published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Pathology / Master / Master of Philosophy

published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

published_or_final_version / Medicine / Master / Master of Philosophy

No description available.

No description available.

Eukaryotic translation initiation factor 5A (eIF-5A) is the only known cellular protein that contains the post-translationally derived amino acid, hypusine. Initially, eIF-5A was named as a translation initiation factor because of its capability to stimulate the formation of methionyl-puromycin, which mimics the first peptide bond formation during protein synthesis, under <em>in vitro</em> conditions. Subsequently, however, this proposed function of eIF-5A has been questioned because a similar effect on translation was not observed <em>in situ</em>. Moreover, eIF-5A appears not to be required for general protein synthesis. Rather, there is evidence that it facilitates the translation of specific subsets of mRNAs required for cell proliferation as well as apoptosis. <br /><br /> There are two isoforms of eIF-5A in the human genome which have designated eIF-5A1 and eIF-5A2. The objective of the present study was to gain an increased understanding of the roles of eIF-5A1 and eIF-5A2 during apoptosis and cell proliferation using human cell lines as a model system. Apoptosis was induced by treating the cells with Actinomycin D or sodium nitroprusside (SNP), which initiate programmed cell death by different mechanisms. It was observed for both normal and cancer cells that eIF-5A1 protein is up-regulated during apoptosis induced by Actinomycin D or SNP, whereas eIF-5A1 mRNA is constitutively expressed and does not change in abundance during this treatment. The up regulation of eIF-5A1 protein levels in the absence of a corresponding up-regulation in eIF-5A1 mRNA suggests that eIF-5A1 may be post-transcriptionally regulated. Moreover, eIF-5A1 protein up-regulation was stronger in normal cells than in cancer cells. By contrast, eIF-5A2 protein was below detection levels during apoptosis in both normal and cancer cells, although the corresponding transcript was detectable by semi-quantitative RT-PCR. This is attributable to inefficient translation of eIF-5A2 mRNA. <br /><br /> The effects of eIF-5A1 and eIF-5A2 on cell proliferation were examined by modulating the levels of serum in cultures of UACC-1598 cells, which are ovarian cancer cells that express high levels of both isoforms of eIF-5A. Serum starvation, which induces cell cycle arrest and ensuing apoptosis, followed by the re-addition of serum had no effect on the transcript levels of either eIF-5A1 or eIF-5A2. However, eIF-5A1 and eIF-5A2 proteins were both up-regulated within 24 hours of the initiation of serum starvation, and this coincided temporally with the onset of apoptosis as measured by TUNEL and a subsequent decline in viable cells. <br /><br /> The data indicate that eIF-5A1 and eIF-5A2 are both post-transcriptionally regulated and that they have functionally redundant roles in apoptosis.

Hyaluronic acid (HA) is one of the major components of the extracellular matrix; and may exhibit different biological functions, dependent on polymer molecular weight (MW). The signalling events performed by HA are mediated through interactions with its main cell receptors: CD44 and RHAMM. However, the direct effect between the HA MW and the expression of CD44 and RHAMM remains unclear. This study aimed to investigate whether different HA polymer MW alters the proliferation of tumour-derived cell lines, and whether different HA-sized has an effect on the regulation of the expression of CD44 and RHAMM. In order to determine size-specific responses of tumour cells of defined fragment MW, this investigation was undertaken using HA-tethered culture surfaces. Four surfaces were constructed, coated with polymers of different MWs. HA (4, 234, 2590 kDa) and an oligomer mixture were tethered onto an aminosilane (AHAPTMS)-treated glass surfaces using a carbodiimide reaction. Surfaces were analysed using a toolbox of in situ characterisation techniques, including wettability measurements, QCM, AFM and confocal microscopy. Using the constructed surfaces was demonstrated that HA-polymer MW modulates cell proliferation of human bladder (RT112 and T24) and prostate (PC3 and PNT1A) cell lines, with low HA MW (HA4) increasing proliferation, whereas a decrease is seen in the presence of medium (HA234) and high MW fragments (HA2590). The proliferation stimulus performed by HA was found to be phenotype dependent, with HA4 surfaces stimulating an increased proliferation in those less invasive cell lines (T24 and PNT1A), while HA234 and HA2590 inducing a sharper decrease in the most malignant tumour cell lines (RT112 and PC3). It was also demonstrated that the regulation of CD44 and RHAMM transcripts expression appears to be phenotype dependent but not HA-MW dependent. HA down-regulates CD44 and RHAMM in the most malignant cell lines; with up-regulation of the expression of the cell receptors in the less invasive cell lines. In addition, the presence of exogenous HA was shown to be involved in the regulation of the expression of CD44 variants expression. The results obtained for the CD44 and RHAMM protein expression were also found to be correlated with the obtained transcripts expression. However, the significance of these findings in tumourigenesis remains unclear. Findings from this investigation may help in the design and development of biocompatible implants with controlled surface properties to be used in cancer therapeutics; with medium and large HA polysaccharides being potential biopolymer candidates, useful for the development of novel therapies for highly invasive cancer. In addition, implications from this work can serve as a base for future research, and can lead to ideas for drugs and methods to be used in cancer therapeutic approaches.