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    Role of N-terminal domains of p400 ATPase in the ATM interaction and DNA damage response : a thesis presented in partial fulfillment of the requirements for a the degree of Master of Science (MSc) in Genetics at Massey University, Manawatū, New Zealand
    (Massey University, 2016) Weber, Lauren Elizabeth
    Efficient repair of damaged DNA and preservation of genomic integrity is integral in the maintenance of proper cellular function and prevention of unrestricted cell proliferation. One critical threat to the stability of the genome is the double strand break (DSB), arguably one of the most cytotoxic lesions to DNA. Interference with the DSB repair mechanism can lead to dysregulation of cellular systems and the prospective development of malignancies. Two critical proteins in DBS repair are the Ataxia Telangiectasia Mutated (ATM) kinase, a serine/threonine kinase from the Phosphatidylinositol 3-Kinase-related Kinase (PIKK) family, and p400, an ATPase chromatin remodeler. ATM is one of the first responders to DSBs and is responsible for the phosphorylation of a multitude of protein substrates including the histone variant H2AX. Beyond its phosphorylation ability, ATM has been proposed as a potential shuttle for other repair machinery, aiding in the early and efficient recruitment of proteins to the DNA damage foci. One such proposed protein is p400. The exact role of p400 in DSB repair is unknown but previous studies show that there is a decrease in repair efficiency in its absence. A prospective interaction is supported by previous studies in which p400 and p400 N-terminal derivatives co-immunoprecipitate with ATM in vivo in HEK293T cells. This study aimed to confirm the interaction of ATM and p400 N-terminal derivatives in vitro and explore the functional implications of the association in vivo in U2OS cells. It was not possible to isolate full-length p400 derivatives in vitro and thus no conclusive results were obtained. Functional assays revealed the ability of one p400 fragment, F1, to inhibit DNA repair and cell proliferation after DNA double-strand break induction with bleomycin. Ectopic expression of the other two p400 N-terminal fragments, F2 and F3, induced an inhibition of cell proliferation under standard growth conditions. Although no conclusive results were acquired, a trend emerged suggesting that N-terminal fragment F1 is able to interfere with ATM protein-protein interactions resulting in a decrease in the efficiency of the DNA damage response and repair. These results implicate F1 as a potential target for further research in both DNA repair and cancer therapy.
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    Development of a tetracycline-inducible lentiviral vector with an instant regulatory system : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science (MSc) in Biochemistry at Massey University, Manawatu, New Zealand
    (Massey University, 2013) Yang, Tian
    Lentiviral vectors, originally derived from human immunodeficiency virus, provide highly efficient viral gene delivery vehicles. Lentiviral vectors often use a constitutive promoter to drive the expression of a therapeutic gene. To regulate the expression of a therapeutic gene, a regulatory system such as Tet-On needs to be established in the target cell lines to produce a regulatory protein, reverse Tet-responsive transcriptional activator (rtTA). The expressed rtTA binds to the tetracycline responsive element (TRE) in the promoter in response to doxycycline and activates transcription of gene of interest. A hypothesis in this study is based on the speculation that a basal leaky expression of rtTA in the bi-directional TRE vectors allows instantly inducible expression of a gene of interest and thereby avoids the time-consuming procedures for generating Tet-On cell lines. Based on this hypothesis, a novel lentiviral vector has been developed to examine an instant induction of PP2Cβ as a target gene. Three instantly inducible bicistronic lentiviral vectors [pLenti-Bi-TRE-Tet-on (V), pLenti-Bi-TRE-Tet-on-PP2Cβ WT (WT), pLenti- Bi-TRE-Tet-on-PP2Cβ MUT (MUT)] were constructed and characterised to assess the usefulness of these vectors. Transient transfection of both WT and MUT vectors into HEK293T cells showed a great induction of PP2Cβ expression upon 24 h of 1 μM doxycycline treatment. The result promises the use of these vectors as a mammalian expression plasmid with a feature of inducible target gene expression. However, viral infection studies involving lentiviral packaging and infection procedures did not show a reproducible expression of rtTA or PP2Cβ in HEK293T cells. Therefore, the inducibility of viral transduction needs to be improved for the future studies of PP2Cβ in primary cells.