Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.617798
Title: Testing the in vivo role of actin cytoskeleton regulators on immune cell behaviour by live imaging studies in Zebrafish larvae
Author: Jones , Rebecca Amy
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2013
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Abstract:
Our immune system protects us from infection and abnormal cell growth. But what happens when the function of immune cells is perturbed by the absence of key cytoskeletal regulators? In the immunodeficiency disorder, Wiskott-Aldrich syndrome, patients express dysfunctional variants of WASp, a haematopoietically restricted regulator of the actin cytoskeleton. The disease manifests in recurrent bacterial infections, thrombocytopenia and a severely defective inflammatory response. In this thesis I have utilised a TILLed zebrafish WASp mutant to characterise the effect of WASp knockout on innate immune cell function using a number of assays and live imaging techniques in transparent larvae. I observe defective neutrophil and macrophage migration to a fin wound in the WASp mutant, and increased larval death from systemic bacterial infection as a result of defective phagocytosis and clearance. I have gone on to use the WASp mutant as a null background to screen an allelic range of human WASp patient mutant genes expressed specifically in neutrophils. I show that expression of wild-type hWASp protein can rescue the neutrophil recruitment defect in WASp mutant larvae. Likewise, expression of two point mutants reveals that Cdc42 binding is not essential for hWASp rescue capability, whereas tyrosine phosphorylation of the protein is required. Moreover, expression of a constitutively active hWASp mutant results in larval neutropenia, that mirrors the WASp-related disorder, X-linked neutropenia. Alongside my WASp mutant study, I have also characterised the role of zebrafish Myosin IXb, a haematopoietically restricted atypical RhoGAP, through use of targeted morpholino. I observe decreased neutrophil recruitment to a fin wound in Myosin IXb morphant larvae, whilst live imaging studies demonstrate a contracted morphology and failure to form a polarised leading edge in morphant cells. In summary, I report a live imaging zebrafish study to assess the role of two key actin regulators in vivo, and describe the first instance whereby an allelic range of human mutations are tested in zebrafish to gain a unique insight into immunodeficiency disorders at both a cellular and whole organism level.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.617798  DOI: Not available
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