Project 1: Investigating the role of PBF in the nucleus and nucleolus and Project 2: An investigation into the role of adiposity on colorectal tumourigenesis

Poole, Vikki Louise (2012). Project 1: Investigating the role of PBF in the nucleus and nucleolus and Project 2: An investigation into the role of adiposity on colorectal tumourigenesis. University of Birmingham. M.Res.

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Abstract

Project 1: Pituitary tumour transforming gene (PTTG) binding factor (PBF or PTTG1IP), is a poorly characterised protein found to be upregulated in thyroid cancer. The protein has previously been determined to contain several predicted signal sequences within its 180 amino acids, and previous studies have shown the nuclear localisation signal (NLS) to be functional. However, it is unknown whether the predicted nuclear export signal (NES) is functional. Use of nuclear/cytoplasmic fractionation and immunofluorescence, in this study, established that when PBF is C-terminally tagged with haemagglutinin (HA), PBF can no longer translocate to the nucleus, implying that the HA tag interferes with recognition of the NLS. The study also attempted to determine functionality of the predicted NES. Homology data revealed the NES is not conserved among six other mammalian species, suggesting it is not evolutionary important and therefore may not functional; however, when exportin-1 (CRM1) was inhibited/knockdowned in vitro, immunofluorescence revealed reduced cytoplasmic and nucleolar PBF staining, suggesting the NES may, in fact, have a role in Homo sapiens.

Project 2: Colorectal cancer (CRC) is a leading cause of cancer death, causing 16,000 deaths each year in the UK alone. Obesity is a major risk factor for CRC, and with a quarter of the UK being classified as clinically obese, it is important to establish how obesity affects CRC. Recently, adipose tissue has been described as an endocrine organ, secreting hormones that may be mediators of CRC proliferation. In this study, human adipocytes were cultured and their secretomes collected prior to and after adiopogenesis. The adipocyte conditioned media was analysed to identify the components and their effect on CRC cells. The study showed that differentiated ACM was capable of increasing both the viability and proliferation of CRC cells. Antibody arrays and ELISA detected the presence of the adipokines, leptin, IL-6 and hepcidin, in differentiated ACM. All three adipokines were individually observed to increase proliferation and viability of CRC cells. Mechanistic analysis, using hepcidin inhibition and reporter assays, suggest that there is a trend towards leptin and IL-6 functioning by increasing hepcidin expression in CRC cells. There is also emerging evidence to suggest that IL-6 and leptin are capable of functioning through the Wnt signalling pathway.

Type of Work:

Thesis (Masters by Research > M.Res.)

Award Type:

Masters by Research > M.Res.

Supervisor(s):