| 1. |
- Nordigården, Amanda
(författare)
-
The FLT3 Tyrosine Kinase in Leukemia : Deciphering the Downstream Signaling Events and Drug-Escape Mechanisms
- 2013
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Acute myeloid leukemia (AML) is a severe disease, which originates in blood-forming cells. Although major advances in understanding the biology of AML, the majority of patients eventually succumb to the disease. The tyrosine kinase receptor FLT3 has become an attractive therapeutic target AML for two major reasons; 1) It is one of the most frequently mutated genes in AML (about 30%). 2) Most of these mutations (FLT3-ITDs) correlate with an increased risk of relapse and poor overall survival. Small targeting inhibitors towards FLT3 have been designed and evaluated in clinical trials. However, the experiences from clinical trials are that drug resistance develops in a substantial number of patients. To overcome these resistance-associated problems it its important to improve the understanding of how FLT3 mutations function and how they respond to targeting drugs. This was addressed in this thesis by elucidating FLT3-ITD cell transformation mechanisms, identifying key downstream target molecules of mutated FLT3 and exploring the effect of various targeting inhibitors. The major finding of my thesis is that FLT3-targeting drugs elicit apoptosis through a FOXO3a-dependent upregulation of proapoptotic BH3-only protein Bim via inactivation of the PI3K/AKT signaling pathway. Furthermore, we have identified an interesting apoptotic mechanism, linked to increased ROS levels caused by expressing hyperactivated AKT in hematopoietic stem cells and bone marrow progenitor cells from FLT3-ITD transgenic mice. These findings are interesting from a therapeutic point of view. We have also shown that canertinib, an inhibitor of the ERBB receptor family, targets mutated FLT3 in vitro and in vivo. The irreversible binding mechanism of canertinib, as well as its multikinase activity, is attractive features. Overall, the results presented herein could provide basis for future directions in treatment of FLT3 mutant positive AML patients. Finally, we studied nine different FLT3-ITD mutations ranging in length from 6-33 amino acids. Data from this study suggest that different FLT3-ITDs may induce distinct degrees of transformation and that they respond differentially to FLT3-targeting drugs. These differences were not associated with size of the duplication but rather the mutational composition. In conclusion, this thesis explores the biologic features of FLT3 mutations and therapeutic targeting opportunities.
|
|
| 2. |
- Chand, Damini, 1986-
(författare)
-
Mechanistic Implications and Characterization of Anaplastic Lymphoma Kinase (ALK) mutations in Neuroblastoma
- 2015
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that was first reported as a fusion partner of nucleophosmin in Anaplastic large cell lymphoma in 1994. ALK is involved in myriad of cancers including neuroblastoma which is the most common extracranial solid tumor affecting young children. It arises in the neural crest cells of sympathetic nervous system origin and is responsible for 12% of all childhood cancer deaths. Several point mutations in ALK have been described in both familial and sporadic neuroblastoma.With the aim to understand the role of ALK in neuroblastoma further, we investigated the point mutations in ALK reported in patients. Using cell culture based methods and Drosophila as a model organism; we first characterized these mutations under three broad categories: 1) Ligand independent mutations that were constitutively active, 2) Kinase dead mutation and 3) Ligand dependent mutations that behaved as inducible wild type. Further, to understand the activation mechanism of ALK, we constructed mutations that could potentially alter ALK’s conformation based on the available crystal structure. From the data generated, we were able to provide a new perspective to the activation of full length ALK receptor. This was more in line with activation mechanism of insulin receptor and different from that suggested for ALK fusion protein. From a clinical point of view, all the mutations in the study were blocked to different degrees using the ALK inhibitor, crizotinib. Lastly, we identified potential downstream targets of ALK using phosphoproteomics. From the various targets identified, we focused on STAT3 and confirmed its role as a mediator in ALK initiated MYCN transcription. We showed that STAT3 inhibition led to reduction of MYCN levels and thereby identifying it as a potential therapeutic target in neuroblastoma. Overall, our study highlights clinical relevance of ALK mutations in neuroblastoma and from a basic biology viewpoint; it reveals important mechanistic insight into receptor activation.
|
|
| 3. |
- Hallberg, Mathilda
(författare)
-
Barn till beskådan : Familj, välfärdsstat och nation i fototävlingar och fotoböcker 1930-1944
- 2017
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- I fokus för den här avhandlingen står åskådningskulturen under 1930- och 40-talen, närmare bestämt fototävlingar och fotoböcker med bilder på barn som bärande element. Den här studien granskar hur fototävlingar och fotoböcker var en del i förhandlingen om välfärdens organisering rörande barn och familj. I studien undersöks hur familj, välfärdsstat och nation konstruerades genom representationer av barn, vilka budskap som därigenom etablerades och vilka politiska visioner som kommunicerades. Analyser av bilder och texter visar att fototävlingar och fotoböcker presenterade ett enhetligt budskap om att det behövdes fler och bättre barn, men också hur fototävlingar och fotoböcker förmedlade skilda visioner om hur detta skulle uppnås. Studien bidrar till att synliggöra hur användbara representationer av barn var i bygget av välfärden. De hade potential att förmedla både skilda och gemensamma visioner om välfärdssamhället.
|
|
| 4. |
- Karlsson, Susann
(författare)
-
T-Cell Protein Tyrosine Phosphatase, a Regulator of the PDGF Signaling Pathway
- 2009
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Platelet-derived growth factor (PDGF) is a potent stimulator of cell growth, survival and motility. PDGF exerts its function by binding to specific tyrosine kinase receptors, initiating receptor auotphosphorylation and initiation of specific signaling pathways that regulates the cellular response. It is critical that these signals can be modulated and terminated, since over-activation of signaling pathways are often found in diseases, such as cancer. Protein tyrosine phosphatases (PTPs) counteract the tyrosine kinases by dephosphorylating proteins, thereby playing a crucial role in the control of signaling events. The aim of this thesis has been to study the regulation of PDGF receptor signaling by the T-cell protein tyrosine phosphatase (TC-PTP). In the first two studies, we demonstrated that loss of TC-PTP specifically redirected the PDGF β-receptor towards a rapid Rab4a-dependent recycling after ligand-induced internalization. Furthermore, we found that the sorting of activated PDGF β-receptor into the recycling pathway was dependent on sequential PKCα and Rab4a activation. Since the PDGF α-receptor did not recycle in the absence of TC-PTP, this study displays the first evidence of differences in trafficking of the PDGF receptor family members. PDGF β-receptor recycling was also induced by activating PKCα through the LPA receptor. The LPA-induced PDGF β-receptor recycling correlated with increased receptor phosphorylation and cell migration at low concentrations of PDGF-BB. The data suggests that PKCα activation could serve as a point of cross-talk between receptor families, regulating the duration and magnitude of PDGF β-receptor signaling. In the last study, we searched for novel substrates for TC-PTP downstream of the PDGF β-receptor, and identified the pyruvate kinase M2, PK-M2, as a possible substrate. PK-M2 is expressed in cells that proliferate rapidly, including tumor cells. Our data suggests that TC-PTP can interact with the glycolytic complex, affecting the activity of PK-M2 and hence, altering the glucose metabolism for proliferating tumor cells.
|
|
| 5. |
- Riaz, Anjum
(författare)
-
Adhesion Dependent Signals : Cell Survival, Receptor Crosstalk and Mechanostimulation
- 2013
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The integrin family of cell surface receptors is evolutionary conserved and found in all multicellular animals. In humans 8-alpha and 18-beta integrins are non-covalently associated into 24 dimers. Integrins mediate cell-extracellular matrix and cell-cell interactions and participate in cell signalling. This ideally places integrins to regulate vital processes such as cell adhesion, migration, differentiation and cytoskeleton dynamics. Integrins also play a fundamental role in regulating cell survival and anoikis. In this thesis molecular mechanisms employed by integrins to induce signal transduction, independently or through crosstalk with other receptors, were characterised.Rictor-mTOR (mTORC2) was required for Akt Ser473 phosphorylation in response to β1 integrin-mediated adhesion as well as EGF-, PDGF- or LPA-stimulation of MCF7 cells. ILK and PAK were dispensable for Akt Ser473 phosphorylation upon β1 integrin-engagement or EGF-stimulation. PAK was needed when this phosphorylation was induced by PDGF or LPA. β1 integrin-promoted cell survival during serum starvation conditions was mTORC2 dependent, indicating the importance of Akt Ser473 phosphorylation.mTORC2 was also required for Akt Ser473 phosphorylation induced upon heparanase treatment of cells. Heparanase preferred PI3K catalytic subunit p110α for the upstream lipid phosphorylation required for Akt activation. Interaction between this subunit and Ras was needed for optimal Akt phosphorylation upon heparanase exposure. Cell adhesion strongly promoted heparanase signalling, which was more efficient in β1 integrin-expressing fibroblasts compared to cells lacking this subunit. The cooperative signalling between integrins and heparanase involved FAK and PYK2 since simultaneous silencing of these kinases suppressed heparanase-triggered Akt activation. Furthermore, the resistance of cells to apoptosis induced by H2O2 or serum starvation was promoted by heparanase. Integrin stimulation by adhesion or cyclic stretching showed divergent downstream signalling responses. Cell attachment on integrin-specific ligands lead to robust phosphorylation of several intracellular integrin-effectors, e.g. p130CAS, FAK, Akt and ERK 1/2. However, mechanical cell stretching only triggered prominent phosphorylation of ERK 1/2. Signalling induced at early stages of integrin-mediated cell adhesion occurred independently of intracellular contraction. Reactive oxygen species (ROS) generated during adhesion and cell stretching influenced integrin signalling. Inhibition of mitochondrial ROS production blocked adhesion-induced Akt phosphorylation. In contrast, stretch-induced ERK 1/2 phosphorylation was elevated when extracellular ROS was scavenged. These results indicate that the two types of integrin stimuli generate signals by different mechanisms.
|
|
| 6. |
- Sarri, Niki
(författare)
-
Mechanisms of modulation of PDGFRβ signaling
- 2023
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Platelet-derived growth factors (PDGF) constitute a family of five functional dimers that bind to two structurally related tyrosine kinase receptors i.e. PDGF receptor α and β (PDGFRα and PDGFRβ, respectively), controlling cell growth, proliferation, and migration in cells of mesenchymal origin. However, the aberrant activation of PDGF-induced intracellular signalling pathways is a frequent event in cancer. Therefore, the aim of this thesis has been to discover novel molecular mechanisms of modulation of PDGFRβ signalling. Since mitogen-activated protein (MAP) kinases are activated in PDGF signalling and their spatiotemporal activity is defined by a balance in phosphorylation and dephosphorylation events, in paper I we focused on dual-specificity MAPK phosphatases (MPKs or DUSPs). We found MKP2/DUSP4 to be induced in response to PDGF-BB stimulation. We then demonstrated that the expression of MKP2/DUSP4 was dependent on ERK1/2 activation and on the STAT3/p53 signalling. Endocytosis of RTKs is another mechanism that serves for signal attenuation and termination and this process can be regulated by ubiquitination or deubiquitination of cell-surface receptors. In paper II, we have identified that ubiquitin specific proteases USP4 and USP17 act as deubiquitinases (DUBs) for PDGFRβ. Both deubiquitinases impacted the timing of PDGFRβ trafficking and prolonged STAT3 activation. Consequently, high transcriptional activity of STAT3 led to the increased expression of STAT3-inducible genes c-MYC, CSF1, JUNB and CDKN1A. USP4 deletion attenuated cell proliferation in response to PDGF-BB stimulation. The family of Cbl E3 ligases is essential for ubiquitination of PDGFRβ upon ligand stimulation, followed by the receptor internalization from the cell surface and downregulation of signalling. In paper III, we have identified a new E3 ligase, i.e. tripartite motif-containing protein TRIM21, that deubiquitinates PDGFRβ and regulates its basal levels and its availability on the cell surface in a PDGF-BB independent manner. In paper IV, we described a regulatory role of the endoribonuclease Ras GTPase-activating protein-binding protein 1 (G3BP1) in PDGF signalling. G3BP1 was identified as a PDGFRβ interacting protein which also interacts with BAF155, a core component of SWI/SNF chromatin remodelling complex. G3BP1 depletion upregulated c-FOS, c-MYC and c-JUN mRNA and negatively affected STAT3 and ERK1/2 mRNA and protein levels, stalling cell proliferation. Collectively, we present new mechanisms that regulate PDGF signalling by controlling either PDGFRβ protein levels, availability on the cell surface, subcellular trafficking or activation of downstream signalling affecting regulation of cell proliferation.
|
|
| 7. |
- Vernersson Lindahl, Emma, 1980-
(författare)
-
Investigating the function of Anaplastic Lymphoma Kinase
- 2008
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Anaplastic Lymphoma Kinase (ALK) was discovered in 1994, as a chromosomal translocation, t(2;5)(p23;q35), often seen in Anaplastic Large Cell Lymphomas (ALCL). Since then ALK has been extensively studied in this disease as well as in different model organisms. Due to its expression pattern within the central and peripheral nervous system ALK has been implicated in neuronal development. This hypothesis has been further strengthened by studies from Drosophila which have shown Alk to have an important role in optic lobe development. A recently described ALK mouse knockout model do not indicate an essential role for ALK in development, although a potential role within the central nervous system was strengthened. This since ALK-/- animals has an increased number of progenitor cells in the hippocampus and display altered behavior. The overall aim of the studies included in this thesis was to elucidate the function of ALK in the mouse. As a first step toward this goal we conducted an analysis of ALK mRNA and protein expression patterns during development. The strong expression of ALK in neuronal structures supports a role for ALK in neuronal development during embryogenesis. To further investigate the function of ALK in a physiological context we have developed two different ALK knockout strains, the ALK Kinase knockout (KO) and the ALK exon1 KO. The only visible phenotype in these strains is a reduction of total body weight which is apparent in the ALK-/- population when compared to wild type littermates. This size difference seems to take place after birth and is not due to an alteration in food consumption. We have also extensively studied the ALK Kinase KO with respect to gross development, the gastrointestinal canal and the olfactory system. ALK displays a very distinct expression pattern within the gastrointestinal canal being confined to enteric neuron precursors during embryogenesis and enteric nerves in the adult tissue. From these studies we conclude that ALK is not needed for development and viability in mice although it does play a role in regulation of body weight via a presently unknown mechanism. In addition, we have investigated the relationship between the Drosophila and mouse ALK receptor by examining the ability of the Drosophila Alk ligand Jelly-Belly, Jeb, to activate mouse ALK. Using different in vivo and in vitro techniques, we have shown that activation of mouse ALK cannot be accomplished by Drosophila Jeb. From this study we draw the conclusion that during development ligands for the Drosophila and mouse ALK has diverged to a level at which they can no longer substitute for each other.
|
|
| 8. |
- Vijayaraghavan, Balaje
(författare)
-
Identification and characterization of nuclear envelope protein interactions
- 2015
-
Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The Nuclear envelope which surrounds the chromatin of eukaryotic cells contains more than a hundred transmembrane proteins. Mutations in some genes encoding nuclear envelope proteins give rise to human diseases including neurological disorders. The function of many nuclear envelope proteins is not well established. This is partly because nuclear envelope proteins and their interactions are difficult to study due to the inherent resistance to extraction of nuclear envelope proteins. We have developed a novel method called MCLIP, to identify interacting partners of nuclear envelope proteins in live cells. Using MCLIP, we found three new binding partners of the inner nuclear membrane protein Samp1: the intermediate filament protein Lamin B1, the LINC complex protein Sun1 and the G-protein Ran. Furthermore, using in vitro studies, we show that Samp1 binds both Emerin and Ran directly. We have also studied the interaction between Samp1 and Ran in detail.
|
|
| 9. |
- Yasmin, Lubna, 1973-
(författare)
-
Exoenzyme S of Pseudomonas aeruginosa : cellular targets and interaction with 14-3-3
- 2007
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Pseudomonas aeruginosa is an opportunistic pathogen that is a serious problem for immuno-compromised patients. Toxins such as exoenzyme (Exo) S, ExoT, ExoY and ExoU are secreted and translocated from the bacteria into the eukaryotic cell via the bacterial encoded type III secretion system. Our research focuses on ExoS, a bifunctional toxin comprising a Rho-GTPase-activating protein domain (RhoGAP) and a 14-3-3 dependent ADP-ribosyltransferase domain. In addition, ExoS contains a membrane localization domain termed MLD. In this study, cell lines expressing activated forms of various components of the Ras signaling pathway have been used to understand the functional and mechanical activation of ExoS-ADP-ribosyltransferase activity and to reveal its cellular targets in the cell. Our observations suggested that Ras GTPase is the dominant target by which ExoS mediates cell death and activated Ras is able to protect cells against cell death, regardless of whether it has been ADP-ribosylated by ExoS. It has been reported that the 14-3-3 cofactor protein is required for ADP-ribosyltransferase activity of ExoS and a phosphorylation-independent interaction occurs between 14-3-3 and the C-terminal part of ExoS. We have undertaken a deeper analysis including structural and biological investigation of this interaction. Our results suggested that leucine-428 of ExoS is the most critical residue for ExoS enzymatic activity. Structural analysis showed that ExoS binds to 14-3-3 in a novel binding mode mostly relying on hydrophobic contacts. Our structure was supported by biochemical and cytotoxicity analyses, which revealed that the substitution of important residues of ExoS significantly weakens the ability of ExoS to modify endogenous targets such as RAS/RAP1 and to induce cell death. Further, mutation of key residues within the ExoS binding site for 14-3-3 impairs virulence in a mouse pneumonia model. Leucine residues-422, 423, 426, and 428 of ExoS are important for the interaction with the ″roof″ of the amphiphatic groove of 14-3-3. In conclusion, we show the mechanism of cell signal transduction pathways affected upon ExoS infection and also demonstrate that the hydrophobic residues of ExoS in 14-3-3 interaction motif have a significant role for ExoS enzymatic activity.
|
|
| 10. |
- Henriksson, Maria, 1971-
(författare)
-
Cellular targets of Pseudomonas aeruginosa toxin Exoenzyme S
- 2003
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Pseudomonas aeruginosa is an opportunistic pathogen that can cause life-threatening infections in immunocompromised patients. It uses a type III secretion dependent mechanism to translocate toxic effector proteins directly into the eukaryotic cell. The enzymatic activity of two of these toxins, Exoenzyme S (ExoS) and Exoenzyme T (ExoT), have been studied in this thesis. ExoS is a bi-functional toxin known to contain a C-terminal ADP-ribosyltransferase activity, which has been shown to modify members of the Ras family in vitro. The N-terminal of ExoS contains a GTPase Activating Protein (GAP) domain, which shows specificity towards Rho proteins in vitro. ExoT shows high homology (76%) towards ExoS and has also been reported to contain ADP-ribosyltransferase activity in vitro. To study the biological effect of the two toxins, we inserted ExoS or ExoT into eukaryotic cells using the heterologous type III secretion system of Yersinia pseudotuberculosis. We found that Ras was ADP-ribosylated in vivo and this modification altered the ratio of GTP/GDP bound directly to Ras. We also found that ExoS could ADP-ribosylate several members of the Ras superfamily in vivo, modulating the activity of those proteins. In contrast, ExoT showed no ADP-ribosylation activity towards any of the GTPases tested. This suggests that ExoS is the major ADP-ribosyltransferase modulating small GTPase function encoded by P. aeruginosa. Furthermore, we have demonstrated that the GAP activity of ExoS abolishes the activation of RhoA, Cdc42 and Rap1 in vivo, and that ExoT shows GAP activity towards RhoA in vitro.The ADP-ribosyltransferase activity of ExoS is dependent on the eukaryotic protein 14-3-3. 14-3-3 proteins interact with ExoS in a phospho-independent manner. We identified the amino acids 424DALDL428 on ExoS to be necessary for the specific interaction between ExoS and 14-3-3. Deletion of these five amino acids abolishes the ADP-ribosylation of Ras and hence the cytotoxic effect of P. aeruginosa on cells. Thus the 14-3-3 binding motif on ExoS appears to be critical for both the ADP-ribosylation activity and the cytotoxic action of ExoS in vivo.
|
|
