| 1. |
- Andäng, Michael
(författare)
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Expression and function of ribozymes in transgenic mice
- 2000
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Hammerhead and hairpin ribozymes are short RNAs that act as sequence-specific endoribonucleases by cleaving a target RNA after hybridization to complementary sequences. By changing the specific target hybridization sequence of the ribozyme, virtually any RNA can be targeted, making ribozymes a versatile tool for gene down-regulation (knockdown). This thesis addresses questions regarding expression and function of ribozymes in transgenic mice. A series of hammerhead and hairpin ribozyme transgenic mouse models were generated. The first transgenic mouse model, expressing a hammerhead ribozyme targeting beta-2-microglobulin (ß2-m), proved the principle that ribozymes can efficiently down-regulate the expression specific mRNAs in vivo in a mammalian organism. A second transgenic mouse model expressing hammerhead ribozymes targeting Wilms tumor gene 1 (WT1) generated interesting preliminary data on the involvement of WT1 in male sexual development. However, ribozyme transgene expression was gradually shut-off during breeding, correlating with a disappearance of the observed abnormalities. A third transgenic model showed the feasibility of using ribozyme transgenic mice as a model for ribozyme gene therapy against HIV-1 infection. Transgenic spleen cells expressing an anti-HIV-1 hairpin ribozyme, showed a dose-dependent resistance to infection compared with control spleen cells, when infected with HIV-1/MuLV pseudotype virus. The different trans-cleaving ribozymes targeting ß2-m, WT1 or HIV-1 in transgenic mice were expressed in an expression cassette, in which a 3' cis-cleaving ribozyme, downstream of the trans-targeting ribozyme, was used to terminate the transcript. This cis-cleavage was found to be essential for the activity of the transcleaving ribozyme. Direct proof of hammerhead ribozyme cleavage in mammalian cells in vivo was given by detection of cis-cleavage products in transgenic mouse organs. Steady state levels of cleavage products, relative to the level of the non-cleaved transcript, showed a consistent ranking order: kidney > liver > lung > spleen, which suggests a difference in hammerhead ribozyme cleavage activity in these organs. In vitro analyses of ribozyme - RNA substrate kinetics have primarily relied on indirect detection methods. We have developed a real-time analysis of interactions (i.e. association, dissociation and cleavage) between hammerhead ribozymes and substrates using the surface plasmon resonance (SPR) technology. The SPR technology permits an inclusion of proteins in in vitro analyses, which may improve the capabilities to predict ribozyme function in living cells. In summary, this work has shown that ribozymes, expressed in transgenic mice, can down-regulate the mRNA of specific target genes in a dose-dependent manner, thereby satisfying the general aim of the thesis. It has also shed light on some of the basic aspects of ribozyme expression and function in transgenic mice.
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| 2. |
- Johard, Helena, et al.
(författare)
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HCN Channel Activity Balances Quiescence and Proliferation in Neural Stem Cells and Is a Selective Target for Neuroprotection During Cancer Treatment
- 2020
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Ingår i: Molecular Cancer Research. - 1541-7786 .- 1557-3125. ; 18:10, s. 1522-1533
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Tidskriftsartikel (refereegranskat)abstract
- Children suffering from neurologic cancers undergoing chemotherapy and radiotherapy are at high risk of reduced neurocognitive abilities likely via damage to proliferating neural stem cells (NSC). Therefore, strategies to protect NSCs are needed. We argue that induced cell-cycle arrest/quiescence in NSCs during cancer treatment can represent such a strategy. Here, we show that hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels are dynamically expressed over the cell cycle in NSCs, depolarize the membrane potential, underlie spontaneous calcium oscillations and are required to maintain NSCs in the actively proliferating pool. Hyperpolarizing pharmacologic inhibition of HCN channels during exposure to ionizing radiation protects NSCs cells in neurogenic brain regions of young mice. In contrast, brain tumor-initiating cells, which also express HCN channels, remain proliferative during HCN inhibition.
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| 3. |
- Kitambi, Satish Srinivas, et al.
(författare)
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Small molecule screening platform for assessment of cardiovascular toxicity on adult zebrafish heart.
- 2012
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Ingår i: BMC physiology. - : Springer Science and Business Media LLC. - 1472-6793. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Cardiovascular toxicity is a major limiting factor in drug development and requires multiple cost-effective models to perform toxicological evaluation. Zebrafish is an excellent model for many developmental, toxicological and regenerative studies. Using approaches like morpholino knockdown and electrocardiogram, researchers have demonstrated physiological and functional similarities between zebrafish heart and human heart. The close resemblance of the genetic cascade governing heart development in zebrafish to that of humans has propelled the zebrafish system as a cost-effective model to conduct various genetic and pharmacological screens on developing embryos and larvae. The current report describes a methodology for rapid isolation of adult zebrafish heart, maintenance ex vivo, and a setup to perform quick small molecule throughput screening, including an in-house implemented analysis script.Adult zebrafish were anesthetized and after rapid decapitation the hearts were isolated. The short time required for isolation of hearts allows dissection of multiple fishes, thereby obtaining a large sample size. The simple protocol for ex vivo culture allowed maintaining the beating heart for several days. The in-house developed script and spectral analyses allowed the readouts to be presented either in time domain or in frequency domain. Taken together, the current report offers an efficient platform for performing cardiac drug testing and pharmacological screens.The new methodology presents a fast, cost-effective, sensitive and reliable method for performing small molecule screening. The variety of readouts that can be obtained along with the in-house developed analyses script offers a powerful setup for performing cardiac toxicity evaluation by researchers from both academics and industry.
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| 4. |
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| 5. |
- Prakash, Varsha, et al.
(författare)
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Ribosome biogenesis during cell cycle arrest fuels EMT in development and disease
- 2019
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Ribosome biogenesis is a canonical hallmark of cell growth and proliferation. Here we show that execution of Epithelial-to-Mesenchymal Transition (EMT), a migratory cellular program associated with development and tumor metastasis, is fueled by upregulation of ribosome biogenesis during G1/S arrest. This unexpected EMT feature is independent of species and initiating signal, and is accompanied by release of the repressive nucleolar chromatin remodeling complex (NoRC) from rDNA, together with recruitment of the EMT-driving transcription factor Snai1 (Snail1), RNA Polymerase I (Pol I) and the Upstream Binding Factor (UBF). EMT-associated ribosome biogenesis is also coincident with increased nucleolar recruitment of Rictor, an essential component of the EMT-promoting mammalian target of rapamycin complex 2 (mTORC2). Inhibition of rRNA synthesis in vivo differentiates primary tumors to a benign, Estrogen Receptor-alpha (ER alpha) positive, Rictor-negative phenotype and reduces metastasis. These findings implicate the EMT-associated ribosome biogenesis program with cellular plasticity, de-differentiation, cancer progression and metastatic disease.
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| 6. |
- Trolle, Carl, 1985-
(författare)
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Stem Cell Transplantation in Dorsal Root Injury
- 2014
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- After traumatic injuries to the brachial plexus there is a risk that one or more of the spinal roots are torn from the spinal cord, known as avulsion injury. This often leads to paralysis and chronic pain, notoriously difficult to treat with current pharmacotherapy. Surgical treatment may improve motor function but sensory recovery is usually poor as sensory axons fail to establish functional connections inside the spinal cord. The aims of this thesis were to develop a model for dorsal root avulsion in rodents in order to investigate the potentials of stem cell therapy for enhancing sensory regeneration after spinal root avulsion. Two different types of stem cells, embryonic and neural crest stem cells, have been transplanted to the avulsion model and analysed using immunohistochemical methods. The results indicate that stem cells survive after transplantation to the avulsed dorsal root and associate with regenerating axons. Furthermore, the different stem cells display different phenotypes after transplantation where embryonic stem cells give rise to neurons located outside the spinal cord that could serve as projection neurons whereas the neural crest stem cells form elongated tubes outlining the avulsed dorsal root and are associated with regenerating neuronal fibers. We have also discovered that the neural crest stem cells migrate into the damaged spinal cord as single cells. The neural crest stem cells also display a diversity in generating both neuronal and glial cells that may have different beneficial effects in neural repair following dorsal root avulsion. To improve the survival of stem cell transplants, the potentials of co-transplanting embryonic stem cells together with nanoparticle delivered growth factor mimetics has been investigated. The results indicate that nanoparticle delivered growth factors improve both transplant survival and maturation in comparison to untreated controls and may be a promising strategy in stem cell transplantation.
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| 7. |
- Zanni, Giulia, et al.
(författare)
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Lithium increases proliferation of hippocampal neural stem/progenitor cells and rescues irradiation-induced cell cycle arrest in vitro.
- 2015
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Ingår i: Oncotarget. - : Impact Journals, LLC. - 1949-2553. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Radiotherapy in children causes debilitating cognitive decline, partly linked to impaired neurogenesis. Irradiation targets primarily cancer cells but also endogenous neural stem/progenitor cells (NSPCs) leading to cell death or cell cycle arrest. Here we evaluated the effects of lithium on proliferation, cell cycle and DNA damage after irradiation of young NSPCs in vitro.NSPCs were treated with 1 or 3 mM LiCl and we investigated proliferation capacity (neurosphere volume and bromodeoxyuridine (BrdU) incorporation). Using flow cytometry, we analysed apoptosis (annexin V), cell cycle (propidium iodide) and DNA damage (γH2AX) after irradiation (3.5 Gy) of lithium-treated NSPCs.Lithium increased BrdU incorporation and, dose-dependently, the number of cells in replicative phase as well as neurosphere growth. Irradiation induced cell cycle arrest in G1 and G2/M phases. Treatment with 3 mM LiCl was sufficient to increase NSPCs in S phase, boost neurosphere growth and reduce DNA damage. Lithium did not affect the levels of apoptosis, suggesting that it does not rescue NSPCs committed to apoptosis due to accumulated DNA damage.Lithium is a very promising candidate for protection of the juvenile brain from radiotherapy and for its potential to thereby improve the quality of life for those children who survive their cancer.
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