Pathology - Theses

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A translational analysis of the genomic variation associated with breast cancer in hereditary breast and ovarian cancer families

Sawyer, Sarah Dilys ( 2014)

Breast cancer remains a significant health issue with the highest incidence and the second highest cause of cancer-related mortality amongst women worldwide. A family history is an important risk factor, with women found to be twice as likely to develop the disease if they have one first-degree relative affected with breast cancer. It is the role of familial cancer clinics (FCCs) and genetic testing services to identify ‘high-risk’ individuals and offer genetic testing to eligible individuals looking for mutations within high-penetrance genes, such as BRCA1 and BRCA2. For mutation carriers, cancer risk assessment and management is based on a well-established foundation of evidence. However, up to 80% of individuals have no identifiable BRCA mutation, resulting in risk assessment and management advice that is based only on family history and is no longer personalised but instead directed by summary data from epidemiological studies of the general population. Researchers have continued the search for the other genetic causes of the heritable fraction of breast cancer through large, collaborative genome-wide association studies (GWAS). To date, these studies have identified over 70 common variants associated with breast cancer risk. Individually, the associations for these variants are too weak to be useful (relative risk <1.5), however, when the risks for these variants are combined multiplicatively (termed polygenic risk) the risk becomes more appreciable. It is estimated that up to 28% of familial breast cancer risk can be explained by common variants. Despite this, genetic testing for common genomic variants has yet to be implemented into routine clinical practice. This study explores the clinical significance and implications of common genomic variants to the assessment and risk management of high-risk breast and ovarian cancer families, to generate the data required to facilitate the translation of common variant genetic testing into clinical practice. To conduct this research, the study utilised the Victorian Familial Breast and Ovarian Cancer Cohort (VFBOCC) which consists of individuals (female index cases) who have been assessed as high-risk for breast and/or ovarian cancer based on their personal and family history of cancer. In Chapter 3, the development of a database that systematically incorporated and allowed for the well-defined characterisation of the VFBOCC is discussed. This database framework facilitated a detailed analysis of the cohort; it identified that females in this cohort have an average breast cancer age of onset of 44.9 years, which is much lower than the average age of onset observed in the general population, and that only 22% of the VFBOCC had a BRCA1 or BRCA2 mutation identified by diagnostic genetic testing. The establishment of this database enabled the VFBOCC data to be utilised in common genomic variant research as part of this thesis as well as within several collaborations which are also investigating the potential role of recently discovered rare genetic variations to familial breast cancer risk. In Chapter 4, the association of 62 common genomic variants with a range of clinical features from 1,136 women affected with breast cancer from the VFBOCC is examined. This study found the associations of common variants were stronger in the familial setting when compared to unselected breast cancer cases from the general population, highlighting the significance of hereditary factors in the aetiology of breast cancer. A polygenic risk score (PRS) was then calculated for each woman and the mean PRS was found to be significantly higher in breast cancer cases with no identifiable BRCA mutation when compared to population controls (P= 4.44x10-40) and women who had a BRCA1 or BRCA2 mutation (P=0.0002). These findings illustrate that common variants can help to predict a group of individuals who are at high risk of developing breast cancer but unlikely to harbour a mutation within the BRCA1 or BRCA2 genes. The next step was to investigate the additional effect of the PRS on the outputs from clinical tools used by FCCs to predict the likelihood of a high-risk individual harbouring a BRCA1 or BRCA2 mutation (Chapter 5). This research found that the addition of common variants to the tools did not significantly improve their performance. In Chapter 6, the focus of this study shifts to the clinical implications of introducing common variant genetic testing into clinical practice. It outlines the development of a personalised breast cancer risk report incorporating common variant data, which was evaluated by a sample of health professionals (oncologists, clinical geneticists and genetic counsellors) in a series of hypothetical patient scenarios to determine the potential impact of this additional genetic information on risk assessment and management recommendations of high-risk women with no identifiable BRCA mutation. This study found common variant information assisted in determining an individual woman’s risk of developing a breast cancer, with health professionals recommending increased screening, risk-reducing medications (Tamoxifen) and prophylactic surgery to women with a high PRS. In addition, health professionals identified the need for further research, the development of education programs, and clinical guidelines for risk management recommendations if this new type of genetic information was to be implemented as part of routine clinical practice.
Investigating MYB in the context of mammary gland biology, transformation and as a therapeutic target in breast and colon cancer

Cross, Ryan N S ( 2014)

Breast cancer is the second most common form of malignancy diagnosed in Australian women, imposing an enormous social and economic burden on society. If the cancer spreads to secondary locations, patient survival decreases dramatically. Therapeutic strategies for treatment of metastatic disease are desperately needed in breast cancer. Recently we have shown that when Myb is conditionally deleted from the mammary gland of MMTV-Neu and MMTV-PyMT mice, tumour formation is ablated. To provide further insight into the function of Myb in mammary gland development, cre-mediated conditional knock out of c-myb in the mouse mammary gland was examined. The conditional deletion of c-myb led to a reduction in branching and terminal end bud formation. These data indicate that if MYB could be inhibited for breast cancer therapy, there are potentially few side effects on the normal mammary gland. The ability to inhibit DNA binding transcription factors is a long sort after goal in oncology, as their importance in disease initiation and progression is well documented. To target MYB, we have developed a DNA vaccine. Preclinical studies have largely examined the MYB DNA vaccine in the context of colon cancer models using prophylactic vaccination. However, preliminary data indicate that it may also be effective in reducing the metastatic burden in preclinical breast cancer models. This thesis aims to further investigate the role of MYB during mammary gland development and provide insights into its involvement in tumourigenesis. Furthermore, the MYB DNA vaccine will be assessed for its effectiveness as a therapeutic treatment in clinically relevant surgical models of metastatic breast cancer, as well as further development as a therapeutic in the setting of colon cancer.
Functional genomics analysis of interplay between cell polarity and oncogenic RAS-MAPK signalling

Smith, Lorey Kirsten ( 2014)

The development of cancer is a complex process and progression towards malignancy requires the cooperation of multiple transformation events. This often involves mutations or aberrant expression of various oncogenes and tumour suppressors. Whilst epithelial tumours are inherently heterogeneous, and each tumour type and sub-type is molecularly distinct, one of their pervasive features is disruption to normal epithelial cell polarity and tissue architecture. Indeed it has now emerged that the polarization and differentiation of epithelial cells is innately tumour suppressive, and deregulation of epithelial polarity often correlates with invasive and metastatic disease. Conversely, evidence from experimental model systems indicates that the enforced expression of core polarity proteins such as Scribble is sufficient to potently suppress cellular transformation driven by oncogenes such as RAS. Despite an indisputable link between deregulation of the polarity network and the process of oncogenic transformation, little is known about how this network functions collectively on a systems-level within human epithelial cells, nor how engagement of this network may mediate innate mechanisms of tumour suppression. Here I have undertaken a detailed systems-level analysis to identify and characterize the interface between the global epithelial polarity network and oncogenic RAS-MAPK signalling in both normal and malignant human epithelial cells. This was achieved through a genome-scale RNAi functional genomics screen carried out in human epithelial cells coexpressing both oncogenic RAS and the polarity protein Scribble. Notably, this approach identified a robust network of cell polarity, adhesion and RAS-MAPK signalling proteins that are required for suppression of oncogenic RASV12 transformation mediated by Scribble. In direct support of these findings, a parallel analysis of the transcriptome of cells expressing RASV12 and Scribble, either alone or in combination, revealed that this network of RAS suppression cell polarity proteins are targeted for transcriptional deregulation by RAS via an ERK-FRA1 dependent mechanism. Conversely, enforced Scribble expression was sufficient to restore basal expression of these polarity genes. Together these findings demonstrate for the first time bidirectional feedback loops that serve to integrate cell polarity with RAS-MAPK signalling in the context of oncogenic transformation. Additionally, a novel role was identified for a TPL2-MAPK-JNK2-JUND stress signalling pathway in iv Scribble-mediated suppression of morphological transformation driven by RAS, suggesting that Scribble controls a human cell polarity network that constitutes a novel form of balancing crosstalk between MAPK-ERK and MAPK-JNK signalling to regulate epithelial polarization and suppress the RAS oncogene. Finally, to examine whether regulation of ERK signalling was a common property of all cell polarity regulators, I designed and applied a comprehensive siRNA library corresponding to the known human cell polarity genes and assessed their role in regulation of the sustained phase of ERK signalling using a high-content bio-image analysis screen. This analysis revealed a previously unappreciated degree of complexity and specificity regarding polarized regulation of ERK signalling, and identifies the polarity network as a novel class of highly specific spatiotemporal modulators of the ERK signalling response. Through this combination of systems-level RNAi mediated approaches, I have been able to construct a detailed working map of the cross-regulation and interplay between the RAS-MAPK-ERK signalling pathway and the broad network of genes that regulate epithelial polarity. This map will now serve as a foundation for future work aimed at understanding the functional landscape of human epithelial polarity networks in normal and malignant cells. Such future studies will serve to bridge the gap in our understanding of how polarity networks and oncogenic signalling interact to promote cancer progression.
Safety of a suprachoroidal retinal prosthesis

LEUNG, RONALD ( 2014)

Light is transformed into neural signals by the retina. Certain conditions, such as retinitis pigmentosa, can cause extensive degeneration of the outer retinal layers, resulting in profound vision impairment. Prosthetic devices have the potential to restore visual percepts in these patients by electrically stimulating the remaining retinal neurons. One such device, the suprachoroidal retinal prosthesis, is placed between the vascular choroid and sclera. It is currently under development and is the focus of this thesis. Safety is an important aspect of medical device design. This thesis focuses on several key aspects of suprachoroidal retinal prosthesis safety. Retinal prostheses must be designed to allow for the ability to safely remove and replace the device in the case of infection, device malfunction, or a device upgrade. This thesis explores the safety and feasibility of explanting or replacing prototype suprachoroidal electrode arrays using clinical and laboratory analysis techniques in a feline model. The results indicate that suprachoroidal electrode arrays can be safely removed or replaced with minimal damage to the retina and surrounding tissues. Furthermore, the device replacement procedures were not detrimental to the retinal response to electrical stimulation. However, careful wound closure was required to minimise post-operative complications. Another key requirement of electrically active neural prostheses is that they should not cause damage that could adversely affect the efficacy of the device. The safe stimulation levels of the platinum macroelectrodes used in the prototype suprachoroidal retinal prosthesis is unknown. In addition to the above, this thesis aims to determine the safe stimulation levels of platinum macroelectrodes using electrochemical methods in vitro and in vivo. Furthermore, methods to safely increase the safe stimulation limit by altering the stimulation waveform or by using nitrogen-doped ultra-nanocrystalline diamond as an electrode material are explored. The results indicate that electrochemically safe stimulation limits were lower than stimulation levels likely to cause histologically observable damage and that altering the stimulus waveform has the potential to increase electrochemically safe stimulation limits. Also, nitrogen-doped ultra-nanocrystalline diamond electrodes have a higher safe stimulation limit than platinum and showed no signs of degradation when stimulated in vitro. The results of this thesis have helped to ensure the safety of patients implanted with prototype suprachoroidal retinal prosthesis (ClinicalTrials.gov, NCT01603576). Furthermore, this thesis has important implications on safe suprachoroidal retinal prosthesis design and makes significant contributions towards our understanding of stimulation safety.
Diagnosing Darwin: Charles Darwin's 'mystery illness'

HAYMAN, JOHN ( 2014)

The naturalist Charles Darwin suffered from severe chronic illness for much of his adult life. He consulted many doctors and suffered from their different treatments with little effect. The nature of his illness was never clear to any of these treating doctors, although they proposed several diverse diagnoses, diagnoses that are not recognised today. Since Darwin’s death his illness has been the subject of much speculation and, in all, more than 40 different diagnoses have been proposed. Many of these suggested diagnoses may be discarded as Darwin had clear evidence of his disorder when still a University student; before he sailed on HMS Beagle and long before he had thoughts on Evolution. His symptoms have been described in great detail in his correspondence, in his autobiography and other writings, in the diary of his wife Emma and in the writings of others. In this thesis, it is proposed that these symptoms are consistent with a mitochondrial disorder, a disorder that he presumably inherited from his mother and shared with his maternal uncle, Tom and their youngest sibling, Mary Ann. Charles Darwin’s own siblings would have also inherited the same mutation and some also developed symptoms in later life. Darwin’s symptoms most closely fit the diagnosis of the Cyclic Vomiting Syndrome with an overlap of symptoms that occur with other poorly defined illnesses– the Chronic Fatigue Syndrome, Abdominal Migraine, the Irritable Bowel Syndrome and Fibromyalgia. He also had symptoms of one other condition, the MELAS syndrome. All these conditions have been shown, at least in some patients, to be associated with mitochondrial dysfunction. As Darwin’s illness appears to have been maternally inherited it is a reasonable assumption that this dysfunction was due to an inherited mitochondrial DNA abnormality. Contrary to previous commentaries, Darwin’s illness was not psychogenic due to suppressed hostility to his father, it was not due to any primary psychological cause, it was not due to arsenic poisoning or systemic lactose intolerance, and it was not due to some parasitic infection acquired while exploring in South America. It is hypothesised that Darwin’s illness was due to a mutation in his mitochondrial genome and further hypothesised that this mutation affected the function of neuroendocrine cells in his gut and brain as well as other cells that have high--‐energy requirements.
Generation of novel chimeric antigen receptors to enhance the specificity and activity of T cells for the adoptive immunotherapy for cancer

DUONG, CONNIE ( 2014)

Adoptive immunotherapy is a promising treatment for cancer, with response rates of up to 70% in metastatic melanoma. To broaden this approach, T cells have been genetically modified to express chimeric antigen receptors (CARs) to endow T cells with anti-tumour activity capable of recognising a range of different cancer types. This approach has shown encouraging results in recent clinical trials for the treatment of haematological malignancies, however it has shown only moderate activity against solid cancers. To date, only a small number of molecules involved in T cell signaling have been incorporated into CARs, resulting in their suboptimal activity. Therefore improvements in CARs are needed in order to realise the full potential of adoptively transferred T cells. We proposed that using multiple or alternate signaling domains could enhance CAR-mediated T cell function. In this thesis, we describe the use of a DNA library of signaling molecules to investigate novel combinations of signaling molecules that could mediate enhanced CAR activity in the Jurkat T cell line and primary human T cells. A novel single-chain variable receptor was discovered comprising DAP10, CD3ζ and CD27 signaling domains that was able to trigger enhanced T cell activity in vitro and in an adoptive transfer mouse model. Clinical trials utilising CAR modified T cells have in some cases resulted in resulted in severe autoimmunity due to T cell recognition of tumour-associated antigens expressed on normal tissues. It is anticipated that as the application and efficacy of adoptive immunotherapy increases, toxicity against normal tissue will become increasingly common. To address this, we proposed that a T cell will respond less against normal tissue if endowed with a tumour-associated antigen-specific activating CAR co-localised with a chimeric inhibitory receptor (CIR) that is capable of turning off the T cell following engagement of antigen on normal tissue. We generated several novel chimeric inhibitory receptors and demonstrated expression of both CAR and CIR in T cells, which were then characterised for function against tumour-associated and normal tissue antigen expressing cell lines. In conclusion, the combination of these novel chimeric receptors may lead to a more efficacious but safer therapy for cancer.
Genome-wide changes conferred by trichostatin A, glucose and metformin in human vascular endothelial cells

RAFEHI, HALOOM ( 2014)

Endothelial dysfunction is a precursor of cardiovascular disease. It is characterised by the impaired production of the vasodilator nitric oxide and the development of a pro-thrombotic and pro-inflammatory endothelial state. Hyperglycaemia is a condition of elevated blood glucose levels as seen in diabetes and is a contributing factor to the development of endothelial dysfunction. The diabetes drug metformin reverses damage to the vascular endothelium, whereas trichostatin A (TSA), a prototypical HDAC inhibitor, has anti-inflammatory properties. While glucose, and TSA have been the focus of extensive research for decades, the genome-wide effects of these compounds in endothelial cells have not been adequately characterised. Recent advances in high throughput sequencing (HTS) technologies have fast-tracked advancements in the genome-wide study of transcriptional regulation. The ability to look beyond single genes and loci is now readily accessible and affordable. This study utilised HTS to study genome-wide gene changes induced by hyperglycaemia and TSA in human endothelial cells. This genome-wide approach led to the identification of novel mechanisms of action by both compounds, while qRT-PCR was used to show that metformin attenuates changes in expression in hyperglycaemic endothelial cells. HDAC inhibitors such as TSA, a diverse group of clinically relevant compounds, are thought to induce global histone hyperacetylation and therefore increased gene expression. In endothelial cells stimulated with TSA, the integration of gene expression changes (RNA-seq) with global histone acetylation data (ChIP-seq) identified that HDAC inhibition induces equal amounts of increases and decreases in gene expression and genome-wide histone acetylation. Histone deacetylation partially accounted for the anti-inflammatory effects of TSA. Deacetylation was dependent on the loss of histone acetyltransferases (HAT) binding at gene promoters. Indeed, the inhibition of structurally related HATs p300 and CBP prevented TSA-dependent reductions in expression at deacetylated genes such as the inflammatory cytokine IL6. A 50% reduction in CBP protein binding was also observed at the IL6 promoter. Transcription factors YY1, STAT2 and IRF3 were also associated with deacetylated promoters. The use of HTS also identified novel pathways in hyperglycaemic endothelial cells that may be involved in the development of endothelial dysfunction. Hyperglycaemic endothelial cells expressed interferon-response pathway genes such as MX1 and IFI27. Transcription factor analysis implicates the activation of STAT1 and IRF1. Co-treatment of hyperglycaemic cells with metformin prevented glucose-dependent changes in gene expression, including interferon-response genes. Indeed, the effects of metformin in endothelial cells were dependent on glucose levels. In normoglycaemic cells, metformin subtly regulated changes in gene expression. In contrast, metformin was strongly associated with the reversal of gene expression changes induced by hyperglycaemia. In this respect, metformin and TSA differ substantially in their anti-inflammatory properties. Metformin did not suppress pro-inflammatory gene expression in unstimulated endothelial cells, whereas TSA did. Interestingly, TSA and metformin were associated with the suppression of genes regulated by pro-inflammatory STAT and IRF family transcription factors. In conclusion, the use of HTS facilitated the identification of novel responses in endothelial cells that were previously under-appreciated. This has provided new insights into the mechanism of action of HDAC inhibitors. It also suggests a potential mechanism for the development of endothelial dysfunction associated with hyperglycaemia and its reversal by metformin. The use of HTS has clear applications in attaining a deeper understanding of disease mechanisms as well as drug action, which, coupled together, have the potential to improve rational drug design.
Proteomic profiling of altered copper homeostasis

Du, Tai ( 2014)

Altered copper (Cu) metabolism is often the hallmark of some diseases of neurodegeneration and cancers. The primary aim of this thesis was to identify proteins that may be associated with Cu metabolism using a high-throughput antibody microarray. We examined the global changes in protein expression in human fibroblast cell lines resulting from altered Cu homeostasis. We identified a number of novel proteins that showed altered expression levels in response to changes in cellular Cu that have not been previously reported. In the process of analysing and validating the array data, we have applied techniques in signal processing, validation and statistical analysis not previously reported in the analysis of antibody arrays. We analysed a panel of 11 proteins identified as being highly ranked as differentially expressed by the antibody array and found that 8 of the 11 proteins could be confirmed by an alternative detection method (Western blotting). Moreover, the richness of the information of the Ab array data supported evidence for cellular processes of cell DNA replication and repair processes that were underpinning the changes observed in protein expression. We identified Ku80 as a protein of interest in relation to altered Cu homeostasis due to its previously known functions and confirmed observations that its metabolism is associated with Cu homeostasis in vitro. We gained insight into the mechanism of Ku80 metabolism in response to altered cellular Cu levels and observed that changes to Ku80 occurred mainly in the cytosol of the cell. We showed that Ku80 expression was inducible by Cu as well as other oxidative stress inducers and that this induction of Ku80 by Cu in these cells showed protective effects against oxidative cell damage in terms of oxidised DNA. We hypothesised that cytosolic interactions with intermediary proteins such as p53 and ATOX1 were involved in the Ku80 response to Cu because of compensatory metabolism to accommodate increased Cu levels and potential protein-protein binding interactions. This thesis demonstrated the progression from using of a multiplexed Ab microarray to identify candidate proteins of interest, to validating of the proteins that were changed, and then exploring a protein of interest (Ku80) in vitro to understand the molecular mechanisms behind the changes observed due to altered cellular Cu levels. We demonstrated the reliability and specificity of the techniques in determining protein expression changes in the cell. Ku80 and some of these proteins identified may have potential for further studies to determine their viability as biomarkers of the Cu associated diseases.
Image guided and adaptive radiotherapy for muscle invasive bladder cancer

Foroudi, Farshad ( 2014)

Introduction: Bladder cancer is one of the ten most frequent cancers in Australia. It is also the only common cancer for which survival has decreased over the last twenty years. The two curative treatment options for muscle invasive bladder cancer are radical surgery requiring removal of the bladder, or radical radiotherapy (alone or in combination with chemotherapy). Radical radiotherapy allows many patients to keep their natural bladder. As a dynamic soft tissue organ, the bladder size, shape and position vary with bladder and rectal filling, requiring traditional radiotherapy fields to have large margins around the target. With conventional radiation treatment such large margins increase the risk of normal tissue side effects and yet there still remains the risk of missing the bladder cancer on some treatment fractions. Methods: I have developed an innovative technique with a new device, cone beam computed tomography to match radiation fields and volume on a daily basis to the bladder position and size. This technique reduced the margin of the radiation fields required around the bladder. I have led a number of training programs and their evaluation to teach radiation therapists to conduct such treatments. In addition to the development work, I have conducted a prospective pilot study in 27 participants, of this adaptive radiotherapy technique. Following further refinement I led the multi-centre clinical trial that established the technique as standard of care in a number of institutions. Results: I found that cone beam computer tomography was of sufficient quality to be used to match radiation fields to the bladder on a daily basis. I determined that such daily matching prior to treatment was better than an ‘offline” process where an average radiation treatment plan was created after several radiation treatments. I found in our pilot study that adaptive radiation treatment decreased surrounding normal tissue irradiation. Separate studies showed that both workshop and e-Learning based radiation therapist training increased confidence and decreased variation from the gold standard (radiation oncologist results). I conducted a number of studies examining appropriate margins for bladder cancer radiation treatment with different imaging techniques. Through a multi-centre feasibility study of 54 participants, I introduced the image guided adaptive radiotherapy technique into a number of Australia centres, and demonstrated that, while technically possible, the margin around the bladder in this protocol was too small. Conclusions: Image guided and adaptive radiotherapy is possible in many radiation therapy departments with likely benefits for patients in terms of cancer control and reduced normal tissue side effects. Through my work the technique has been established as standard of care in a number of Australian and New Zealand centres. However such radiotherapy techniques can continue to be optimised. Future phase III studies are required to conclusively prove their benefit.
Cellular mechanisms underlying the cognition-enhancing properties of metal-complexes

Bica, Laura ( 2014)

Alzheimer’s disease (AD) is caused by a myriad of complex pathological factors that contribute to Amyloid-beta (Aβ) accumulation and oxidative stress as well as synaptic damage and dysfunction that result in cognitive decline. Metal dyshomeostasis is a key factor in these processes and is therefore an attractive therapeutic target. Clioquinol (CQ) was initially investigated as an AD therapeutic due to its copper- and zinc-chelating properties. It inhibited Aβ accumulation and enhanced cognitive performance in an AD mouse model as well as humans with AD in a clinical trial; however, issues with purification led to development of an alternative candidate, PBT2. PBT2 has been tested in AD mouse models and trialled in humans with AD, it improved cognition and reduced cerebrospinal fluid (CSF) Aβ levels. Bis(thiosemicarbazonato) metal-complexes (mII(btsc)s), previously used in applications such as cancer imaging, have been examined as potential treatments for AD as well as other neurodegenerative disorders such as Parkinson’s disease and Amyotrophic Lateral Sclerosis. Their structure contains a Cu or Zn molecule and is able to cross the cell membrane into the cytosol where metals are released, making them bioavailable. Like CQ and PBT2, the mII(btsc) CuII(gtsm) enhanced cognition while lowering CSF Aβ in a mouse model of AD. CQ and PBT2 don’t introduce more metals into the body or cell but may help remove excess metals from outside cells and redistribute them into a metal-depleted intracellular environment. However, mII(btsc)s allow a more controlled delivery of bioavailable metals that has proven to have therapeutic effects in models of AD as well as other neurodegenerative disorders. Despite these advances, little is known of the cellular metal delivery and neurotherapeutic action of these metal-binding compounds. This thesis investigated the potential mechanisms of action of metal delivery agents PBT2 and CuII(gtsm), potential therapeutic compounds for AD. While CQ is not currently being pursued as an AD therapeutic, its effects were also examined. PBT2 enhanced dendritic spine density of Tg2576 mice compared to sham treated controls and had no effect on wild-type controls. Several biomarkers of synaptic plasticity were examined and found to be increased with PBT2 treatment. In vitro, neurite elongation was also increased by exposure to PBT2 with a significantly stronger effect with the addition of equimolar Cu or Zn. When a chelator with high affinity for Cu and Zn was present, the effect of PBT2 on neurite elongation was blocked, indicating that bioavailable Cu and Zn is necessary for this effect. CuII(gtsm) also enhanced neurite elongation in vitro and similar compounds with different Cu-binding affinity or with a Zn molecule instead of Cu required 10-fold higher concentrations to elicit a similar neurogenerative effect. The neurogenerative effect of CuII(gtsm) was examined further and found to require JNK phosphorylation and was associated with inhibition of cellular phosphatase activity, in particular, calcineurin. Furthermore, specific inhibition of calcineurin with FK506 enhanced neurite elongation. While there were subtle differences in the effects of the metal delivery agents examined, this thesis supports the use of metal delivery agents as a potential AD therapeutic and demonstrated that the effects of these agents involves neurogenerative actions requiring the activity of phosphatases such as calcineurin.

Pathology - Theses

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