diss/z2004/0393/.
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Yan, Yi. "The role of Akt in AMPA receptor insertion and LTP." Thesis, University of British Columbia, 2007. http://hdl.handle.net/2429/31741.
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It has been widely accepted that long-term potentiation (LTP) in the hippocampal CA1
region mostly results from increased insertion of post-synaptic α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptors (AMPARs). The previous
study in our lab has shown that activation of phosphatidylinositol 3-kinase (PI3K) by
selective stimulation of synaptic N-methyl-D-aspartate receptors (NMDARs) is required
for the increased cell surface expression of AMPARs and the consequent LTP. However,
the following signaling pathways still remain unknown. In the present study, the
involvement of Akt, the primary downstream protein kinase of PI3K, was examined with
a combination of electrophysiological, biochemical and molecular biological techniques.
The study found that Akt is required for the post-synaptic AMPAR insertion and LTP.
Furthermore, the threonine 840 (Thr840) on GluRl C-tail was identified as a novel Akt
phosphorylation site, suggesting a potential mechanism by which Akt contributes to
AMPAR incorporation and LTP.Medicine, Faculty of
Graduate
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Lyons, Traci Renae. "Analysis of potential substrates for the pro-survival kinase AKT /." Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2006.
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Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2006.Typescript. Includes bibliographical references (leaves 194-209). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;
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Boudreau, Mathieu. "Substrates and biochemical mechanisms by which Akt promotes cellular survival." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=85051.
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Apoptosis is a phenomenon conserved from flies to humans, crucial in development and in the maintenance of cellular homeostasis. Throughout the years various molecules have been identified as critical regulators of apoptotic signaling. Among these, the serine/threonine kinase Akt has been demonstrated to play a pivotal role in the regulation of cell survival by phosphorylating a broad range of effectors. This thesis looks at the regulation of anti and pro-apototic Akt substrates, and examines the survival-promoting activity of Akt in neural tumor cells and neurons.In the first section of this thesis I demonstrated, using a recombinant adenovirus-based approach, that Akt is necessary for the NGF-dependent survival of differentiated neuronal-like PC12 (pheochromocytoma) cells and undifferentiated PC12 cells. Furthermore, I suggested that Akt possibly performs this function via the repression of the c-jun N-terminal kinase (JNK) pathway, upstream of JNK, as overexpression of kinase-inactive Akt activates JNK.
In the second section of this thesis, I identified mixed-lineage kinase-3 (MLK-3) as a substrate of Akt. I showed that Akt associates with the JIP/MLK-3/JNK scaffold complex, and that it inactivates MLK-3 by phosphorylating T477. This study strongly suggests that in certain cellular systems, Akt represses the JNK signaling pathway and apotosis via the inhibition of MLK-3.
Finally, in the third section, we discovered that Akt phosphorylates and interacts with the caspase inhibitor, X-linked inhibitor of apoptosis (XIAP). The phosphorylation of XIAP occurs on serine 87. We also showed that PI3-K/Akt synergize with XIAP to promote sympathetic neuron survival.
These studies contribute to the understanding of Akt's anti-apoptotic mechanisms and might also help design highly specific therapeutic approaches.
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Dudley, Alix. "DRR regulates the activation of AKT kinase in brain cancer." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=110452.
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Brain cancer invasion is a leading cause of treatment failure yet there are no therapies to prevent invasion. In developing therapeutic strategies, we must first identify the molecular mechanisms that regulate this event. We previously identified downregulated in renal cell carcinoma (DRR) as a promoter of invasion that augments focal adhesion turnover in malignant glial cells. We continued to investigate the molecular mechanisms that underlie this invasion by examining cell signaling in the context of DRR. We found that AKT phosphorylation (Ser473, Thr308) is elevated in DRR-over-expressing cells compared to control. Through a series of molecular and pharmacologic techniques, we selectively targeted AKT inputs in order to identify how DRR regulates its activation. We demonstrated that phospho-AKT is recruited to focal adhesions and is activated in an adhesion, SRC-family kinase (SFK), phosphoinositide-3-kinase (PI3K)-dependent and epidermal growth factor receptor (EGFR)-independent manner. We concluded our work by testing the relevance of our model in a 3D invasion assay. Our results demonstrated that SFK but not EGFR inhibition significantly prevents DRR-induced invasion. Together, these findings support a model by which DRR regulates AKT activity to drive malignant glial cell invasion.L'invasion des cellules cancéreuses est la principale cause d'échec des traitements des gliomes car il n'existe actuellement aucune thérapie permettant de bloquer ce processus. Afin de développer des stratégies thérapeutiques efficaces, il apparaît donc essentiel d'identifier les mécanismes moléculaires régulant la migration de ces cellules. Nous avons précédemment montré que la protéine 'downregulated in renal cell carcinoma' (DRR) contribue à l'invasion des cellules gliales malignes en augmentant le renouvellement de leurs complexes d'adhésion focaux. Nous avons poursuivi cette étude par l'analyse des voies de signalisation impliquées dans ce processus et nous avons tout d'abord mis en évidence une augmentation de la phosphorylation d'AKT (Ser473, Thr308) dans les cellules surexprimant DRR. Par une combinaison d'approches moléculaires et pharmacologiques, nous avons alors étudié spécifiquement le rôle de DRR dans l'activation d'AKT et avons démontré que la forme phosphorylée d'AKT est localisée au sein des complexes d'adhésion focaux. Nous avons également mis en évidence que son activation est régulée par SRC, membre de la famille des protéines tyrosine kinase (PTK), et par phosphatidylinositol-3-kinase (PI3K), indépendamment du récepteur à l'EGF. Enfin, nous avons validé notre modèle dans un système d'invasion en trois dimensions ou nous avons montré que l'inhibition spécifique de SRC bloque significativement l'invasion des cellules induite par DRR.L'ensemble de ces résultats nous permet finalement de proposer un modèle selon lequel l'invasion des cellules malignes gliales est régulée par l'activation de la protéine AKT par DRR.
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Sampson, Oliver J. "The role of AKT in tumour cell survival post-irradiation." Thesis, University of Oxford, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.542968.
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Middel, Ines Kristin [Verfasser]. "Quantitative Untersuchung der Proteinkinase AKT am Ovarialkarzinom / Ines Kristin Middel." Köln : Deutsche Zentralbibliothek für Medizin, 2010. http://d-nb.info/1000727416/34.
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Heupel, Christian [Verfasser], and Guido [Akademischer Betreuer] Sauter. "AKT Amplifikationen im humanen Lungenkarzinom / Christian Heupel. Betreuer: Guido Sauter." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2013. http://d-nb.info/1045024082/34.
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Piscitello, Desiree. "Activated Akt pathway promotes genome instability through suppression of Mre11." Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/6507/.
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Activating mutations in RAS are often found in different human cancers. The expression of an oncogene is called a driver mutation because it provides the bases for tumour initiation, but it still requires additional mutations to achieve tumour progression. 90% of invasive pancreatic ductal adenocarcinoma (PDAC) present activating KRAS mutation, in conjunction with inactivation of various tumour suppressor genes such as BRCA1, TP53, SMAD4 and CDKN2A. The most important effectors of RAS are PI3K and its downstream kinases. These function as mediators of RAS-induced cell survival and proliferation. Interestingly, concurrent mutations of RAS and PI3K/PTEN/Akt pathway have been described in the same human tumour types. Endometrial cancer, thyroid cancer and acute lymphoblastic leukemia have all been shown to harbor the simultaneous mutation of RAS gene and those encoding various members of the PI3K signalling pathway. Published data suggest that 25% of human colon cancers contain mutations in both K‐RAS and PI3K-associated genes. Moreover, 60% of human PDAC show PTEN loss, due to deletions, mutations or epigenetic silencing. Despite this prevalence, the molecular mechanism for the cooperation between RAS and PI3K pathway in tumourigenesis is poorly understood. A fundamental barrier for tumourigenesis is senescence. The activation of an oncogene such as RAS in a primary cell line drives cells into unscheduled DNA synthesis, resulting in a high frequency of stalled replication forks and DNA double strand breaks (DSBs). A DSB is one of the most deleterious lesions if unrepaired, and it is the primary trigger of oncogene-induced senescence (OIS). DSBs activate the ATM/ATR signalling pathway and senescence-associated cell cycle arrest. However, various oncogenes differ in their ability to induce senescence, for example activated Akt is a weak inducer of senescence compared to RAS. Previous work from our lab and others has suggested that the co-activation of these two oncogenes may serve to bypass certain aspects of the senescence program, but the precise mechanism by which this is achieved remains unclear. Surprisingly, detailed cell cycle analyses in this study demonstrate that the simultaneous activation of Akt in primary fibroblasts expressing oncogenic RAS reinforces RAS-induced senescence. This correlates with an increased accumulation of unrepaired damage, which is known to directly contribute to establishment of senescence. Interestingly, the expression of activated Akt in these cells correlates with reduced expression of MRN complex components, which in presence of RAS-induced damage impairs the activation of the checkpoint kinases. The inhibition of Mre11, the nuclease component of the MRN complex, in RAS expressing cells recapitulates the phenotype of RAS/Akt cells. Thus, Akt downregulates Mre11 to exacerbate RAS-induced DNA damage and induce a qualitatively stronger senescence. Multiple studies have previously reported the negative regulation of DDR by Akt, however a mechanism for this has not been described. Experiments on two colon cancer lines, HCT116 and DLD1, have revealed that inactivation of PTEN/activation of Akt suppresses DDR via a reduction in MRN complex expression and activity. In these cells, the components of the MRN complex display low protein stability and are rapidly degraded by an unknown mechanism. MRN complex is central in the DNA damage response to DSBs. Its suppression impairs the activation of the two checkpoint kinases Chk1 and Chk2, which mediates the G2/M arrest, and also impairs HR repair. The inhibition of these two events severely affects cell survival in presence of DSBs, and the surviving fraction present high levels of genome instability. The use of specific inhibitors targeting S6K1 activity rescues the levels of MRN complex in these cells, suggesting a role of this kinase in DDR suppression. Thus, the enhanced RAS-induced senescence in cells caused by Akt can be ascribed to the high levels of unrepaired damage due to the suppression of MRN complex. Despite compounding senescence, the simultaneous mutation of RAS and Akt allow the cells to acquire genome instability, which in vivo significantly contributes to bypassing senescence and promotes tumour progression.
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Rato, Leila Sofia Coelho. "Vimentin interacts with the Akt/mTOR pathway mediating cell growth." Master's thesis, Universidade de Aveiro, 2017. http://hdl.handle.net/10773/22372.
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Mestrado em Bioquímica - Bioquímica ClínicaA vimentina é uma proteína da classe III dos filamentos intermédios que promove processos tais como proliferação, migração e invasão celular através da interação com diferentes vias de sinalização. No entanto, o papel da vimentina no crescimento celular é ainda pouco conhecido. Neste estudo, observamos que fibroblastos isolados de embriões de ratinhos sem vimentina (Vim -/- MEFs) eram mais pequenos que o tipo normal (WT). Assim, o objetivo deste estudo era entender de que forma a vimentina regula o crescimento celular. Com recurso a modelos in vitro, técnicas de microscopia e técnicas bioquímicas descobrimos que Vim -/- MEFs tinham menor volume e concentração de proteínas quando comparadas com WT MEFs. Adicionalmente, a síntese proteica e ativação de mTORC1 estavam significativamente reduzidas em Vim -/- MEFs. Através de co-imunoprecipitação, descobrimos que a vimentina interage com os complexos mTORC2 e TSC. Assim, postulamos que a vimentina regula o crescimento celular por interação com proteínas da via de sinalização AKT/mTO
Vimentin is a type III intermediate filament protein that takes part in cell proliferation, migration and invasion, by acting as a signalling scaffold. The role of vimentin in cell growth, however, is poorly understood. We observed that vimentin knockout mouse embryonic fibroblasts (Vim -/- MEFs) were smaller than the wild type (WT). Therefore, this work aimed to understanding how vimentin regulates cell growth. Using in vitro models, imaging techniques and biochemical approaches, we have found that the volume and protein concentration of Vim -/- MEFs is lower when compared to WT MEFs. Further, protein synthesis and mammalian target of rapamycin complex 1 (mTORC1) activation was attenuated in Vim -/- MEFs. By co-immunoprecipitation we found that vimentin interacts with mammalian target of rapamycin complex 2 (mTORC2) and tuberous sclerosis protein complex (TSC) after insulin stimulation. Consequently, we postulate that vimentin regulates cell growth by interacting with proteins of the AKT/mTOR pathway
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Tavelis, Christodoulos. "Investigating the potential role of PIP4Ks in PI3K/Akt signalling." Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/investigating-the-potential-role-of-pip4ks-in-pi3kakt-signalling(e70d9473-5932-468a-bdad-01668a68db58).html.
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Class I phosphoinositide 3-kinases (PI3Ks) generate the essential lipid second messenger PtdIns(3,4,5)P3 which plays a key role in the regulation of numerous cellular processes including cell growth and survival, gene transcription, cytoskeletal organisation and glucose metabolism through its downstream effectors and in particular the serine/threonine protein kinase Akt. Therefore, the PI3K/Akt signalling plays a critical regulatory role in diverse cellular processes and its dysregulation is implicated in the pathogenesis of many human diseases including cancer and type 2 diabetes. Overexpression of phosphatidylinositol-5-phosphate 4-kinase β (PIP4Kβ), a lipid kinase which phosphorylates the poorly understood phosphoinositide phospholipid PtdIns5P to produce PtdIns(4,5)P2, has been demonstrated to lead to the attenuation of PtdIns(3,4,5)P3 levels and decreased Akt phosphorylation in insulin-stimulated cells. Conversely, mice lacking PIP4Kβ exhibit increased insulin-induced Akt phosphorylation, suggesting a potential role of PIP4Ks in the regulation of PI3K/Akt signalling. The aim of this project was to investigate the potential role of PIP4Kα, the most active isoform among PIP4Ks, in the regulation of PI3K/Akt signalling and whether its substrate, PtdIns5P, is involved in this regulation.While YM201636, an inhibitor of PIKfyve an enzyme believed to be involved in PtdIns5P production, markedly reduced Akt phosphorylation in PDGF-stimulated NIH/3T3 cells, contrary to expectations, overexpression of PIP4Kα in NIH/3T3 and HeLa S3 cells had no effect on PtdIns(3,4,5)P3 levels or Akt phosphorylation upon stimulation with PDGF and IGF-1 respectively. However, PtdIns(3,4,5)P3 levels were significantly decreased in insulin-stimulated L6 myotubes overexpressing PIP4Kα, indicating a possible cell type specific modulation of PI3K signalling by PIP4Kα. Interestingly, despite the decreased PtdIns(3,4,5)P3 levels, insulin-stimulated Akt phosphorylation remained unaffected in PIP4Kα expressing L6 myotubes. PIP4Kα overexpression also resulted in increased levels of PtdIns(3,4)P2. This suggests an increased 5-dephosphorylation of PtdIns(3,4,5)P3 through the action of one or more 5-phosphatases. Although the precise 5-phosphatase(s) are not known, the data indicate that this cannot be SHIP2 which has previously been implicated in the regulation of PtdIns(3,4,5)P3 levels in insulin signalling. Taken together, the data presented in this thesis indicate a role for PIP4Kα in PI3K signalling in a cell type specific manner. This might have important physiological implications.
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Gunn, Richard Martin. "Synthesis and evaluation of novel PI3-K-Akt-mTOR modulators." Thesis, Imperial College London, 2010. http://hdl.handle.net/10044/1/9015.
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The phosphoinositide 3-kinase / Akt / mammalian target of rapamycin (PI3-K-AktmTOR) signalling pathway is a regulator of critical cellular functions including apoptosis, metabolism and survival. Its deregulation is involved in numerous human diseases. This thesis describes the synthesis and biological evaluation of a series of analogues of the PI3-K-Akt-mTOR inhibitor E1, a homo-dimeric diarylmethane. Several structurally diverse hetero-dimeric E1 derivatives were discovered that inhibited PI3-K-Akt-mTOR signalling in human cancer cells. Chapter 1 provides an overview of the PI3-K-Akt-mTOR signalling pathway and its biological significance. Chapter 2 discusses the use of small molecules for the investigation of PI3-K-Akt-mTOR signalling, with examples given by structural class. It concludes with a profile of lead compound E1. Chapter 3 outlines the proposed approach to analogue synthesis by the coupling of functionalised building blocks, and describes the development of building block compounds via the orthofunctionalisation of phenol derivatives. Chapter 4 describes efforts towards the derivatisation of E1 with a linker group in order to allow the conjugation of biotin for affinity chromatography, or the incorporation of other groups useful for biological characterisation. In Chapter 5, the coupling of building blocks via C-C, C-O, C-N and N-S bond-forming reactions to generate homo- and hetero-dimeric E1 derivatives is discussed. Several of these compounds were capable of inducing cellular Akt inhibition. Chapter 6 focuses on the synthesis of hetero-dimeric analogues based on these new lead compounds. The biological evaluation of E1 derivatives in a cellular assay is described in Chapter 7. Finally, detailed experimental procedures are described in Chapter 8.
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Yan, Weisi. "Functional proteomic study of Akt reveals novel substrates in translation /." Access full-text from WCMC, 2008. http://proquest.umi.com/pqdweb?did=1619246521&sid=9&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Zhang, Yun. "Activation of Erk1/2 and Akt in astrocytes under ischemia /." View Abstract or Full-Text, 2002. http://library.ust.hk/cgi/db/thesis.pl?BIOL%202002%20ZHANGY.
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Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2002.Includes bibliographical references (leaves 98-115). Also available in electronic version. Access restricted to campus users.
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Mammana, Santa. "Evaluation of the PI3K/Akt/mTOR pathway in Multiple Sclerosis." Doctoral thesis, Università di Catania, 2017. http://hdl.handle.net/10761/3951.
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Background
The PI3K/AKT/mTOR pathway is an intracellular signaling pathway that regulates cell activation. proliferation, metabolism and apoptosis. Increasing body of data suggests that alterations in the PI3K/AKT/mTOR pathway may result in an enhanced susceptibility to autoimmunity. Aim of our work was to evaluate the involvement of the mTOR pathway in the pathogenesis of autoimmune diseases, with particular focus on Multiple Sclerosis (MS). Multiple sclerosis (MS) is one of the most common chronic inflammatory diseases of the central nervous system leading to demyelination and neurodegeneration. Drugs targeting the PI3K/AKT/mTOR pathway are currently under extensive investigation for their possible use as cancer chemotherapics and as immunosuppressive agents, but no clinical trial has been so far approved for the evaluation of their efficacy in the context of immunological disorders.
Methods
In the current study, we have firstly evaluated in silico the involvement of the mTOR network on the generation and progression of MS, making use of currently available whole-genome transcriptomic data. Also, the involvement of the mTOR network on oligondendrocyte function was studied, in order to ascertain whether treatment with drugs targeting the PI3K/Akt/mTOR pathway may be useful to promote the remyelination process, so to reverse disability in MS patients.
Then, the data generated in silico were subjected to an ex-vivo evaluation. To this aims the involvement of mTOR was validated on a well-known animal model of MS and in vitro on Th17 cells.
Results
Our data indicate that there is a significant involvement of the mTOR network in the ethiopathogenesis of MS and that Rapamycin treatment may represent a useful therapeutic approach in the clinical setting. Ex vivo analysis showed that treatment of MOG-specific T cells from EAE affected mice with the mTOR inhibitor, Rapamycin, and the dual PI3K/mTOR inhibitor, BEZ-235, was able to significantly reduce antigen-specific proliferation. In addition, we show that Rapamycin treatment of murine T cell stimulated under Th17 conditions, was able to significantly inhibit the expression of some of the genes previously identified in the in silico analysis. On the other hand, our data showed that a significant involvement of the mTOR network could be observed only in the early phases of oligodendrocyte maturation, but not in the maturation process of adult oligodendrocytes and in the process of remyelination following demyelinating injury.
Conclusions
Overall, our study suggest that targeting the PI3K/mTOR pathway, although it may not be a useful therapeutic approach to promote remyelination in MS patients, it can be exploited to exert immunomodulation, preventing/delaying relapses, and to treat MS patients in order to slow down the progression of disability.
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Zeybek, Ayça. "Essai du traitement pré-clinique du carcinome hépatocellulaire sur la cirrhose dans le modèle de rat." Thesis, Université Grenoble Alpes (ComUE), 2016. http://www.theses.fr/2016GREAV054/document.
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Hepatocellular carcinoma (HCC) is the second most common cause of cancerrelated mortality worldwide. AKT pathway has been found activated in 50% of HCC cases, making it promising target. Therefore we assess efficacy of the allosteric AKT inhibitor or the combination of Sorafenib with AKT inhibitor compared to untreated control and to standard treatment, Sorafenib, in vitro and in vivo. AKT inhibitor blocked phosphorylation of AKT in vitro and strongly inhibited cell growth with significantly higher potency than Sorafenib. Similarly, apoptosis and cell migration were strongly reduced by AKT inhibitor in vitro. To mimic human advanced HCC, we used diethylnitrosamine-induced cirrhotic rat model with fully developed HCC. MRI analyses showed that AKT inhibitor significantly reduced overall tumor size. Furthermore, number of tumors was decreased by AKT inhibitor, which was associated with increased apoptosis and decreased proliferation. Tumor contrast enhancement was significantly decreased in the AKT inhibitor group. Moreover, on tumor tissue sections, we observed a vascular normalization and a significant decrease in fibrosis in surrounding liver of animals treated with AKT inhibitor. Finally, pAKT/AKT levels in AKT inhibitor treated tumors were reduced, followed by down regulation of actors of AKT downstream signalling pathway: pmTOR, pPRAS40, pPLCγ1 and pS6K1. In conclusion, we demonstrated that AKT inhibitor blocks AKT phosphorylation in vitro and in vivo. In HCC-rat model, AKT inhibitor was well tolerated, showed anti-fibrotic effect and had stronger antitumor effect than Sorafenib. Our results confirm the importance of targeting AKT in HCCHepatocellular carcinoma (HCC) is the second most common cause of cancerrelated mortality worldwide. AKT pathway has been found activated in 50% of HCC cases, making it promising target. Therefore we assess efficacy of the allosteric AKT inhibitor or the combination of Sorafenib with AKT inhibitor compared to untreated control and to standard treatment, Sorafenib, in vitro and in vivo. AKT inhibitor blocked phosphorylation of AKT in vitro and strongly inhibited cell growth with significantly higher potency than Sorafenib. Similarly, apoptosis and cell migration were strongly reduced by AKT inhibitor in vitro. To mimic human advanced HCC, we used diethylnitrosamine-induced cirrhotic rat model with fully developed HCC. MRI analyses showed that AKT inhibitor significantly reduced overall tumor size. Furthermore, number of tumors was decreased by AKT inhibitor, which was associated with increased apoptosis and decreased proliferation. Tumor contrast enhancement was significantly decreased in the AKT inhibitor group. Moreover, on tumor tissue sections, we observed a vascular normalization and a significant decrease in fibrosis in surrounding liver of animals treated with AKT inhibitor. Finally, pAKT/AKT levels in AKT inhibitor treated tumors were reduced, followed by down regulation of actors of AKT downstream signalling pathway: pmTOR, pPRAS40, pPLCγ1 and pS6K1. In conclusion, we demonstrated that AKT inhibitor blocks AKT phosphorylation in vitro and in vivo. In HCC-rat model, AKT inhibitor was well tolerated, showed anti-fibrotic effect and had stronger antitumor effect than Sorafenib. Our results confirm the importance of targeting AKT in HCC
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Peluso, Antonio Augusto Bastos. "Efeito comparativo entre a angiotensina-(1-7) e o novo agonista do receptor MAS, CGEN 856S, nas vias AKT/eNOS e AKT/FOXO1 utilizando modelos celulares." Universidade Federal de Minas Gerais, 2014. http://hdl.handle.net/1843/BUBD-9NBKEF.
Full textAbstract:
Biotechnology studies based on a computational platform developed by Compugen, Tel Aviv, Israel - allowed the discovery of a potential Mas agonist, CGEN 856S, which produces several effects resembling those produced by Angiotensin-(1-7) [Ang-(1-7)], including anti-hypertensive and cardioprotective effects, such as decrease of ischemia, renal and cardiac fibrosis reduction and vasodilation nitric oxide (NO) dependent. Moreover, CGEN 8566S attenuates isoproterenol-induced cardiac remodeling and myocardial infarction injury. Studies using phosphoproteomics have shown that Ang-(1-7) promotes FOXO1 activation and nuclear translocation in human aortic endothelial cells (HAEC). It is not clear, however, whether CGEN 856S is capable to activate the same intracellular pathways than those previously demonstrated for Ang-(1-7). This study evaluated and compared the effect of CGEN 856S and Ang-(1-7) on AKT/eNOS and AKT/FOXO1 activation, using different cell types. Mas-transfected CHO (Chinese Hamster Ovary - CHO-Mas) cells were stimulated with CGEN 856S and Ang-(1-7). Non-transfected CHO cells were used as control. The protein extract was used for, AKT, phospho-AKT, phospho-eNOS, phospho-FOXO1, Mas and GAPDH detection by western blot. CHO-Mas and CHO cells were also exposed to both peptides for NO release measurement, through DAF-FM incorporation technique. HAEC, A549 and DU145 (human tumoral linages from lungs and prostate, respectively) cells were treated with CGEN 856S and Ang-(1-7) with or without previous A779 treatment. The cells were used for immunolocalization of FOXO1, analyzed by confocal microscope and the nuclear fluorescence intensity was quantified. A549 and DU145 cells were also used for cytotoxicity test, being treated with a combination of PI3 kinase inhibitors wortmannin or LY294002 in different concentrations and CGEN856S or Ang-(1-7). The results showed a significant increase phosphorylation of AKT, eNOS as well as the dephosphorylation of FOXO1 only in CHO-Mas cells treated with CGEN856S and Ang-(1-7). CHO-Mas transfect cells treated with CGEN 856S and Ang-(1-7) also promoted a significant release of NO compared with CHO-Mas non-treated cell. No differences were observed in CHO non-transfected cells. HAEC, A549 and DU145 cell types treated with CGEN 856S and Ang-(1-7) showed a significant FOXO1 nuclear translocation. A779 blocked this effect, at least partly. In both A549 and DU145 cells, the combination of the PI3 kinase inhibitors wortmannin and LY294002 with CGEN 856S or Ang-(1-7) showed a potentiated anti-proliferative effect. These data suggest that CGEN 856S and Ang-(1-7) as agonists of Mas receptor had similar effects, and thus represent a potential therapeutic target in cancer and cardiovascular pathologies.Estudos em biotecnologia baseados em uma plataforma computacional desenvolvida pela empresa Compugen, Tel Aviv Israel permitiram a descoberta de um potencial agonista do receptor Mas, o CGEN 856S, o qual produz vários efeitos semelhantes àqueles produzidos pela Ang-(1-7), incluindo efeitos anti-hipertensivos e cardioprotetores, como diminuição de isquemia, redução de fibrose renal e cardíaca e vasodilatação dependente de óxido nítrico (NO). Além disso, o CGEN 856S é capaz de atenuar o remodelamento cardíaco induzido por isoproterenol e lesão por infarto do miocárdio. Estudos utilizando fosfoproteômica mostraram que a Ang-(1-7) promove ativação e translocação nuclear do fator de transcrição FOXO1 em células de endotélio de aorta humana (HAEC). No entanto, ainda não é claro se o CGEN 856S é capaz de ativar as mesmas vias de sinalização intracelular anteriormente já demonstradas para Ang-(1-7). Este trabalho avaliou e comparou os efeitos do CGEN 856S e da Ang-(1-7) sobre a ativação das vias AKT/eNOS e AKT/FOXO1, utilizando diferentes modelos celulares. Células de ovário de hamster chinês (CHO) transfectadas com o receptor Mas (CHO-Mas) foram estimuladas com CGEN 856S e Ang-(1-7). Células CHO não transfectadas foram usadas como controle. O extrato proteico foi utilizado em Western Blot para detecção das proteínas AKT, AKT fosforilada, eNOS fosforilada, FOXO1 fosforilado, Mas e GAPDH. Células CHO-Mas e CHO foram expostas a ambos os peptídeos para medida da liberação de NO, através da técnica de incorporação de DAF-FM. Células HAEC, A549 e DU145 (as duas últimas, linhagens tumorais de câncer de pulmão humano e próstata humana, respectivamente) foram tratadas com CGEN 856S e Ang-(1-7) com ou sem tratamento prévio com A779, utilizadas para imunolocalização de FOXO1, analisadas por microscopia confocal e a intensidade de fluorescência nuclear foi quantificada. Células A549 e DU145 também foram utilizadas em teste de citotoxicidade, sendo tratadas com uma combinação dos inibidores de PI3K wortmannin ou LY294002 em diferentes concentrações e CGEN 856S ou Ang-(1-7). Os resultados mostraram uma diferença significativa no aumento da fosforilação da AKT, eNOS, bem como na desfosrorilação de FOXO1 apenas em células CHO-Mas tratadas com CGEN 856S e Ang-(1-7). Células CHO-Mas tratadas com ambos os peptídeos também promoveram um aumento significativo na liberação de NO comparado com células CHO-Mas não tratadas. Não foram observadas diferenças em células CHO não transfectadas. Células HAEC, A549 e DU145 tratadas com CGEN 856S e Ang-(1-7) promoveram translocação nuclear significativa de FOXO1. O A779 bloqueou este efeito, pelo menos, parcialmente. Tanto em células A549 quanto em DU145, a combinação dos inibidores de PI3K wortmannin e LY294002 com CGEN 856S e Ang-(1-7) mostrou um efeito anti-proliferativo potencializado. Estes dados sugerem que tanto o CGEN 856S quanto a Ang-(1-7), como agonistas do receptor Mas, possuem efeitos semelhantes e representam um potencial alvo terapêutico no câncer e em patologias cardiovasculares.