Pathology - Theses
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ItemA novel depiction of genome variation data( 2006)The use of locus-specific databases for the storage and dispersal of genetic variation data has become the norm. The Human Genome Variation Society (formerly the HUGO Mutation Database Initiative) have led this field and produced a body of work to define standards for online variation databases. While these databases are available for all to access over the internet, they are often difficult to use and, particularly, to understand. Data presentation techniques and “fancy tools”1 for graphically displaying variation data are yet to receive the attention they deserve. This thesis looks at providing a system for use in a locus specific database, as a complement to an existing user interface, to quickly and easily produce graphical conceptual models of variation data in order to improve understanding by the database users.
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ItemCharacterisation of axon glial interactions in the 2-50 transgenic mouse( 2006-12)The 2-50 transgenic mouse is a mutant containing the functional exons of human c-myc, an oncogene and cell cycle controller, under the control of the minimal sequence of the promoter of myelin basic protein, a component of the sheath surrounding axons of neurons. The transgene complex is expressed only in oligodendrocytes during a limited period of neonatal life, and is not detectable in large amounts. The animals suffer significant loss of oligodendrocyte precursor cells prior to myelination and onset of myelin formation is delayed. These animals elaborate only an incomplete myelin sheath in the central nervous system. Quantitative genetic analysis was used to characterize the transgene insertion and optic nerves from transgenic and non-transgenic animals were used for light and election microscopy, for electrophysiological testing and for immunohistochemical studies of glial cell subpopulations and axonal cytoskeletal components.
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ItemFunctional and molecular changes of mitochondria in human aging: observations in dividing tissues( 2000)Studies in a number of human tissues have revealed that the activities of mitochondrial respiratory chain enzyme complexes decline during the aging process. Other studies have suggested that aging increases the frequency of mitochondrial DNA (mtDNA) mutation and leads to the accumulation of mutant mtDNA species, mainly those with large deletions and point mutations. Although the mitochondrial theory of aging may be more applicable to post mitotic tissues, abnormalities of mtDNA have also been reported in tissues which retain a mitotic capacity. Fresh tissues from elderly patients are difficult to obtain and only a limited number of studies on biochemical examination of respiratory chain enzyme complex activities have been carried out. Prostate tissue is readily available in elderly male subjects because of the high prevalence of benign prostatic hypertrophy in this sub-group of the population, and endoscopic surgery is routinely performed for excision of the diseased prostate. In this study, mitochondrial respiratory function and the mtDNA mutations in prostate tissues of elderly patients (aged from 56 to 92) were studied in 24 subjects. This included the measurement of the activities of the respiratory chain enzyme complexes and screening for mitochondrial point mutations and deletions at sites commonly affected in neurodegenerative diseases. (For complete summary open document)
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ItemModulation of death receptor-induced apoptosis by Hsp72( 2004-12)The inducible heat shock protein Hsp72 inhibits apoptosis and promotes long term survival after a number of stresses but the mechanism by which this is achieved remains unclear. A role for Hsp72 in modulating apoptosis mediated through members of the TNF-receptor super family other than TNF-R1 has not been clearly established. Given the observations of high levels of Hsp72 in tumours of poor prognosis, we set out to determine whether Hsp72 could specifically modulate apoptosis induced through the death receptor pathways mediated by Fas and the TRAIL receptors. Both these pathways are of relevance in tumour surveillance. (For complete abstract open document)
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ItemThe role of TNF-related apoptosis-inducing ligand in immune function( 2004)Tumor necrosis factor (TNF)-related apoptosis-inducing ligand [TRAIL or Apo-2 ligand (L)] is a member of the TNF superfamily of ligands that can induce cellular responses such as activation, proliferation, differentiation, migration and apoptosis. Recombinant (r) soluble TRAIL is currently being developed as a most promising natural immune molecule for trial in cancer patients since it selectively induces apoptosis in transformed or stressed cells, but not in most normal cells. Unlike TNF and FasL (CD95L), that both exert significant systemic toxicities, rTRAIL has been shown to be relatively non-toxic and to exert potent anti-tumor functions when administered in vivo to tumor-bearing mice and non-human primates. Moreover, whilst radiation and most deoxy ribonucleic acid (DNA)-damaging chemotherapeutic drugs induce tumor cell apoptosis in a p53-dependent manner, TRAIL-mediated apoptosis is p53-independent. Treatment with rTRAIL might therefore be expected to circumvent resistance to conventional chemotherapy and radiotherapy in cancer patients lacking p53 function. Despite great interest in TRAIL as a cancer therapeutic, either as a sole agent or in combination with irradiation or chemotherapeutic drugs, what has undeniably been lacking is an in depth understanding of the natural physiological role of TRAIL. This knowledge is fundamental if TRAIL is to be used safely and with efficacy in the clinic. Very recently, constitutive TRAIL expression has been identified on a small subset of liver natural killer (NK) cells in adult mice. TRAIL expression can also be induced on interferon (IFN)-α-stimulated peripheral blood T cells, IFN-stimulated human monocytes and dendritic cells (DCs), and NK cells stimulated with interleukin (IL)-2, IFNs or IL-15. We and others have reported that TRAIL-expressing liver NK cells play a role in suppression of TRAIL-sensitive liver tumor metastasis in vivo, suggesting that TRAIL might play a role in tumor surveillance. In a small number of preliminary studies, TRAIL has also been identified to suppress autoimmune disease induction. The focus of this thesis has been to help elucidate the roles of endogenous host TRAIL in disease. The research described herein provides the first detailed evidence of the leukocyte expression and function of mouse TRAIL. We describe the initial characterization of TRAIL-deficient mice generated by gene-targeting, and use these mice to (I) identify TRAIL as a marker of NK cell differentiation, (2) determine the role for TRAIL in regulation of tumor development, (3) determine the role for TRAIL in T cell development and homeostasis, and (4) determine the importance of TRAIL in controlling the induction of experimental autoimmune encephalomyelitis (EAE).
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ItemMolecular basis of amyloid-β formation: focus on β-secretase( 2003)Aβ amyloid deposition is the pathognomonic feature of Alzheimer's disease (AD) and cytotoxic Aβ oligomers are considered to be responsible for neuronal degeneration. Aβ is proteolytically derived from the type 1 amyloid precursor protein (APP) by the sequential action of β- and y-secretases. This thesis focuses on the characterization of the recently identified β-secretase, BACE1 in human brain. This molecule has been propelled to the forefront of AD research due to its potential as a therapeutic target. The work presented here describes the first report of increased BACE1 protein and activity in AD brain. The BACE1 gene was cloned from human brain total RNA and used to transfect mammalian cells. Expressed protein was detected using three BACE1-specific antibodies generated during this candidature. Protein samples were then prepared from human brain and analyzed using BACE1 C-terminal antibody that revealed a significant 2.7-fold increase in BACE1 protein level in AD frontal cortex compared to age-matched and neurological controls. Examination of the C-terminal membrane-bound stub resulting from BACE1 cleavage demonstrated an ~2-fold increase in β-CTF levels confirming elevated enzyme activity. A more detailed analysis aimed at elucidating the mechanism by which BACE1 protein levels are increased in AD brain showed that this did not occur at the level of the message since BACE1 mRNA levels did not differ between control and AD groups. Analysis of lipid raft fractions prepared from AD and control brains demonstrated the presence of increased levels of amyloid generating machinery as well as amyloidogenic Aβ in rafts. Furthermore, it was also discovered by Western blotting that multiple BACE1 immunoreactive species were present in human brain and that this immunoreactivity was increased in more dense fractions that also contained lipid rafts. The second area of research describes the generation of recombinant BACE1 protein in a mammalian cell expression system. Cloning and characterization of the putative ectodomain construct revealed that a six amino acid sequence previously believed to be part of the ectodomain was sufficient to retain BACE1 within the cell, associated with the membrane as determined by biochemical, enzymatic and microscopic techniques. Removal of these six amino acids resulted in a secreted product that was purified from culture medium by anion chromatography coupled to FPLC. Using techniques developed in this section, lipid raft fractions were examined for BACE1 enzymatic activity and it was discovered that elevated levels of activity were associated with dense fractions. These results add support to those from Western blotting described above that showed increased BACE1 immunoreactivity coupled with that of other amyloidogenic proteins segregating in these dense fractions suggesting possible compartments where Aβ generation may occur. Work presented in the final section describes the first report of BACE1 enzyme activity measured in cerebrospinal fluid. Using a quenched fluorescence enzyme assay BACE1 activity was found to be elevated 3-fold in post-mortem AD CSF compared to control. This finding suggests the possibility of using BACE1 as a biological tool in the diagnosis of Alzheimer's disease. The work presented in this thesis describes the analysis of the β-secretase enzyme BACE1 in human brain. We have shown that BACE1 protein and activity are increased in AD brain and have identified probable compartments where this may occur. We have also shown that BACE1 activity is increased in AD CSF identifying this enzyme as a potential diagnostic marker of the disease.
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ItemMolecular analysis of the estrogen receptor alpha pathway in human breast cancer( 2002)The recognition that estrogen is involved in breast cancer underscores the need for achieving a higher resolution view of signalling by its cellular target the estrogen receptor alpha (ER). Central to this pathway is the ER itself, numerous interacting proteins that modulate ER activity and downstream ER target genes. Although the ER has been extensively studied in breast cancer, further study is required to examine the role of dysfunction in other components of ER signalling. Upon embarking this thesis, the possibility remained that past studies may have underestimated the frequency of somatic ER mutations in breast carcinoma specimens due to the use of whole tumour homogenates and indirect mutation screening techniques. To overcome these limitations direct DNA sequencing of the entire coding region of the ER was performed on microdissected subpopulations of tumour cells of defined ER and progesterone receptor A (PR) protein expression from thirty-four breast carcinomas. No somatic ER mutations were detected indicating that previous studies had not failed to detect ER sequence alterations due to limitations in tissue selection and mutation detection. The steroid receptor coactivator, AIB1 (amplified in breast cancer 1), previously shown to be amplified in breast cancer was therefore used as a candidate to examine the role of alterations in components of the ER transcriptional machinery. In situ hyhridisation and immunohistochemistry of 93 breast carcinomas demonstrated AIB1 mRNA overexpression was associated with high tumour grade, lack of ER and PR protein expression and strong immunoreactivity for p53 and IIER2/neu. These results highlighted the potential complex interplay between components of ER signalling with other non-steroid receptor pathways in breast cancer and stressed the need to examine ER signalling more globally. The microarray analysis of global gene expression patterns in thirty-two primary breast carcinomas or varying ER status demonstrated that ER positive and ER negative tumours display distinct expression profiles. Using the transcriptional program of the ER positive MCF-7 breast cancer cell line model in response to estrogen and the estrogen antagonist ICI 182 780 as a biological framework, the major differences related to the altered expression of downstream ER target genes and genes involved in cellular turnover. The preferential expression of multiple ER target genes in ER positive tumours was attributed to the direct transcriptional effects of ER activation. Stanniocalcin 2 (STC2), a glycoprotein hormone originally found to control calcium homeostasis in bony fish was investigated further as one of the novel ER target genes whose expression closely paralleled ER expression. In situ hybridisation and immunohistochemistry on 236 unselected breast carcinomas independently confirmed the positive association between STC2 mRNA/protein expression with tumour ER and PR status. The distinct molecular profiles associated with tumour ER status appeared to also reflect the result of ER activation or lack thereof on cellular turnover. ER negative tumours expressed relatively higher levels of promitotic genes that correlated with tumour miotic index, whereas the ER positive counterparts expressed relatively higher mRNA levels of specific anti-apoptotic genes. The conclusion drawn was that the primary sequence of the ER gene is maintained functionally intact and that ER signalling is associated with distinct gene expression profiles in ER positive versus ER negative breast tumours. The alterations identified which related to tumour ER protein status included genes that make up part of the transcriptional apparatus used by the ER such as the AIB1 coactivator, downstream ER target genes and genes implicated in the regulation of cellular turnover. A unifying implication is the potential existence of a dualistic model of breast cancer development that incorporates an ER signalling driven pathway and an alternate ER negative pathway.
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ItemThe role of c-Myb in mammary gland development and tumourigenesis( 2009)c-Myb/MYB is an established and key player in hematopoietic malignancies but more recently a strong case for c-Myb as an oncogene in breast cancer has emerged. c-Myb and its transcriptional target genes have direct bearing on tumour initiation and progression and thus this has opened new opportunities to the development of therapeutic approaches in a range of cancer types with the aim of treating cancer at its various stages. In this study, the requirement of c-Myb during mammary gland tumourigenesis is being examined. In addition a direct therapeutic approach to targeting c-Myb-driven gene grp78/GRP78 in the context of primary and metastatic breast cancer was assessed. The first aim of this study is to examine the expression of c-Myb during normal mammary gland development. The expression of c-Myb is extensively characterised in a temporal and spatial fashion. Nuclear staining of c-Myb by immunohistochemisty was found to be most elaborately expressed in the ductal epithelium during early mammary gland development. Mouse mammary gland lacking c-myb showed disorganised ductal structure in virgin mice, but did not affect subsequent pregnancy and lactation. To extend the view that c-Myb is involved in mammary tumourigenesis c-myb-transduced immortalised mammary epithelial cells and two mammary tumour prone transgenic mouse models were examined. NMuMG cells transduced with c-myb showed enhanced proliferation and reduced Annexin V staining consistent with the protection from apoptosis. This reduced apoptosis is consistent with, and perhaps contingent upon, the elevated expression observed for bcl-2 and grp78. The data assembled by expression studies raised the possibility that c-Myb is essential for the establishment of mammary gland tumor in both MMTV-Neu and MMTV-PyMT spontaneous mammary gland tumor models. Loss of c-Myb expression in these models significantly delayed and in most instances completely abolished the onset of mammary gland tumours in both models. Preliminary evidence also indicated that Stat3 phosphorylation may underpin the elevated c-Myb expression in mouse mammary tumour cells. The focus of my thesis then shifted to examining ways to exploit elevated c-Myb target gene GRP78 expression on the cell surface of mammary tumour cells. To do this I employed a GRP78 binding pro-apoptotic chimera peptide that specifically binds to GRP78 where I examined its efficacy against primary and metastatic breast cancer models. My results demonstrated the anti-tumour activity of the GRP78-chimera peptide both in vitro and in vivo. More importantly, the peptide is also effective at prolonging disease-free survival in mice with established metastatic disease. Evidence obtained within these studies suggests that c-Myb plays an important role in mammary gland development and tumourgenesis. Although it may be difficult to directly target c-Myb in malignant disease, alternative anti-tumoural therapy may be developed against c-Myb-regulated target genes that are also implicated in mammary tumours. Collectively my thesis studies have advanced our understanding of c-Myb in mammary cancer initiation, progression and as a direct or indirect therapeutic target.