The focus of this research was to investigate the role of protein kinase C-iota (PKC-é) in the regulation of Bad function, a pro-apoptotic member of the Bcl-2 family and Cdk7 function, a master cell cycle regulator in glioblastoma. The results were obtained from the human glial tumor derived cell lines, T98G and U87MG. In these cells, PKC-é co-localized and directly associated with Bad as shown by immunofluorescence, immunoprecipitation, and Western blotting. Furthermore, in-vitro kinase activity assay showed that PKC-é directly phosphorylated Bad at phospho specific residues, S112, S136 and S155 which in turn induced inactivation of Bad and disruption of the Bad/Bcl-XL dimer. Knockdown of PKC-é by siRNA exhibited a corresponding reduction in Bad phosphorylation suggesting that PKC-é may be a Bad kinase. Since, PKC-é is an essential downstream mediator of the PI (3)-kinase, we hypothesize that glioma cell survival is mediated via a PI (3)-kinase/PDK1/PKC-é/Bad pathway. Treatment with PI(3)-kinase inhibitors Wortmannin and LY294002, as well as PDK1 siRNA, inhibited PKC-é activity and subsequent phosphorylation of Bad suggesting that PKC-é regulates the activity of Bad in a PI (3)-kinase dependent manner. Robust expression of PKC-é is a hallmark of human glioma and benign and malignant meningiomas, however, little is understood about its role in glioma cell proliferation. The cyclin dependent kinase activating kinase complex (CAK), comprises of cyclin dependent kinase 7 (Cdk7), cyclin H and MAT1, is the master cell regulator. Cdk7 phosphorylates its downstream cyclin dependent kinases (cdks) and promotes cell proliferation. Results show that PKC-é directly associated and phosphorylated Cdk7 at T170. Furthermore, Cdk7 phosphorylated its downstream target, cyclin dependent kinase 2 (cdk2) at T160. Purified PKC-é was also observed to phosphorylate endogenous as well as exogenous Cdk7. PKC-é knockdown with siRNA, PDK1 siRNA and (PI) 3-kinase inhibitors, Wortmannin and LY294002 treatment exhibited corresponding reduction in phosphorylation of Cdk7 and subsequently cdk2. In addition, PKC-é knockdown reduced cell proliferation; led to cell cycle arrest and also induced apoptosis. Thus, these findings suggest the presence of a novel PI (3)-kinase/PKC-é/BAD mediated cell survival and PI (3)-kinase/PKC-é/Cdk7 mediated cell proliferation pathway.

Activated receptor tyrosine kinases recruit many signalling proteins to initiate downstream cell proliferation and survival pathways, including phosphatidylinositol 3-kinase (PI3K), a heterodimer consisting of a p85 regulatory protein and a p110 catalytic protein. Our laboratory has previously shown the p85α protein also has in vitro GTPase activating protein (GAP) activity towards Rab5 and Rab4, small GTPases that regulate vesicle trafficking events for activated receptors. Expression of a p85α protein containing an arginine to alanine substitution at position 274 (p85R274A) that affects its GAP activity, caused sustained levels of activated platelet-derived growth factor receptors (PDGFRs), enhanced downstream signalling, and resulted in cellular transformation. Together with other data, this suggested that in p85R274A-expressing cells, PDGFRs are more rapidly trafficked through the endocytic pathway, which reduces opportunities for sorting events necessary for receptor degradation. Our laboratory has observed previously that p85 was capable of binding to both Rab5-GDP, as well as Rab5-GTP, which is an atypical characteristic of GAP proteins, whereas p110β had previously been reported to bind Rab5-GTP selectively. Based on these observations, this thesis project was designed to test the hypothesis that both proteins contributed GAP activity towards Rab5, with p85 providing a catalytic arginine residue (R274) and p110β providing switch stabilization functions specific to the GTP-bound state. To accomplish the thesis objective, cells expressing individual p85 defects (lacking GAP activity, R274A; or lacking p110-binding ability through deletion of residues 478-513, Δ110) were compared to cells expressing a double mutant missing both functions. Stable clonal NIH 3T3 cell lines were generated and selected in G418 and clones expressing similar levels of FLAG-tagged p85 wild type or mutants compared to the control cell lines (NIH 3T3, FLAG-vector control, p85 wild type, and p85R274A) were chosen for analysis. A time-course of PDGF stimulation showed that cells expressing p85R274A or p85Δ110+R274A have sustained phosphorylation levels of the PDGFR, reduced rates of PDGFR degradation and sustained MAPK/Erk signalling. Contrary to the cellular transformation previously reported for p85R274A-expressing cells, expression of p85Δ110+R274A did not lead to cellular transformation. These divergent results suggest that p85-associated p110 serves two functions. As the catalytic subunit of PI3K, one function is the localized generation of PI3,4,5P3 lipids at the plasma membrane for Akt activation, and possibly during receptor endocytosis where it could impact MAPK/Erk activation/deactivation kinetics and cell transformation. These results support a second function for p110 in the regulation of PDGFR activation/deactivation kinetics and PDGFR half-life, both strongly influenced by alterations in PDGFR trafficking. This suggests that p110β may regulate PDGFR trafficking by providing Rab5-GTP switch stabilization that complements the catalytic arginine residue (R274) within p85, and that p85α and p110β work together as a Rab5 GAP. The role of PDGFR in the localization of the RabGAP function of p85 to specific subcellular compartments was also examined. It was hypothesized that PDGFR may help localize the RabGAP function of p85 to vesicles containing Rab5 or Rab4 through the binding of p85 to phosphorylated tyrosine residues on activated PDGFR. Stable cell lines expressing individual p85 defects (lacking GAP activity, R274A; or lacking PDGFR-binding ability through site-directed mutation of residues 358 and 649 from arginine to alanine, ΔR; or a double mutant missing both functions) demonstrated that p85R274A or p85ΔR+R274A expression leads to sustained PDGFR activation and signalling, and to delayed PDGFR degradation in response to PDGF stimulation. The sustained signalling observed resulted in cellular transformation in cells expressing p85R274A or p85ΔR+R274A. The data suggests that PDGFR does not play a role in the localization of the RabGAP activity of p85. The findings of this study elucidates important non-canonical functions of the PI3K heterodimer and contributes to our understanding of how specific mutations in both p85 and p110β within regions implicated in the regulation of RabGAP activity can alter signalling events and lead to enhancement of tumour-associated phenotypes.

Due to the high energetic expenditure for the cell, the protein biosynthesis in eukaryotes is an extensively controlled process predominantly regulated at the ribosomal biogenesis and translation initiation steps. The ribosomal biogenesis defines the global translational aptitude of the cell. It is a mainly nucleolar process which is regulated at multiple steps (e.g. transcription, rRNA processing and modification, ribosomal protein translation etc). However, the most extensively regulated and the rate limiting step of translation is the initiation. Multiple eukaryotic translation initiation factors (eIFs) function to facilitate this priming step of translation. The initial recognition of the mRNA molecule happens through the 5' cap structure found in all mRNAs of nuclear origin. This event is mediated through the recruitment of heterotrimeric complex eIF4F consisting of cap-binding protein eIF4E, scaffolding protein eIF4G and the RNA helicase eIF4A unwinding secondary structures found in 5'UTR of mRNA and thus thought to facilitate the scanning process. The helicase activity of elF4F complex or of eIF4A alone is further potentiated by eIF4B in vitro. The latter protein is at the focus of present thesis. / Signal transduction regulates multiple cellular processes including mitogenesis, differentiation, apoptosis, chemotaxis etc. Signaling pathways also regulate ribosomal biogenesis to coordinate mitogenic cues, nutrient and energy availability with the translational capacity of the cells. Mounting evidence links PI3K-Akt-mTOR and Ras-MAPK cascades to the translational control. In this thesis, I show that PI3K/mTOR and MAP kinase cascades converge to phosphorylate eIF4B on Ser422. This phosphorylation results in an increased interaction with eIF3, an essential factor bridging between eIF4F and the small ribosomal subunit. Physiological significance of eIF4B phosphorylation on Ser422 has been demonstrated by the stimulatory effect of eIF4B Ser422Asp phosphomimetic mutant on cap-dependent translation. Taken together, this represents a new paradigm of translational control mechanism regulated by signaling crosstalk. The function of eIF4B in vitro is well characterized but its in vivoeffects are disputed in literature. To address this I established HeLa cell line stably expressing shRNA targeting eIF4B. eIF4B silencing inhibits proliferation rates and anchorage-independent growth. Expression of luciferase reporter gene containing 5' terminal oligopyrimidine tract (TOP) is selectively repressed in eIF4B-silenced cells and can be rescued by exogenous eIF4B regardless of Ser422 phosphorylation status. Moreover, the de novo synthesis rates of endogenous ribosomal proteins in serum starved cultures recapitulate the luciferase reporter assay data. Utilizing polysomal analysis, I was able to show more significant inhibition of translation initiation in serum starved eIF4B-silenced cells. Our attempt to discover novel eIF4B-interacting proteins by Mass Spectrometry approach led to the identification of nucleolar RNA helicase DDX21. Confocal microscopy has shown partial co-localization of tagged eIF4B and DDX21 in nucleolar periphery. Pulse chase experiments metabolically labeling rRNA show an attenuated 28S rRNA production and concomitant accumulation of 36S intermediates in eIF4B-silenced cells. Since ribosomal biogenesis is highly coordinated process and requires strict stoichiometry maintenance of ribosomal components the observed inhibition of rRNA processing could be consequential to the decreased ribosomal protein expression. However, given the fact that eIF4B is associated with the nucleolar pre-ribosomal particle complexes its direct effect on rRNA processing cannot be ruled out. Regulation of ribosomal biogenesis by translation initiation factor may represent an important control mechanism allowing cells to co-ordinate these two processes.

The aim of our study was to evaluate the effects of a resistance exercise session on the vascular action of insulin in superior mesenteric artery of rats. Wistar rats (250-300g) were divided into 3 groups: control (CT, n = 20), electrically stimulated (ES, n = 5) and resistance exercise (RE, n = 20). The exercise was conducted in the apparatus of the squat, where the animals were subjected to 15 sets of 10 repetitions with 3 minutes rest between sets. The intensity was set at 70% of maximum load established by repetition maximum test performed 48h before the exercise session. The animals were kept suspended in EE squat machine and received the same intensity of electrical stimuli applied to the exercised animals. Immediately after the single resistance exercise session, the animals were anesthetized and killed by exsanguination, the superior mesenteric artery was removed and sectioned into rings (1-2 mm) which were mounted on tanks for isolated organ. The endothelium-dependent relaxation was obtained from concentration-response curves for insulin on rings precontracted with phenylephrine. After that, concentration response curves were obtained for groups CT response and ER, in the absence and/or presence of the following inhibitors: L-NAME (NOS inhibitor), TEA (inhibitor nonselective channel for K+), LY294002 (PI3K inhibitor), BQ123 ( ETA receptor antagonist), and Ouabain (inhibitor of Na+/K+-ATPase), concentration-response curves to KCl in the absence and/or presence of ouabain. According to the data obtained, we found that there was no significant difference in the relaxation induced by insulin between groups EE and CT, however, the animals in the ER showed a significant increase in relaxation when compared to CT group (p<0.001). After use of L-NAME reducing the relaxation was observed in both groups (p<0.001). When evaluating the involvement of K + channels, using TEA, relaxation was inhibited only in the RE group (p <0.001). In the presence of L-NAME+TEA relaxation in the CT group was reduced and there was a group ER contraction (p<0.001). The presence of these rings LY294002 interestingly responded in similar ways in the presence of L-NAME+TEA, promoting and inhibiting the group CT contraction curve ER group (p<0.001). The BQ123 were able to amplify the relaxation in both groups (p<0.001). Using both inhibitors (LY294002+BQ123) was observed an inhibition of contractile and relaxing effects in both groups (p<0.001). To assess the functional activity of the Na+/K+-ATPase curves were made for insulin and KCl in the absence and presence of ouabain. It was observed that ouabain was a decrease of insulin-induced relaxation only in the ER (p<0.001) and reduced the relaxation promoted by KCl in both groups, this reduction being higher in the ER (p<0.001). These data demonstrate that a single session of resistance exercise promotes adjustments in insulin-induced relaxation, which is mediated by NO, the channels for K+ and the activity of Na+/K+-ATPase. In addition, there was an effect mediated by ET-1 (via ETA receptors), necessary for the control of vascular tone. / O objetivo do nosso estudo foi avaliar o efeito agudo de uma sessão de exercício resistido na ação vascular da insulina em artéria mesentérica superior de ratos. Ratos Wistar (250-300g) foram divididos em 3 grupos: controle (CT, n = 20), eletroestimulado (EE, n = 5) e exercício resistido (ER, n = 20). O exercício foi realizado no aparelho de agachamento, onde os animais foram submetidos a 15 séries de 10 repetições, com 3 minutos de intervalo entre as séries. A intensidade foi fixada em 70% da carga máxima estabelecida pelo teste de repetição máxima, realizado 48 h antes da sessão de exercício. Os animais EE foram mantidos suspensos no aparelho de agachamento e receberam a mesma intensidade de estímulo elétrico aplicadas aos animais exercitados. Imediatamente após a única sessão de exercício resistido, os animais foram anestesiados e mortos por dessangramento, a artéria mesentérica superior foi removida e seccionada em anéis (1-2 mm), os quais foram montados em cubas para órgão isolado. O relaxamento dependente de endotélio foi obtido a de curvas concentração-resposta para a insulina, em anéis pré-contraídos com fenilefrina. Após isto, foram obtidas curvas concentração-respostas para os grupos CT e ER, na ausência e/ou na presença dos seguintes inibidores: L-NAME (inibidor da NOS); TEA (inibidor não seletivo dos canais para K+), LY294002 (inibidor da PI3K); BQ123 (antagonista do receptor ETA); e Ouabaína (inibidor da Na+/K+-ATPase); curvas concentração-resposta para KCl na ausência e/ou presença de ouabaína. De acordo com os dados obtidos, constatamos que não houve diferença significativa no relaxamento induzido por insulina entre os grupos CT e EE, entretanto, os animais do grupo ER apresentaram um aumento significativo do relaxamento quando comparado ao grupo CT (p<0,001). Após a utilização do L-NAME, foi observada redução do relaxamento em ambos os grupos (p<0,001). Quando avaliamos a participação dos canais para K+, utilizando TEA foi observado inibição do relaxamento apenas no ER (p<0,001). Na presença de L-NAME+TEA o relaxamento no grupo CT foi reduzido e no grupo ER houve uma contração (p<0,001). A presença do LY294002 interessantemente estes anéis responderam de formas similares quando na presença de L-NAME+TEA, promovendo uma inibição no grupo CT e uma contração da curva no grupo ER (p<0,001). O BQ123 foi capaz de amplificar o relaxamento em ambos os grupos (p<0,001). Utilizando ambos os inibidores (LY294002+BQ123) foi observada uma inibição tanto dos efeitos contráteis quanto dos efeitos relaxantes em ambos os grupos (p<0,001). Para avaliar a atividade funcional da Na+/K+-ATPase foram feitas curvas para insulina e KCl na ausência e na presença de ouabaína. Foi observado que a ouabaína promoveu uma redução do relaxamento induzido pela insulina apenas no grupo ER (p<0,001) e reduziu o relaxamento promovidos pelo KCl em ambos os grupos, sendo esta redução maior no grupo ER (p<0,001). Estes dados demonstram que uma sessão de exercício resistido promove ajustes no relaxamento induzido por insulina, que é mediado pelo NO, pelos canais para K+ e pela atividade da Na+/K+-ATPase. Além disso, houve um efeito mediado pela ET-1 (via receptores ETA), necessária para o controle do tônus vascular, durante o exercício.

Abstract Toll-like receptors (TLRs) are transmembrane proteins involved in the recognition of specific microbial structures and thus the activation of signaling cascades of innate immunity. Regulation of the innate immune response is a complex biological process involving the combined synergistic and antagonistic effects of distinct signaling mediators. Although TLR signaling has been widely studied in recent years, there remain many unexplored unique features of each TLR signaling pathway. The present study evaluated the activation and regulation of TLR4 and TLR2 signaling with the aim of better understanding the molecular mechanisms that control these inflammatory signaling pathways. In the present study, the signal transduction mechanisms of TLR4 and TLR2 in response to Escherichia coli LPS and Staphylococcus aureus LTA were evaluated in mouse macrophages. The inductions, interactions, and activations of the signaling molecules and mediators in the TLR pathways were studied by using several molecular biology and protein chemistry methods. In addition, the role of TLR4 and TLR2 in the regulation of the hepatic inflammatory reaction during endotoxemia was studied. Mouse macrophages were found to induce central proinflammatory mediators in response to LPS and LTA stimulation. Specific roles for PI 3-kinase and Btk were described. These kinases were found to be activated by LPS and LTA; moreover, PI 3-kinase and Btk were found to form specific interactions with TLRs and their intracellular signaling mediators. In addition, a unique IRF2 signaling pathway for LTA-induced TLR2 was found, resulting in the activation of signal transducers and activators of transcription (Stats) and IFN-α secretion. The secreted IFN-α was shown to regulate the LTA-induced inflammatory responses, thereby combining the LTA-induced IRF proteins into NF-κB pathway. The present study provides insight into the signal transduction mechanisms of TLRs. The understanding of these molecular mechanisms that control the activation of TLR signaling cascades will in the future help to predict predisposition and outcome in infectious diseases, and to control the course of disease at an earlier stage. / Tiivistelmä Toll:n kaltaiset reseptorit (TLR) ovat solukalvon proteiineja, jotka tunnistavat taudinaiheuttajien eli patogeenien spesifisiä rakenteita johtaen elimistön puolustusjärjestelmän, immuniteetin, aktivoitumiseen. Immuniteetin säätely on monimutkainen biologinen prosessi, joka tapahtuu kudosten, solujen ja erilaisten synnynnäiseen immuniteettiin liittyvien molekyylien vuorovaikutuksina. Tulehdusvasteen säätelyssä tasapaino positiivisten ja negatiivisten säätelysignaalien välillä on erittäin tärkeää, jotta autoimmuunisairauksien, akuuttien tai kroonisten tulehdusten sekä infektiosairauksien synty voitaisiin välttää. Tämän tutkimuksen tavoitteena oli saada lisätietoa TLR2 ja TLR4 proteiinien säätelemistä signaalireiteistä, niiden vasteista tiettyjä patogeenirakenteita vastaan ja ymmärtää paremmin synnynnäisen immuniteetin puolustusmekanismeja. Patogeenirakenteiden aiheuttamaa tulehdusvastetta tutkittiin pääosin soluviljelymallissa. Lisäksi selvitettiin immuunivasteen luonnetta fysiologisessa kokonaisuudessa ja sen korrelaatiota solutasolla nähtyihin vasteisiin käyttäen in vivo hiirimallia. Tutkimus tehtiin käyttäen useita molekyylibiologian ja proteiinikemian menetelmiä proteiini- ja mRNA-ekspressioiden sekä proteiini-interaktioiden tutkimiseen ja erilaisten aktiivisuuksien määrityksiin. Tulehdusvastetta tutkittiin etenkin sytokiinivastetta määrittämällä ja signaaliketjujen toimintaa analysoitiin estämällä spesifisesti niiden toimintaa. Tarkoituksena oli selvittää, mitkä tekijät ovat välttämättömiä kyseisten tulehdusta aiheuttavien bakteerien tunnistuksessa ja puolustusreaktiossa niitä vastaan. Tutkimuksessa havaittiin kahden kinaasin, PI 3-kinaasin ja Brutonin tyrosiinikinaasin, liittyvän oleellisesti TLR signaalireitteihin. Nämä TLR:ien stimulaation seurauksena aktivoituneet kinaasit muodostivat spesifisiä sidoksia TLR:ien ja niiden signaaliketjuihin liittyvien solunsisäisten signaalivälittäjien kanssa. Lisäksi TLR2 signaalireitillä havaittiin aktivoituvan tekijöitä, jotka johtivat interferoni-α välitteiseen tulehdusvasteen säätelyyn. TLR signaalireittien selvittäminen auttaa ymmärtämään tulehdussairauksien patofysiologiaa ja voi siten tulevaisuudessa johtaa parempien hoitomenetelmien kehittämiseen.

Carvacrol is a phenol monoterpene found in aromatic plants specially in Lamiaceae family, which has been evaluated in an experimental model of breast cancer. However, any proposed mechanism based on its antitumor effect has not been reported. In our previous study, carvacrol showed a protective effect on 7,12-dimethylbenz[α]anthracene- (DMBA-) induced breast cancer in female rats. The main objective in this research was to evaluate by using in silico study the carvacrol on HER2, PI3Kα, mTOR, hERα, PR, and EGFR receptors involved in breast cancer progression by docking analysis, molecular dynamic, and drug-likeness evaluation. A multilevel computational study to evaluate the antitumor potential of carvacrol focusing on the main targets involved in the breast cancer was carried out. The in silico study starts with protein-ligand docking of carvacrol followed by ligand pathway calculations, molecular dynamic simulations, and molecular mechanics energies combined with the Poisson–Boltzmann (MM/PBSA) calculation of the free energy of binding for carvacrol. As result, the in silico study led to the identification of carvacrol with strong binding affinity on mTOR receptor. Additionally, in silico drug-likeness index for carvacrol showed a good predicted therapeutic profile of druggability. Our findings suggest that mTOR signaling pathway could be responsible for its preventive effect in the breast cancer. / Revisión por pares

Epidermal growth factor (EGF) previously has been shown to stimulate short-term survival in vitro of cells derived from the native amphibian retinal pigment epithelium (RPE). In the present experiments, we have examined intracellular signaling pathways responsible for mediating these survival-specific growth factor effects, distinct from proliferative effects, using the human epithelial cell line RPE D407. When maintained as single cells in suspension culture in the absence of serum and exogenous survival factors, RPE D407 cell viability gradually declined over a 3-4 day period as a result of apoptotic cell death, a pattern similar to that seen for eye-derived RPE cells. Exposure to EGF (50 ng ml-1) enhanced cell survival by nearly 40% and caused a parallel increase in the tyrosine phosphate content of the EGF receptor (EGFR), as determined by immunoprecipitation and Western blotting. Both effects were completely blocked by 1μM AG1478, an EGFR-selective tyrosine kinase inhibitor. EGF also stimulated phosphorylation of the phosphatidylinositol 3′-kinase (PI3K)-dependent effector kinase Akt, as well as that of the MEK-dependent mitogen-activated kinase (MAPK), extracellular signal-regulated kinase (ERK). Furthermore, EGF-induced protection was substantially reduced by either the PI3K inhibitor LY294002 (25μM) or the MEK inhibitor U0126 (10μM), under conditions in which phosphorylation of Akt and ERK1/2, respectively, was blocked. Our results indicate that EGF-stimulated survival of RPE D407 cells takes place as a result of signaling through both PI3K and ERK/MAPK pathways. Further, residual anti-apoptotic activity stimulated by EGF in the presence of both blockers suggests that additional as yet unidentified growth factor-dependent survival pathways exist.

No description available.

No description available.

7 Abstract Protein kinase C alpha (PKCα) is a serine/threonine protein kinase. PKCα is an important protein regulating cell polarity, protein secretion, apoptosis, cell proliferation and differentiation and tumorogenesis. Previous research has shown a role of PKCα also in a cancer cell migration and cancer cell invasion. The aim of this study was to investigate the role of protein kinase C alpha (PKCα) played in amoeboid mode of cancer cell invasion. We showed that higher expression of PKCα resulted in mesenchymal-amoeboid transition of K2 and MDA mesenchymal cancer cell lines, which was accompanied with decreased cancer cell invasive capability in 3D collage matrix. PKCα overexpression had no effect on the cell morphology of A375m2, however, the results showed a trend in increased invasive potential of A375m2 cells. Conversely, the expression of dominant-negative PKCα resulted in amoeboid-mesenchymal transition of A375m2 cells, and it was associated with decreased invasive potential of K2 and MDA cell lines. Furthermore, a linkage between PKCα and phosphatidylinositol 3-kinase (PI3K) was tested. The results revealed that increased activity of PKCα was accompanied with decreased level of active Akt in K2 cell line. To summarize, our results suggest a probable role of PKCα in regulation of amoeboid...