| 1. |
- Varshney, Gaurav, 1977-
(författare)
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Identification of downstream targets of Alk signaling in Drosophila melanogaster
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The Drosophila gene Anaplastic lymphoma kinase (Alk) is homologous to mammalian ALK, a member of the Alk/Ltk family of receptor tyrosine kinases. In Drosophila Alk is crucial for development of the embryonic visceral mesoderm, where it is the receptor for Jelly Belly (Jeb) ligand. Jeb binding stimulates an Alk-driven, extracellular signal-regulated, kinase-mediated signaling pathway, which results in the expression of the downstream gene duf/kirre. The visceral mesoderm is made up from two different cell types, founder cells and fusion competent myoblasts. The Jeb-Alk signal transduction pathway drives specification of the founder cells of the Drosophila visceral muscle. In this work we aimed to identify genes specifically expressed in the founder cells and/or fusion competent myoblasts. Four genes from a number of candidiates were investigated further. These genes are Hand, expressed in founder cells, goliath, parcas and delilah, which are expressed in fusion competent myoblasts. Hand is a basic helix-loop-helix (bHLH) transcription factor. Alk-mediated signal transduction drives the expression of the bHLH transcription factor hand in vivo, and loss of Alk function results in a complete lack of hand expression in the visceral mesoderm, while Alk gain of function results in an expansion of hand expression. There are no obvious defects in the visceral muscle fusion process hand mutant animals, suggesting that Hand is not critical for visceral muscle fusion per se. I have studied another molecule Goliath, a putative RING finger E3 ligase. goliath is specifically expressed in the FCM of visceral and somatic muscles. goliath mutant animals do not display any obvious muscle phenotypes, perhaps reflecting a redundant role with CG10277, which encodes a second Goliath family protein in Drosophila. Deletion mutation of the CG10277 locus does not result in muscle defects either, and generation of double mutants of goliath and CG10277 will be required to determine their function in vivo. In addition, I have studied another bHLH transcription factor Delilah and its role in muscle development. We show that delilah is expressed in visceral muscle, somatic muscles and in tendon cells. Delilah mutant animals display a held out wing phenotype and are unable to fly. Inducible RNAi against delilah results in a similar phenotype. Delilah is transcriptionally regulated by mef2 and biniou, early regulators of muscle development. While delilah appears to function in tendon cells, we were unable to find any obvious phenotype in either visceral or somatic muscles. In order to further investigate the underlying mechanism of Delilah function we have used Tandem affinity purification (TAP) methodology followed by mass spectrometry to identify Delilah binding partners. This analysis suggests a number of candidate functional partners for the Delilah protein.
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| 2. |
- Vernersson Lindahl, Emma, 1980-
(författare)
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Investigating the function of Anaplastic Lymphoma Kinase
- 2008
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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.
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| 3. |
- Kanai, M, et al.
(författare)
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- 2023
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swepub:Mat__t
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| 4. |
- Kowanetz, Katarzyna, 1977-
(författare)
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Adaptor Proteins in Regulation of Receptor Endocytosis
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ligand-induced endocytosis of receptor tyrosine kinases (RTKs) is a dynamic process governed by numerous protein-protein and protein-lipid interactions. This is a major mechanism of signal termination and is also frequently impaired in cancer. The Cbl family of ubiquitin ligases has been shown to play a key role in downregulation of RTKs, by directing their ligand-induced ubiquitination and subsequent lysosomal degradation. My thesis work has led to the identification of novel, ubiquitin-ligase independent, functions of Cbl in receptor endocytosis. We demonstrated that the adaptor protein CIN85 links Cbl with epidermal growth factor receptor (EGFR) internalization. The three SH3 domains of CIN85 interact with Cbl/Cbl-b in a phosphotyrosine dependent manner, whereas its proline-rich region constitutively binds endophilins, known regulators of plasma membrane invagination. The SH3 domains of CIN85 recognize an atypical proline-arginine (PxxxPR) motif present in Cbl and Cbl-b. Moreover, we showed that numerous endocytic regulatory proteins, among them ASAP1 and Dab2, interact with CIN85 via their PxxxPR motifs. The SH3 domains of CIN85 are able to cluster and exchange its effectors at subsequent stages of EGFR endocytosis, thus participating in the control of receptor internalization, recycling and degradation in the lysosome. We proposed that CIN85 functions as a scaffold molecule implicated in control of multiple steps in downregulation of RTKs.Furthermore, we identified two novel Cbl- and ubiquitin-interacting adaptor proteins named Sts-1 and Sts-2 (Suppressors of T-cell receptor signaling). Ligand-induced and Cbl-mediated recruitment of Sts-1/Sts-2 into activated EGFR complexes led to inhibition of receptor internalization and subsequent block of receptor degradation followed by prolonged mitogenic signaling pathways. Our results indicate that Sts-1 and Sts-2 represent a new class of negative regulators of Cbl functions in receptor endocytosis.In conclusion, this thesis describes novel mechanisms by which Cbl, coupled to its effectors, orchestrates trafficking of RTKs. Detailed understanding of how these processes are controlled under physiological as well as under pathological conditions may be important for future therapeutic approaches.
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| 5. |
- Schönherr, Christina, 1980-
(författare)
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Anaplastic Lymphoma Kinase mutations and downstream signalling
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The oncogene Anaplastic Lymphoma Kinase (ALK) is a Receptor Tyrosine Kinase (RTK) and was initially discovered as the fusion protein NPM (nucleophosmin)-ALK in a subset of Anaplastic Large Cell Lymphomas (ALCL). Since then more fusion proteins have been identified in a variety of cancers. Further, overexpression of ALK due to gene amplification has been observed in many malignancies, amongst others neuroblastoma, a pediatric cancer. Lately, activating point mutations in the kinase domain of ALK have been described in neuroblastoma patients and neuroblastoma cell lines. In contrast, the physiological function of ALK is still unclear, but ALK is suggested to play a role in the normal development and function of the nervous system. By employing cell culture based approaches, including a tetracycline-inducible PC12 cell system and the in vivo D. melanogaster model system, we aimed to analyze the downstream signalling of ALK and its role in neuroblastoma. First, we wished to analyze whether ALK is able to activate the small GTPase Rap1 contributing to differentiation/proliferation processes. Activated ALK recruits a complex of the GEF C3G and CrkL and activates C3G by tyrosine phosphorylation. This activated complex is able to activate Rap1 resulting either in neurite outgrowth in PC12 cells or proliferation of neuroblastoma cells suggesting a potential role in the oncogenesis of neuroblastoma driven by gain-of-function mutant ALK. Next, we could show that seven investigated ALK mutations with a high probability of being oncogenic (G1128A, I1171N, F1174L, F1174S, R1192P, F1245C and R1275Q), are true gain-of-function mutations, respond differently to ALK inhibitors and have different transforming ability. Especially the F1174S mutation correlates with aggressive disease development. However, the assumed active germ line mutation I1250T is in fact a kinase dead mutation and suggested to act as a dominant-negative receptor. Finally, ALK mutations are most frequently observed in MYCN amplified tumours correlating with a poor clinical outcome. Active ALK regulates mainly the initiation of MYCN transcription in human neuroblastoma cell lines. Further, ALK gain-of-function mutants and MYCN synergize in transforming NIH3T3 cells. Overall, somatic mutations appear to be more aggressive than germ line mutations, implying a different impact on neuroblastoma. Further, successful application of ALK inhibitors suggests a promising future for the development of patient-specific treatments for neuroblastoma patients.
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| 6. |
- Ulvklo, Carina
(författare)
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Genetic mechanisms controlling cell specification and cell numbers in the Drosophila CNS
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A central theme in developmental neurobiology pertains to how the diversity of different cell types is generated. In addition, it is equally important to understand how the specific numbers of each cell type is regulated. The developing Drosophila central nervous system (CNS) is a widely used system in which to study the genetic mechanisms underlying these events. Earlier studies have shown that a small number of progenitors produce the daunting number of cells that builds the mature CNS. This is accomplished by a series of events that in an increasingly restricted manner results in different combinatorial transcription factor codes that act to specify the different cell types in the CNS. However the factors controlling the progressive restriction in developmental potential and the ultimate fate of cells have not been completely elucidated.My PhD project has been focused on a specific stem cell in the embryonic Drosophila CNS, the neuroblast 5-6 (NB 5-6), and the lineage of neural cells that is produced by that stem cell. Earlier work have provided both a lot of knowledge and a multitude of genetic tools regarding this specific stem cell, which allowed us to address these issues at single cell resolution in an identifiable lineage. In particular, a late-born group of neurons expressing the apterous gene, the Apterous neurons, had been extensively studied in the past. One particular Apterous neuron, Ap4, expresses the neuropeptide gene FMRFamide (FMRFa), and the selective expression of this gene makes it a powerful marker for addressing many aspects of NB 5-6 development.To identify novel genes acting to control neuronal development, a large scale forward genetic screen was performed utilizing an FMRFa-GFP transgenic reporter construct, thereby using a marker that reports perturbations of NB 5-6-lineage development. Flies were treated with EMS, a chemical that induces random point mutations and the progeny where screened for aberrant FMRFa-GFP expression. From a total of ~ 10,000 mutated chromosomes ~600 mutants where isolated and further characterized. One group of mutants displayed additional Apterous neurons when compared to wild type, and a number of them represented new alleles of three previously known genes: neuralized (neur), kuzbanian (kuz), and seven up (svp). Neur and Kuz are parts of the Notch signaling pathway and Svp is the Drosophila COUP-TF1/2 ortholog; an orphan member of the steroid/thyroid receptor superfamily. These findings initiated two separate studies regarding the roles of these genes in the NB 5-6 lineage.Mutants in the Notch pathway i.e., neur and kuz displayed an excess number of Apterous neurons, born from NB 5-6. We initiated detailed studies regarding the origin of these ectopic neurons and could show that Notch signaling is critical for controlling a switch in proliferation mode in the latter part of the NB 5-6 lineage. With this new mechanism we could independently and simultaneously manipulate cell proliferation and temporal progression, and thereby predictable control cell fate and cell numbers born from the NB 5-6.The screen further identified additional mechanisms acting to specify the Ap cluster neurons. During NB 5-6 lineage development several temporal transitions acts to specify neurons born in different time windows. The temporal gene castor is expressed in a fairly large temporal window and the Ap neurons are sub-specified during that window by several combinatorial feed forward loops of transcription factors. In the screen, we identified a novel allele of the svp gene. We found that svp acts as a sub-temporal factor, fine-tuning the castor window into three different temporal parts. Previous studies have shown a role for svp earlier in the temporal cascade and we could confirm this in the NB 5-6 lineage. Together these data for the first time identify dual temporal roles of the same gene in a single NB lineage.In summary, my thesis has helped identify novel genetic mechanisms controlling neuron subtype specification and numbers.
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