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Non-canonical TGFb signaling pathways in prostate cancerSong, Jie January 2016 (has links)
Prostate cancer is the second leading cause of cancer-related death in men in the Western world. Deregulation of transforming growth factor β (TGFβ) signaling pathway is frequently detected in prostate cancer and contributes to tumor growth, migration, and invasion. In normal tissue and the early stages of cancer, TGFβ acts as a tumor suppressor by regulating proliferation, differentiation, and apoptosis. In later stages of cancer, TGFβ acts as a tumor promoter by inducing angiogenesis, tumor invasion, and migration. Thus, it is important to investigate the molecular mechanisms behind the tumor-promoting effects of TGFβ, which is the topic of this thesis. The tumor necrosis factor receptor–associated factor 6 (TRAF6) controls non-canonical TGFβ signals due to its enzymatic activity, causing polyubiquitination of the cell membrane–bound, serine/threonine kinase TGFβ type I receptor (TβRI) and its subsequent cleavage in the extracellular domain by tumor necrosis factor a–converting enzyme (TACE) in a protein kinase C ζ (PKCζ)-dependent manner. TRAF6 also recruits the active g-secretase complex to the TβRI, resulting in a second cleavage in the transmembrane region and the liberation of the TβRI intracellular domain (TβRI-ICD), which enters the nucleus, where it associates with the transcriptional co-regulator p300. In Paper I, the aim was to elucidate by which mechanisms TβRI-ICD enters the nucleus. We found that the endocytic adaptor protein APPL1 interacts with TβRI and PKCζ. APPL proteins are required for TβRI translocation from endosomes to the nucleus via microtubules in a TRAF6-dependent manner. Moreover, APPL proteins are important for TGFβ-induced cell invasion, and high levels of APPL1 are detected by immunohistochemistry in prostate cancer. Finally, we demonstrated that the APPL1–TβRI complex visualized with the in situ proximity ligation assay (PLA) correlates with Gleason score, indicating that it might be a novel prognostic marker for aggressive prostate cancer. In Paper II, the aim was to explore by which mechanisms TGFβ causes activation of the AKT pathway, which regulates migration and therapy resistance of cancer cells. We found that the E3 ligase activity of TRAF6 induces Lys63-linked polyubiquitination of p85α upon TGFβ stimulation, resulting in plasma membrane recruitment, Lys63-linked polyubiquitination, and subsequent activation of AKT. Moreover, the TRAF6 and PI3K/AKT pathway were found to be crucial for the TGFβ-induced migration. Importantly, we demonstrated, by PLA, a correlation between Lys63-linked polyubiquitination of p85α and aggressive prostate cancer in tissue sections from patients with prostate cancer. In Paper III, the aim was to investigate the mechanisms for TGFβ-induced activation of PKCζ and the role of PKCζ in tumor regression. We found that TRAF6 caused Lys63-linked polyubiquitination of PKCζ. By using two novel chemical compounds that inhibit PKCζ, we demonstrated that PKCζ is crucial for prostate cancer cell survival and invasion. In Paper IV, the aim was to investigate further the target genes for the nuclear TβRI-ICD-APPL1 complex identified in Paper I. We provide evidence that APPL proteins and the TGFβ signaling pathway are important for cell proliferation. In summary, the results reported in this thesis suggest the potential usefulness of the identified signaling components of the tumor-promoting effects of TGFβ as drug targets and biomarkers for aggressive prostate cancer.
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Protein expression analysis of PI3K/AKT pathway components in cells expressing INPP5K and MYO1CMehrbani Azar, Yashar January 2012 (has links)
In an Experimental Rat model for endometrial carcinoma (EC) a minimal region of recurrent deletion/allelic loss at the neighborhood of the Tp53 gene has been identified. A similar observation of deletion at the homologous position on human chromosome 17 unassociated with TP53 mutation has been reported in several human cancer types. Thus an important tumor suppressor activity located close to, but distinct of TP53 is suggested. Detailed molecular analysis of this candidate region in a tumor model suggested Myo1c (myosin 1C) and Inpp5k (inositol polyphosphate-5-phosphatase K), also known as Skip (skeletal muscle and kidney enriched inositol polyphosphate phosphatase), as the best candidates. These two genes are suggested to be involved in glucose metabolism through PI3K/AKT signaling and neither of them has earlier been reported as a tumor suppressor gene. The present work aimed to investigate the potential correlation of MYO1C and/or INPP5K proteins with components of PI3K/AKT signaling pathway involved in cell growth and survival. Cells were transfected with increasing amounts of MYO1C- or INPP5K- gene expression constructs and protein extracts of the cells were subjected to Western Blot analysis for 13 important components of the signaling pathway: p110β\α\δ, p85, pAkt308&473, 14-3-3β, PTEN, Akt, pErk, Erk, Ras, p4EBP1 and 4EBP1. The analysis showed dose-dependent changes in the expression levels of several of these proteins, and the observed changes for the most part were directed towards negative regulation of cell proliferation and survival. The presented data further extended the initial hypothesis for potential tumor suppressor activities of MYO1C and INPP5K proteins through PI3K/AKT pathway.
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Plasticita buněk karcinomu prostaty indukovaná zářením / Radiation-induced plasticity of prostate cancer cellsKyjacová, Lenka January 2015 (has links)
Resistance of various cancers to conventional therapies including radio- and chemo- therapy is one of the most investigated phenomena in the molecular and clinical oncology. Recurrent disease is characterized by the presence of metastases, which are responsible for 90% of cancer-related mortality. Fractionated ionizing radiation (fIR) combined with surgery or hormone therapy represent the first-choice treatment for medium to high risk localized prostate carcinoma (PCa). In PCa, the failure of radiotherapy (RT) is often caused by radioresistance and further dissemination of escaping (surviving) cells. To investigate the radioresistance-associated phenotype, we exposed four metastasis- derived human PCa cell lines (DU145, PC-3, LNCaP, and 22RV1) to clinically relevant daily fractions of ionizing radiation (fIR; 35 doses of 2 Gy) resulting in generation of two surviving populations: adherent senescent-like cells expressing common senescence-associated markers and non-adherent anoikis-resistant stem cell-like cells with active Notch signaling and expression of stem cell markers CD133, Oct-4, Sox2, and Nanog. While the radioresistant adherent cells were capable to resume proliferation shortly after the end of irradiation, the non- adherent cells started to proliferate only after their reattachment...
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EFFECTS OF REPEATED ARIPIPRAZOLE TREATMENT ON THE cAMP AND AKT PATHWAYS IN THE DORSAL STRIATUM OF PREADOLESCENT AND ADULT RATSBecker, Megan Leigh 01 December 2016 (has links)
The positive symptoms of schizophrenia primarily result from an excess of high affinity D2-like receptors (i.e. D2High receptors). First-generation antipsychotics, such as haloperidol, are D2-like antagonists that can cause severe extrapyramidal effects. Aripiprazole, a dopamine and serotonin partial agonist, has fewer side effects, making it tolerable for adults and children. Extrapyramidal effects (e.g. Parkinsonism, dystonia, and akathisia) are among the most problematic side effects produced by antipsychotic compounds, which likely result from an excess of D2-like receptors in the dorsal striatum. In order to examine the effects of repeated antipsychotic treatment on dopamine system functioning, this thesis compared the molecular effects of repeated haloperidol and aripiprazole administration on D2High receptors, as well as various indices of dopamine second messenger system functioning. Preadolescent and adult rats were pretreated with haloperidol or aripiprazole for 11 consecutive days. After either a 4- or 8-day drug abstinence period dorsal striatal tissue was extracted. [35S]GTPγS binding assays were conducted to assess the effects of repeated haloperidol and aripiprazole treatment on the efficacy and potency of D2-like receptors. PKA subunits and components of the Akt pathway were measured using Western Blots. Results showed that repeated treatment with haloperidol or aripiprazole did not significantly affect D2-like receptor efficacy or potency in young or adult rats. In both age groups, haloperidol significantly increased the expression of PKA-Cα, PKA-Cβ, and PKA-RII, but not p-PKA. Haloperidol also significantly increased PKA-Cβ and PKA-RII levels relative to aripiprazole. Repeated administration of haloperidol significantly increased p-GSK-3β levels in young and adult rats, but neither haloperidol nor aripiprazole significantly affected GSK-3β, Akt, or p-Akt levels. Overall, the results of this thesis indicate that repeated aripiprazole and haloperidol treatment differentially affects D2 signaling pathways in the dorsal striatum. Aripiprazole has less extreme or prolonged effects on D2 receptor signaling pathways than haloperidol, as evidenced by the lack of post-treatment upregulation in the cAMP and Akt pathways. Upregulation of D2-like receptors and, in turn, upregulation of proteins in the cAMP and Akt pathways may be partially responsible for the side effects induced by long-term antipsychotic treatment.
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Teknikdidaktikens vad och hur. : En kvalitativ studie om hur förskolepedagoger synliggör teknik i förskolan. / Technology educations what and how. : A qualitive study about how preschool teachers visible technology in preschool.Algotsson, Cecilia January 2019 (has links)
Abstrakt Titel- Teknikdidaktikens vad och hur. En kvalitativ studie om hur förskolepedagoger synliggör teknik i förskolan. Title Technology educations what and how. A qualitive study about how preschool teachers visible technology in preschool. Enligt tidigare studie har pedagoger svarat att teknikundervisning är svårt och komplicerat att göra. Ibland förväxlas teknik med andra ämnen på grund av att teknikintresset är litet bland pedagoger samtidigt står det i styrdokumentet Läroplanen för förskolan att barnen ska få möjlighet att urskilja teknik i vardagen och skapa med olika redskap och material. Syftet med denna studie är att studera om hur förskolepedagoger synliggör teknik i förskolan. Studien genomfördes på två förskolor i samma kommun i Sverige. Empirin bygger på observationer som sträcker sig från planerade aktiviteter till barnens fria lek till rutinsituationer i förskolans vardag. Observationerna genomfördes på tre avdelningar: två med äldre barn och en med yngre barn. Resultatet analyserades med utgångpunkt från utvecklingspedagogikens lärandets objekt och lärandets akt. Resultatet visar att förskolepedagoger i förskolan synliggör användandet av artefakter, lösningars variationer, skapande i konstruktionslek och tekniska system genom att förklara ställa frågor och visa. Förskolepedagogernas teknikundervisning ägde rum i olika situationer under dagen såsom i planerade aktiviteter, fri lek och rutinsituationer.
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Wirkung und Wirkmechanismus von AEZS 126 auf verschiedene Subentitäten des Mammakarzinoms / Anti-tumour activity of phosphoinositide-3-kinase antagonist AEZS 126 in models of triple-negative breast cancerSchmidt, Heike January 2013 (has links) (PDF)
Untersuchung des Wirkmechanismus von AEZS 126 auf drei triple negative Mammakarzinomzelllinien HCC1937, HCC1806 und MDA-MB468 und eine Oestrogenrezeptor positive Zelllinie MCF-7 mittels Kristallviolett assay, FACS und Western Blot. Es konnte gute Antitumorwirkung des Inhibitors in vitro gezeigt werden. / Of more than one million global cases of breastcancer diagnosed each year, a high percentage are characterized as triple-negative, lacking the oestrogen, progesterone and Her2/neu receptors. The incidence exceeds the incidence of malignancies like CML by far. Lack of effective therapies, younger age at onset and early metastatic spread have contributed to the poor prognosis and outcomes associated with these malignancies. Here, we investigate the ability of the PI3 K/AKT inhibitor AEZS 126 to selectively target the triple negative breast cancer (TNBC) cell proliferation and survival in vitro by MTT-assays and FACS-based analysis. Furthermore, the mechanism of cytotoxicity is analysed by FACS-based assays and Western blots. Results AEZS 126 showed good antitumour activity in in vitro models of TNBC as well as in MCF-7 cells. We demonstrated the highly efficient antitumour activity of AEZS 126 in in vitro models of TNBC. Due to the good anti-tumour activity and the expected favourable toxicity profile, AEZS 126 in combination with chemotherapy seems to be a promising candidate for clinical testing in TNBC especially in the basal-like subgroup of TNBC.
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Angiotensin IV and the Molecular Mechanisms Involved in the Development of Insulin Resistance in 3T3-L1 AdipocytesJungwirth, Julie Anne 01 August 2011 (has links)
This study explored angiotensin IV’s (Ang IV) affects on the signaling pathways involved in the development of insulin resistance in 3T3-L1 adipocytes. Ang IV, working through the AT4 receptor, interferes with insulin signaling through the blockade of the phosphatidylinositol 3-kinases (PI3K)/Akt pathway and through activating mitogen activated protein kinases (MAPK): extracellular signal regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK) which are known to impair insulin receptor substrate-1 (IRS-1) signaling. The expression of AT4 receptors was confirmed by reverse transcription polymerase chain reaction. Ang IV’s effects were found by treating adipocytes with combinations of Ang IV, AT4 receptor inhibitor Norleual, and insulin. Cell lysates were resolved by SDS-PAGE electrophoresis and immunoblotted. Ang IV down-regulated the PI3K/Akt pathway. Insulin exerts its effects on adipocytes by activating this pathway, phosphorylating Akt (S473 and T308) residues. Pre-treatment with Ang IV blocked insulin’s effects, reversing Akt activation. Addition of Norleual blocked Ang IV’s inhibitory actions, leading to the phosphorylation of Akt residues. Studies show elevated MAPK levels produced by angiotensin peptides catalyze the phosphorylation of serine residues on IRS-1, impairing insulin signal transduction. Ang IV increased the activation of ERK 1/2 and JNK. This was reversed by pretreatment with Norleual. Ang IV’s effects on IRS-1 residues were found by immunoprecipitation of IRS-1 followed by SDS-PAGE immunoblotting. Ang IV increased the phosphorylation of IRS-1(S307 and S612). Pre-incubation with Norleual attenuated Ang IV’s effects. Ang IV’s stimulation of adipocytes quickly caused the phosphorylation of MAPK corresponding to serine residue phosphorylation on IRS-1, which may implicate Ang IV activation of MAPK in the development of insulin resistance. Ang IV is involved in the down-regulation of the insulin-signaling cascade by inhibiting insulin’s phosphorylation on Akt (S473 and T308). Ang IV increased phosphorylation of ERK 1/2 and JNK, corresponding with increases in serine phosphorylation of IRS-1. Pre-treatment with Norleual inhibited Ang IV’s negative effects on insulin signaling. This study elucidates a new mechanism that may lead to the development of insulin resistance in adipose tissue.
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Integrin Signaling in Cell Adhesion and Mechanotransduction : Regulation of PI3K, AKT, and ROSZeller, Kathrin Stephanie January 2012 (has links)
Integrins are a family of conserved cell surface receptors found throughout the animal kingdom. They comprise 24 dimers in mammals, and regulate a number of processes including cell survival, differentiation, and migration. These complex cellular responses involve processes such as cell attachment, spreading, and various signaling pathways, which in turn depend on the composition of the extracellular environment, on its mechanical properties, and involved integrin types. This thesis focuses on identifying molecules that signal downstream of integrins and how integrin-induced signals may differ dependent on the type of mechanical stimulus that is given. In Paper I, we show that cell spreading and the activation of AKT is regulated by the catalytic PI3K isoform p110α. An intact β1 integrin cytoplasmic tail and actin polymerization was needed for spreading, whereas the presence of FAK or SRC, or the interaction between p110α and RAS was dispensable. Paper II reports that the RICTOR-mTOR complex (TORC2) acts as the kinase downstream of β1 integrins in order to phosphorylate AKT on Ser473, which was functionally linked to cell survival. β1 integrins activated both AKT1 and AKT2, but seemed to prefer AKT2. The investigation of several receptor types with regard to their requirement of TORC2, PAK, and ILK for AKT Ser473 phosphorylation revealed that different kinds of receptors engage specific enzyme combinations depending on cell type and context. In the third paper, we demonstrate that adhesion- and mechanical stretch-induced integrin signaling lead to divergent protein phosphorylation patterns, and that most signals from cell adhesion were not dependent on intracellular contractility. This indicates that integrin ligand binding and mechanical stretch induce signaling via distinct mechanisms. Reactive oxygen species (ROS) derived from different cellular sources modulated these responses. Stretching primarily induced phosphorylation of ERK1/2, and this signal was markedly increased by a derivative of the antioxidant ascorbate and extracellularly administered catalase. The robust AKT phosphorylation in response to adhesion was almost completely abolished with an inhibitor targeting mitochondrial ROS, whereas phosphorylation levels were only marginally affected in stretch assays. Similar results were obtained with siRNA knock-down of a critical subunit of ROS-producing NADPH oxidases.
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Bcl-xL/xS phosphorylation regulates the sensitivity of PC12 cells to apoptosisQi, Ji 19 January 2010
The Bcl-2 family of proteins contains both anti-apoptotic (e.g.Bcl-2, Bcl-xL) and pro-apoptotic (e.g.Bad, Bcl-xS) proteins. The Bcl-xL and Bcl-xS are splice variants, but have different functions during apoptosis. The pro-survival kinase Akt can phosphorylate certain Bcl-2-related proteins, specifically on serine residues, to regulate their function and localization. This is an extension of the work from our laboratorys finding that haloperidol induces PC12 cell death by inducing Bcl-xS which then translocates from cytosol to mitochondria where it facilitates the release of cytochrome c. The toxicity induced by Bcl-xS is reversed by expression of constitutively active Akt. I hypothesized that Akt-mediated post-translational modification may be important for regulating the function of Bcl-xS and Bcl-xL.<p>
Three specific serine residues were ultimately chosen for the characterization of Bcl-xS/xL function: Ser62 (inactivation mutant), Ser106 (putative Akt phosphorylation motif), and Ser165 in Bcl-xS (and the corresponding Ser228 in Bcl-xL) (immediately upstream of hydrophobic tail). The individual substitution of all three Serines with Alanines (which precludes phosphorylation at that site) in Bcl-xS did not affect the expression of the protein, but they did induce varying degrees of cytotoxicity in both PC12 and HEK cultures. I focused on the Ser106 substitution mutant given my hypothesis that Akt targeted this site. Overexpression of Bcl-xS(S106A) was toxic in both PC12 and HEK cultures, as expected, and this coincided with the appearance of the Bcl-xS(S106A) protein in the mitochondrial fraction. The release of cytochrome c from PC12 cell mitochondria coincided with the co-immunoprecipitation of the Bcl-xS protein with VDAC (voltage-dependent anion channel), a channel-forming protein that is known to mediate cytochrome c release, and with the initiation of caspase-dependent events. This was not the case in HEK cells, where the mitochondrial VDAC seemed to be diminished and the toxicity was cytochrome c-independent as well as caspase-independent. In addition, I was able to demonstrate that the S106A substituted protein was not able to co-immunoprecipitate with Akt, supporting Ser106 as a potential target for the Akt protein. I then studied the effects of the homologous substitutions in Bcl-xL on cell function. I chose to use treatment with the potent inducer of apoptosis, staurosporine, as a model of cytotoxicity. Again, substituted proteins exerted toxicity, but they did not potentiate the effects of staurosporine, at least not on MTT conversion. I did notice, however, that there was a clear morphological change with certain concentrations of staurosporine, and subsequently demonstrated that the Bcl-xL(S106A) protein potentiated PC12 cell differentiation induced by staurosporine. This protein also co-immunoprecipitated better with Akt, which was unexpected given my results with the Bcl-xS(S106A) protein described above. Perhaps the extra amino acids in Bcl-xL account for this.<p>
It is clear that the phosphorylation of Bcl-xS and Bcl-xL proteins is an important means of regulating their function and localization within the cell. These data support the S106 residues in both Bcl-xS and Bcl-xL as novel targets for the pro-survival Akt kinase, and indicate a role for this/these residue(s) in cellular functions as diverse as apoptosis and differentiation.
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Scaffolding functions of MAGI-2 in the PTEN mediated attenuation of the PI3K/Akt signalling pathwayPoland, Sharon Franceska 24 September 2009
Activated receptor tyrosine kinase (RTK), such as the epidermal growth factor (EGF) receptor (EGFR) and the platelet-derived growth factor (PDGF) receptor (PDGFR), recruit downstream signalling proteins, including phosphatidylinositol 3-kinase (PI3K). PI3K, composed of a regulatory p85 subunit and a catalytic p110 subunit, phosphorylates phosphatidylinositol 4,5-bisphosphate at the 3 position to generate phosphatidylinositol 3,4,5-trisphosphate. This lipid second messenger activates Akt, which promotes cell growth, cell cycle entry and progression, as well as cell survival and cellular migration. PTEN, a tumor suppressor protein, dephosphorylates phosphatidylinositol 3,4,5-trisphosphate at the 3 position, turning off Akt signalling. PTEN contains a C-terminal PDZ binding motif that binds to the PDZ2 domain of MAGI-2, a scaffolding protein that localizes signalling molecules to the plasma membrane. MAGI-2 has ten domains that potentially mediate multiple protein-protein interactions simultaneously. A PTEN associated-complex (PAC) has been described and may contain MAGI-2, PTEN and p85. The PAC is hypothesized to form at the plasma membrane at appropriate sites for PTEN to gain access to its lipid substrates, since the binding of PTEN to MAGI-2 has been shown to enhance the suppression of PI3K-mediated Akt signalling. In order to better understand the role of the PAC in attenuation of the Akt signalling pathway, regions of the MAGI-2 scaffolding protein were mapped to identify the interactions taking place in the PAC. MAGI-2, and its individual domains, were expressed as GST fusion proteins. These were immobilized onto beads and allowed to bind to cellular proteins including PTEN, p85, PDGFR and EGFR using a GST pull-down experiment. The proteins bound to GST-MAGI-2 were identified using an immunoblot analysis. In vitro pull-down experiments revealed that MAGI-2 PDZ2 and PDZ5 domains bind to PTEN, and both MAGI-2 WW domains were shown to bind to p85. EGFR and PDGFR did not bind to the PDZ domains of MAGI-2 under the conditions studied. In order to study protein-protein interactions in cells, immunoprecipitation assays were also performed. Full length MAGI-2 was expressed tagged to a Myc epitope. This was used in immunoprecipitation assays to determine if MAGI-2 could co-immunoprecipitate with proteins involved in the Akt signalling pathway, such as PTEN, p85, PDGFR and EGFR. MAGI-2 can co-immunoprecipitate with PTEN upon 5 min EGF stimulation however, this result was inconclusive because replicate experiments did not verify this initial observation. MAGI-2 does not co-immunoprecipitate with the EGFR nor p85, under the conditions tested. We examined for these interactions after 5 min of growth factor stimulation and more experiments that test different time points after growth factor stimulation would reveal if these interactions are present at shorter time points. MAGI-2 has been shown to bind to PTEN and p85 in vitro and therefore has the potential to regulate the attenuation of the PI3K/Akt signalling pathway in response to activated EGFR and/or PDGFR.
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