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Identifier 000439995
Title Molecular and cellular mechanisms activated in response to organismal and cellular stress / Kleopatra Avrampou.
Alternative Title Μοριακοί και κυτταρικοί μηχανισμοί απόκρισης στο συστημικό και στο κυτταρικό στρες
Author Αβράμπου, Κλεοπάτρα
Thesis advisor Ζαχαρίου Βενετία
Αϋφαντή, Ιωάννη
Reviewer Τσατσάνης, Χρήστος
Βενυχάκη, Μαρία
Βεργίνη, Παναγιώτη
Ταβερναράκη, Νεκτάριο
Abstract In the presence of unfavorable conditions cells activate molecular pathways to maintain cellular and consequently organismal homeostasis. Dysregulation of these pathway may drive maladaptation and disease. To study stress at the organismal level, we focused on chronic pain. Regulator of G protein signaling 4 (RGS4) is a potent modulator of G protein-coupled receptor (GPCR) signal transduction that is expressed throughout the pain matrix. Here, we use genetic mouse models to demonstrate a role of RGS4 in the maintenance of chronic pain states in male and female mice. Using paradigms of peripheral inflammation and nerve injury, we show that prevention of RGS4 action leads to recovery from mechanical and cold hypersensitivity and increases motivation for wheel running. Similarly, RGS4KO eliminates the duration of nocifensive behavior in the second phase of the formalin assay. Using the Complete Freud’s adjuvant (CFA) model of hindpaw inflammation we also demonstrate that downregulation of RGS4 in the adult ventral posterolateral thalamic nuclei (VPL-THL) promotes recovery from mechanical and cold allodynia. RNA sequencing analysis of THL from RGS4WT and RGS4KO mice points to many signal transduction modulators and transcription factors that are uniquely regulated in CFAtreated RGS4WT cohorts. Ingenuity Pathway Analysis suggests that several components of glutamatergic signaling are differentially affected by CFA treatment between RGS4WT and RGS4KO groups. Notably, western blot analysis shows increased expression of metabotropic glutamate receptor 2 (mGluR2) in THL synaptosomes of RGS4KO mice at time points at which they recover from mechanical allodynia. Overall, our study provides information on a novel intracellular pathway that contributes to the maintenance of chronic pain states and points to RGS4 as a potential therapeutic target (Part A). To study cellular stress responses, we focus on T-cell acute lymphoblastic leukemia (TALL). Protein folding demands and genomic instabilities in tumor cells result in proteotoxic stress. To survive and counterbalance stress, cancer cells activate heat shock pathways and HSF1 factor. While NOTCH1-driven leukemic cells are critically addicted to heat shock factor 1 (HSF1) and maintain high heat shock protein 90 (HSP90) -epichaperome levels, it remains largely unexplored whether HSF1 is required for tumor initiation and maintenance. Here, we generate a transgenic mouse model with an accumulation of nuclear HSF1 protein, termed as HSF1S303/7A, and we demonstrate a similar disease trajectory between HSF1WT and HSF1S303/7A, suggesting that augmented HSF1 does not impact T-ALL initiation or progression upon NOTCH1 oncogenic induction; albeit, established tumors show HSF1 addiction. Given that the epichaperome supports tumor growth, we target the HSP90-epichaperome complexes using a tumor-specific HSP90 inhibitor, PU-H71. While we demonstrate that PU-H71 in vitro induces apoptosis preferentially in NOTCH1-driven T-ALL patient samples, we further reveal that PU-H71 is cytostatic irrespective of NOTCH1 status in TALL patient samples. Using patient-derived xenografts with increased NOTCH1 and elevated epichaperome levels, we show that PU-H71 can effectively attenuate T-ALL progression and increase animal survival. Yet, the PU-H71 treated mice still succumb to the disease, indicating the potential acquisition of resistance in the T-ALL cells. To study the molecular mechanisms underlying the PU-H71 resistance, we generated resistant T-ALL cell lines to PU-H71. Although human T-ALL cell lines display cell cycle arrest and undergo apoptosis following PU-H71 treatment, the resistant cells are still able to proliferate upon PU treatment. Interestingly, PU-H71 resistant cells show diminished HSF1 protein levels, while maintaining relatively lower HSP90-epichaperome levels compared to the initial sensitive populations. However, we found a significant upregulation of HSP70 in all our resistant clones, suggesting that the HSP70 chaperone might compensate for the HSP90 loss in epichaperome complexes and the regulation/response of the proteotoxic stress. The expression profile unravels vast transcriptional alterations in genes encoding regulators of cell cycle and metabolism upon the gain of PU-H71 resistance. Pathway analysis predicts mainly metabolic rewiring in PU-H71-resistant T-ALL cells, possibly mediated by the hypoxia-induced factor-alpha (HIF-1α) pathway. Overall, our study highlights the critical role of HSF1 mainly in established tumors, demonstrates the beneficial targeting of HSP90-epichaperome complexes in human T-ALL therapy, and provides novel information regarding the adaptations of the leukemic cells undergo after the acquisition of PU-H71 resistance (Part B). In conclusion, this comprehensive study unveils mechanistic details of stress regulation both at the cellular and organismal level. At the systemic level, we demonstrate, for the first time, that RGS4 function in the thalamus contributes to pain chronification, while interventions in RGS4 activity promote recovery from chronic pain symptoms. These results provide molecular insights for the design of novel targeted therapies to overcome chronic pain, which is associated with multiple pathologies including rheumatoid arthritis, osteoarthritis, cancer pain, spinal cord injury, and multiple sclerosis. At the cellular level, we provide evidence that targeting the cellular stress response in acute leukemia is a promising therapeutic strategy. In addition, we illuminate the molecular adaptations that occur in leukemic cells after targeting regulators of stress response for long periods. Hence, this study suggests ways to circumvent drug resistance in leukemia, which could lead to the development of innovative and efficient combinational therapies. Collectively our findings point to novel avenues for efficient chronic pain medications and leukemia treatments to ultimately improve the lives of patients.
Language English, Greek
Subject Brain
Chronic pain
Molecular pathways
Μοριακά μονοπάτια
Χρόνιος πόνος
Issue date 2021-07-30
Collection   School/Department--School of Medicine--Department of Medicine--Doctoral theses
  Type of Work--Doctoral theses
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