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    Interactions between MCF-7 and MDA-MB-231 breast cancer cell lines and neutrophils : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Anatomy and Physiology at Massey University, Albany, New Zealand
    (Massey University, 2019) Holland, Sherina
    The tumour micro-environment (TME) has an essential role in tumour development and progression. Immune cells recruited to the site of the tumour secrete soluble factors such as proteinases, growth factors, survival factors and angiogenic factors into the TME. The secretion of these factors is up-regulated via inflammatory mediators secreted by tumour cells, resulting in a pro-malignant cycle between the cancer and immune cells. A greater understanding of the molecular mechanisms underpinning these interactions is required, as this will assist towards identifying potential new drug targets for cancer and ultimately, will aid in the long-term development of targeted and effective treatments for breast cancer and MBC. The activities of certain immune cells, such as tumour associated macrophages, have been reasonably well characterised in cancer, however, until recently, less was known regarding the role of neutrophils in tumour progression. The goal of the research described in this thesis was to determine whether soluble factors secreted by breast cancer cells might alter the phenotype or lifespan of neutrophils. The latter may allow neutrophils sufficient time to participate in activities within the TME that may either help or hinder tumour progression, while soluble factors released by the neutrophils might influence the invasiveness of breast cancer cells. To investigate whether soluble factors released by breast cancer cells could delay neutrophil apoptosis, neutrophils were cultured in conditioned medium (CM) prepared from highly metastatic MDA-MB-231 or poorly metastatic MCF-7 cells. Flow cytometry experiments showed a delay in apoptosis for neutrophils cultured in MDA-MB-231 CM, but not MCF-7 CM. Quantitative RT-PCR was used to measure neutrophil mRNA expression of pro- versus anti-apoptosis peptides; neutrophils incubated in MDA-MB-231 CM, but not MCF-7 CM, demonstrated a significantly higher expression of the anti-apoptosis peptide BCL2 (A1) and significantly lower expression of the pro-apoptosis peptide BAK compared to control. Western blots showed extensive caspase-8 activation for neutrophils cultured in MCF-7 CM, consistent with apoptosis, whilst neutrophils cultured in MDA-MB-231 CM showed little activation of caspase-8, indicating low levels of apoptosis. The soluble factor contained within the MDA-MB-231 CM, responsible for the delay in neutrophil apoptosis was found to be heat stable and have a molecular weight of between 10-100kDA. Prostaglandin E2 (PGE2) was identified as a potential candidate molecule, as it is a heat stable lipid, and when bound to plasma proteins, fits the molecular weight criteria. In addition, neutrophils cultured with 10μM native or heat treated PGE2 demonstrated a delay in apoptosis, however, this was to a lesser extent compared to neutrophils cultured in MDA-MB-231 CM. Cycoloxygenase-2 (COX-2), the enzyme responsible for PGE2 synthesis, was shown to be expressed in MDA-MB-231 cells but not MCF-7 cells, which is in agreement with the results demonstrating a delay in apoptosis for neutrophils cultured in MDA-MB-231 CM but not MCF-7 CM. Freshly isolated human neutrophils, obtained from the peripheral blood of healthy volunteers, cultured in MDA-MB-231 or MCF-7 CM for 7hrs were not polarised toward a pro or anti-tumour phenotype, as determined via the expression of ICAM-1 and MMP-9. Finally, to investigate whether neutrophils could influence the process of EMT and alter the migration of breast cancer cells, neutrophils were indirectly cultured, via transwell plates, with MDA-MB-231 or MCF-7 cells. Neutrophils were not found to enhance the migration of the cancer cells, as determined via a wound scratch assay. Likewise, neutrophils were not shown to influence the process of EMT in the cancer cells, as determined by changes to cell morphology or the expression of EMT markers.
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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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    Synthesis of cyclodextrin composites incorporating targeting and drug carrying capabilities : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry, Massey University, Turitea Campus, Palmerston North, New Zealand
    (Massey University, 2010) White, Rachel Jane
    A selective, versatile, and robust methodology for the bi-functionalisation of - cyclodextrin has been developed which allows for the attachment of peptides and/or sulfonamides in varying C- and N-terminal combinations on resin using NFluorenylmethoxycarbonyl (Fmoc) Solid Phase Peptide Synthesis (SPPS). Mono-6Afluorenylmethyloxycarbonylamino- mono-6X-succinyl-b-cyclodextrin, an amino acid based bi-functionalised derivative of b-cyclodextrin, has been functionalised with the bioactive peptide, bradykinin, and/or sulfonamides using Fmoc SPPS on Rink resin. The all-in-one molecule contains a carrier (cyclodextrin), targeting agent (bradykinin) and/or drug (sulfonamide). Varying combinations of these bradykinin-focused molecules have been synthesised in an attempt to determine the structure-function relationship against cancer cell lines using cell-based screening in vitro. This study commenced with the synthesis of two linkers on to cyclodextrin. This enabled selective binding directly on to the resin, or a peptide attached to the resin. Peptide growth and/or cleavage from the resin followed allowing for the synthesis of peptide-cyclodextrin species in various combinations. Fmoc SPPS techniques have been employed to allow for the addition and synthetic extension of peptides on to cyclodextrin. Peptide purification was achieved by reverse phase high pressure liquid chromatography, and nuclear magnetic resonance spectroscopy and mass spectrometry were used to determine the success of the coupling reactions and identification of cyclodextrin regio isomers. Sulfonamide additions to the cyclodextrin and/or peptide compounds were obtained after numerous studies investigating the optimal reaction conditions. 4-Fluorenylmethyloxycarbonylaminobenzenesulfonyl chloride was found to give the highest yields for the synthesis of C-terminal peptide sulfonamides with 4- carboxybenzenesulfonamide giving the highest optimal yields for N-terminal peptide sulfonamides. Peptide coupling efficiency of cyclodextrin and sulfonamides were investigated and optimised by comparing different SPPS resins and solvents. The incorporation of spacers between the peptide/cyclodextrin and/or resin have also been investigated in an attempt to improve overall reaction yields. Preliminary bioassay testing against tumour cell lines HT-29 Human Duodenum, Hs700T Human pancreatic adenocarcinoma, and MA-104 Human pancreatic adenocarcinoma were performed. The MTT assay and the flow cytometry assay were used to show the effect of varying combinations of these cyclodextrin-peptidesulfonamide molecules against the three cell lines and compared to a known anticancer drug, 5-Fluorouracil. Despite employment of simple entities in the construction of these compounds, an increase in cell proliferation (ca 10-20%) was seen for some cyclodextrin-bradykinin complexes. In addition, an exposed C-terminus on the bradykinin-sulfonamide moiety and an exposed N-terminus on the cyclodextrinbradykinin sulfonamide moiety both gave positive results. Mixed results were obtained with the addition of a linker between the cyclodextrin and the bradykinin molecules (less then 5% increase or decrease) compared to their non-linker counterparts.