Empirical thesis.

Chapter 1. Introduction  – Chapter 2. Literature review  – Chapter 3. Specific aims and experimental design  – Chapter 4. Inhibition of glycosphingolipid synthesis ameliorates atherosclerosis and arterial stiffness in Apo E(-/-) mice and rabbits fed a high fat and cholesterol diet  – Chapter 5. Prevention of cardiac hypertrophy by the use of a glycosphingolipid synthesis inhibitor in ApoE(-/-) mice  – Chapter 6. Improved Intervention of atherosclerosis and cardiac hypertrophy through Biodegradable polymer-encapsulated delivery of glycosphingolipid inhibitor  – Chapter 7. Molecular imaging of inflammation in the ApoE (-/-) mouse model of atherosclerosis with IodoDPA  – Chapter 8. General discussion  – Appendices.

Background. Vascular dysfunction of conduit arteries is manifest as atherosclerosis and vascular stiffness, which are significant contributors to cardiovascular disease. Vascular dysfunction is characterized by thickening and stiffening of the vessel walls, and is accelerated by a sedentary lifestyle and advancing age. Despite the heavy public health burden of vascular dysfunction, no cure has been found. A recent study by Chatterjee et al., using a dietary model of atherosclerosis in rabbits, showed that atherogenesis was inhibited by D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), a glycosphingolipid synthesis inhibitor. The work presented in this thesis focusses on determining the molecular mechanism by which D-PDMP affects vascular and cardiac function through modification of wall properties, by inhibition of atherosclerosis and associated pathological cardiac hypertrophy (LVH), in murine models of atherosclerosis. Novel imaging methods of assessing atherosclerosis are explored, as well as drug delivery systems of D-PDMP with the aim of improving therapeutic efficacy.

Methods. Experiments were performed in ApoE (-/-) mice fed a high cholesterol and high fat diet, with and without D-PDMP, from 12 to 36 weeks, during the prevention phase and treated from 20 to 36 weeks for the intervention phase. Plaque area, intima-media wall thickness, cardiac hypertrophy and contractility were assessed using high-resolution ultrasound. Blood pressure (tail cuff) and aortic pulse wave velocity (PWV, measure of arterial stiffness) were measured. Tissues were harvested for further molecular and histopathological studies. In separate cohorts of mice, molecular imaging of inflammation was conducted using the radiotracer Iodo-DPA-713 and SPECT. Drug delivery of D-PDMP was explored using biodegradable polymer encapsulation.

Results. Apo E(-/-) mice fed a hyperlipidemic diet showed marked accumulation of atherosclerotic plaque, increased PWV independent of blood pressure, decreased cardiac contractility, and increased LVH and fibrosis, compared to control and treated mice These effects were largely attenuated during the prevention phase, and reversed during the intervention phase of D-PDMP. Measurement of glycolipid glycotransferases showed that a hyperlipidemic diet…

Thesis by publication.

Bibliography: pages 210-230.

Introduction  – Chapter 1. Literature review  – Chapter 2. Materials and methods  – Chapter 3. Microbial biofilms associated with healthcare infections  – Chapter 4. Microbial biofilms and their role in implantable device-associated infections  – Chapter 5. Microbial biofilms in diabetic foot infections  – Chapter 6. Discussion and conclusions  – References  – Appendix.

Background - A better understanding of the microbial communities in medical environments is crucial for improving human health, given the increasing problem of biofilm-related infections. With the rapidly expanding molecular methods opening new horizons in the study of the presence of microbial biofilm from samples collected within medical settings. The aims of this project were therefore to better understand the microbiome and the role of biofilm in (i) providing a protected source of pathogens that can cause healthcare-associated infections (HAIs), (ii) causing granulomatous reactions and its possible role in potentiating cancer, and (iii) chronic wound infections.

Methods - We employed advanced molecular methods, including next-generation DNA sequencing, fluorescence in situ hybridisation (FISH) and real-time quantitative polymerase chain reaction (qPCR), along with confocal laser scanning and scanning electron microscopy, to investigate the project aims.

Results(i) The majority of ICU surfaces sampled from three hospitals were contaminated with polymicrobial biofilms, which contained MDR strains, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE) and extended spectrum beta-lactamase (ESBL) Sphingomonas paucimobilis. Similarly, a high number of patient-ready endoscopes were contaminated with biofilms composed of potentially pathogenic Gram-negative bacteria, including Shigella dysenteriae, Escherichia coli and Klebsiella pneumonia. (ii) Soft-tissue fillers support the growth of Staphylococcus epidermidis biofilm in vitro. While soft-tissue filler clinical samples all demonstrated multi-species biofilm with a predominance of Pseudomonas, Staphylococcus and Propionibacterium. In breast implant-associated anaplastic large-cell lymphoma (BIA-ALCL) clinical samples, a high bacterial load, present as a biofilm was identified. Moreover, a distinct microbiome in BIA-ALCL specimens was identified, with a significantly greater proportion of Ralstonia spp. compared to non-tumour contracted capsules. (iii) Diabetic foot ulcer (DFU) clinical samples all contained biofilm, with multi-species communities comprising of both strict anaerobes and aerobic species. In addition, chronic DFUs were found to be associated with a highly polymicrobial microbiome with greater species richness and diversity. In the treatment of chronic non-healing DFUs complicated by biofilm, cadexomer iodine was found to significantly reduce the microbial load. However, short exposure times to topical antimicrobial solutions (commonly utilised by clinicians) were found to…

Theoretical thesis.

Bibliography: pages 154-163.

Chapter 1. Introduction and literature review  – Chapter 2. Non-contrast MR angiography techniques used for renal artery imaging  – Chapter 3. Non-contrast respiratory-gated MR angiography of renal artery in hypertensive patients using true fast imaging with steady-state precession technique compared with contrast-enhanced MR angiography  – Chapter 4. Hemodynamic computational fluid dynamic techniques and their application in renal artery  – Chapter 5. Hemodynamic analysis of renal artery stenosis using computational fluid dynamics technology based on non-contrast steady-state free precession MR angiography  – Chapter 6. Analysis of various virtual angioplasty operation of renal artery stenosis in hypertension using MR angiography-based computational fluid dynamics technology  – Chapter 7. Conclusions and future works.

Renovascular hypertension is caused by renal artery stenosis (RAS) . The relationship . The relationship among RAS, hypertension, and renal function varies from patient to patient is difficult to assess, but the severity of association increases risks for patient. An accurate, reliable and non-invasive method to evaluate severity and hemodynamics of RAS is mandatory.

MRA is a well-known method in the angiographic illustration of RAS in current clinical application. However, there exists limitation in its application, especially lack of capability in blood pressure measurement. On the other hand, the technology of the patient-specific computational fluid dynamics (CFD) is another novel technology to to enable quantitatively estimate vascular vascular hemodynamics. To date, there is no report no report to determine the RAS vascular hemodynamics using CFD.

The objective of this study is to establish a non-invasive methodology for morphological and hemodynamic assessment of RAS. The hypothesis is that there exists an internal relationship between hemodynamic change and morphological transformation of renal arteries. Thus, evaluating the hemodynamic change of different grade stenotic renal arteries can reflect on the morphologic status. Non-contrast MRA was used in this thesis to assess the morphological status of renal arteries and CFD was used to calculate the hemodynamic parameters of various stenoses.

Newly developed non-contrast MRA technique, named steady state free precession (SSFP) state free precession was investigated and assessed as an accurate method in visualization of renal artery. The results of CFD simulation demonstrated its potential ability for understanding the relationship between hemodynamics and morphology of renal artery stenosis. The application of CFD technology on modified renal artery stenosis improved prediction of the hemodynamics of renal artery after simulated percutaneous angioplasty and stenting.

It was found that CFD simulation can provide useful information for patient stratification and strategy for further treatment decision, and it may also be able to support be able to support the clinical…

Thesis by publication.

Bibliography: pages 211-282.

1. Introduction  – 2. Methods  – 3. Transient CCNF overexpression  – 4. Constitutive overexpression of CCNF  – 5. Inducible overexpression of CCNF  – 6. Discussion  – Appendix  – References.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterisedby the death of upper and lower motor neurons. Approximately 10% of ALS patients have a known family history of the disease and genetic analysis of ALS-affected families has identified causal mutations in multiple genes. The identification of these mutations has provided the opportunity to develop models of ALS, essential tools for studies investigating the biology of the disease and for preclinical testing of potential therapeutics. While many of the mechanisms underlying ALS have been elucidated, these mechanisms remain poorly understood. Further studies using both established and novel models of ALS are required to enhance the current understanding of these mechanisms. A greater understanding of disease biology will lead to the identification of potential therapeutic targets.

Mutations in CCNF linked to both familial and sporadic ALS were recently reported. Patients who carry these CCNF mutations develop TDP-43 positive protein aggregates within the their motor neurons - pathology considered to be the hallmark of the disease in over 95% of cases. Therefore, the identification of ALS-linked mutations in CCNF provides an opportunity to develop novel models that reflect the most common pathology seen in ALS patients. This project aimed to develop these novel models in the zebrafish.

Zebrafish have emerged as useful tools to identify and investigate mechanisms of human disease. As vertebrates, they share significant genetic, anatomical and physiological similarities with humans, while their speed of development, their high fertility and the relative ease of manipulating their genome contribute to efficient development of disease models. This project investigated the suitability of zebrafish to model ALS-linked mutations in CCNF by characterising the zebrafish CCNF homologue and its encoded protein, cyclin F. Comparison of zebrafish and human cyclin F identified significant structural similarities between the proteins, suggesting that they perform similar functions in the two species. Further, cyclin F was found to be persistently expressed in the zebrafish central nervous system throughout development. This suggests that models in which cyclin F is artificially expressed in the central nervous system will have physiological relevance. These findings supported the hypothesis that zebrafish are a suitable species in which to model cellular changes associated with ALS-linked mutant CCNF.

Based on these findings, generation of the CCNF-based zebrafish commenced. A variety of model paradigms were explored to identify strategies that produced models suitable for investigative studies. Several strategies failed to generate viable models, including persistent embryonic…

Thesis by publication.

Bibliography: pages 252-308.

Introduction. So here the tale begins!  – Chapter 1. Literature review  – Chapter 2. Materials and methods  – Chapter 3. Transmission of bacteria from biofilms on dry surfaces  – Chapter 4. Is combination therapying the way forward to biofilm eradication?  – Chapter 6. Impact of positive pressure use combined with disinfectants on eradicating bacterial biofilms in healthcare settings  – Chapter 7. Use of hospital grade chlorine to eradicate bacterial biofilms from healthcare settings  – Chapter 8. General discussion and conclusion  – References  – Appendix.

It is estimated that more than 99% of bacteria form biofilm where they survive safely in the slime taking refuge from antimicrobials, host immune responses and environmental stress conditions.

Scientific literature demonstrates that more than 85% of chronic infections are currently attributed to biofilm development in human tissues (chronic otitis media, non-healing skin ulcers, persistent perionditis, chronic osteoarthritis) on prosthetic medical devices (contact lenses, implants etc.) and on medical equipment (endoscopes, urinary catheters etc.)

Transmission and control of bacterial biofilm to reduce these infections has been quite a challenge for clinicians and scientists worldwide, especially owing to the tough resistive life style of bacteria inside biofilms. This study investigated if bacteria residing in environmental biofilms are a potential source for healthcare associated infections by determining:1) The transferral rate of bacteria from dry-surface biofilms via gloved and un-glovedhands ; 2) Determining the killing efficacy of commonly used disinfectants and sterilisation protocols against dry-surface and traditional hydrated biofilms ; 3) Investigating the killing efficacy of combining a physical stress (topical negative pressure and positive pressure ≤10 atmospheres) with a chemical stress (disinfectants/antiseptics) on dry and hydrated biofilms.

To study, in-vitro bacterial biofilm was cultured in PC2 laboratory on coupons using Communicable Disease Control and Prevention biofilm reactor and then tested for transmission and various treatments.

We found that dry-surface biofilm bacteria are highly transmissible. Although gloved hands transferred up to six folds fewer bacteria than bare hands, total bacterial numbers transferred was more than the dose required to cause infection.

Heat treatment as per the prevailing infection control guidelines, successfully eradicated bacteria in hydrated biofilm, but failed to eradicate dry-surface biofilm when autoclaved at 121°C for up to 30 minutes. Similarly, commonly used disinfectants failed to kill dry surfacebiofilm. However, enhanced killing of biofilm bacteria was achieved when compression pressure treatment was combined with biocides.

The current thesis establishes high transmissibility of bacteria from dry surface biofilm affirming it to be a potential source of hospital acquired infections whilst demonstrating that current…

Empirical thesis.

Bibliography: pages 286-315.

Introduction  – Chapter One. Review of literature  – Chapter Two. Material and methods  – Chapter Three. Biofilm : transmission and inactivation by heat  – Chapter Four. Developing novel chemistries for removing environmental surface biofilms to reduce hospital acquired infections  – Chapter Five. Development of multispecies dry surface biofilm model  – Chapter Six. Comparative proteomic profile : new insight of Staphylococcus aureus dry surface biofilm  – Chapter Seven. General discussion and conclusion  – References  – Appendix.

Environmental surface cleaning and disinfection is crucial for preventing HAI and maintaining patient safety. However, biofilms incorporating multiple bacterial species form on dry hospital surfaces. These biofilms are tolerant to biocides making their eradication difficult. Persistence of pathogens on surfaces increases the risk of transmission and development of HAI.

Aims:1) To determine if bacteria can be transmitted from dry surface biofilm (DSB) to healthcare workers' hands and through cotton sheets. 2) To develop and test new removal chemistries against dry surface biofilm utilising laboratory models. 3) To develop a mixed species biofilm model analogous to the hospital surface DSB to determine its composition, the effect of multiple species on biofilm's susceptibility to detergents and disinfectants and analyse proteomics and transcriptomics technique. 4) To investigate protein regulatory changes in DSB compared with traditional hydrated biofilm and confirmation with RT PCR.

This study revealed that: 1) DSB is highly transmissible. The total bacterial number transferred are sufficient to cause disease. 2) Soil inactivates the commonly used disinfectants. Peracetic acid reduces 64% of the biofilm mass and >6 Log10 viability in single species DSB, 48% of the biofilm mass in mixed DSB in the presence of soil. 3) This study developed a mixed species DSB model with reproducible cell number within and between repeated experiments. This mixed biofilm model was employed to assess the effectiveness of disinfectants. 4) These findings clearly imply that S. aureus biofilms represent a unique growth condition in terms of overall numbers of differentially expressed distinct proteins and genes.

This study establishes the transmissibility of bacteria from DSB affirming it to be a potential source of HAI and determined that currently surface disinfectants and methods are insufficient to completely eradicate DSB. This study also established different changes in protein and genetic expression in biofilm, this might act as target group to invent new effective disinfectant for complete eradication of DSB.

1 online resource (xviii, 318 pages) illustrations (some colour)

Empirical thesis.

Bibliography: pages 127-140.

Chapter 1. Literature review  – Chapter 2. Temporal trends on endoscope reprocessing  – Chapter 3. Material and methods  – Chapter 4. Assessing manual cleaning of flexible endoscope by measuring soil and microbial contamination levels  – Chapter 5. Evaluation of endoscope reprocessing : high level disinfection  – Chapter 6. Assessing cleaning and bacterial load on endoscopy unit surfaces  – Chapter 7. Endoscope channel contamination evolution  – Chapter 8. A preliminary study on the contribution od endoscope channel damage to biofilm formation  – Chapter 9. General discussion and conclusion  – References.

The difficulties in reprocessing gastrointestinal endoscopes and risk of patient to patient transmission of infectious organisms are recognized challenges in medicine. This thesis investigated gastrointestinal endoscope reprocessing both in the clinic and in the laboratory. Analysis of clinical data was performed for assessing soil level, contamination level and biofilm formation in four different settings: before and after manual cleaning, directly after endoscope reprocessing (12 to 48 hours after disinfection), following internal channels extraction for repair (clinically used endoscope from Australia and Brazil) and environmental sampling of endoscopy unit surfaces. Experimental analysis involved investigation of endoscope internal channels for surface damage and its relationship to frequency of use.

The tests used for the analysis were adenosine triphosphate (ATP) bioluminescence for presence of soil, polymerase chain reaction (PCR) for bacterial load, microbial culture for viable bacterial numbers, scanning electron microscopy (SEM) for biofilm presence and stylus profilometer for surface roughness. Statistical analysis was performed by descriptive analysis, Mann-Whitney, Wilcoxon and Spearman tests, p<0.05, using IBM SPSS Statistics version 23.0.

Before and after cleaning analysis of 99 endoscopes showed cleaning effectively reducing soil (p<0.001) and microbial contamination level (p=0.03).

After complete reprocessing, all 75 endoscopes tested showed reduced soil contamination (all samples < 50RLU, from internal and external area); however the median microbial load was 3 Log10 and 10% of samples were culture positive. Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus capitis, Roseomonas gilardii and Micrococcus luteus were isolated after endoscope reprocessing.

Environmental samples were obtained from the nursing station and procedure room. Nine of the 24 (38%) samples had ATP values > 100 RLU and were then samples for microbial load by culturing. The areas with more contamination were the procedure room´s pipe and nursing station chair´s lift. Some potential pathogenic microorganisms were isolated (i.e. Shigella spp. and E.coli).

Endoscope biopsy channel analysis showed that samples from Brazil had higher contamination levels than Australian samples (p<0.001). All channels analyzed were…

Empirical thesis.

Bibliography: pages 566-598.

Chapter 1. General introductionn  – Chapter 2. Material and methods  – Chapter 3. PDI is protective against ALS-associated mutant cyclin F expressed in neuronal cells  – Chapter 4. Characterising the protective properties of PDI against misfolded proteins in ALS  – Chapter 5. The role of redox activity in the protective function of PDI  – Chapter 6. The role of PDI and its redox activity in FUS-linked ALS  – Chapter 7. Peptides based on PDI's active site are protective in neuronal cells expressing mutant SOD1  – Chapter 8. General discussion  – References  – Appendices.

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disorder characterised by the degeneration of motor neurons. Numerous genes are linked to ALS both genetically and pathologically. Approximately 10% of ALS cases are familial, and of these cases, approximately 20% are due to mutations in the gene encoding superoxide dismutase 1 (SOD1), and approximately 10% are due to mutations in TAR DNA binding protein 43 (TDP-43) and fused in sarcoma (FUS). A major hallmark observed in ALS is the accumulation of misfolded proteins, containing either SOD1, TDP-43 and FUS, which form aggregates in the cytoplasm of degenerating motor neurons. However, the pathogenic mechanisms of disease in ALS remain poorly understood. Recent evidence suggests that dysfunction to the Endoplasmic Reticulum (ER), resulting in ER stress, is increasingly implicated in ALS pathogenesis. Protein Disulphide Isomerase (PDI) is an ER chaperone which functions as an oxidoreductase, utilising its disulphide interchange activity to oxidise, reduce and isomerase disulphide bonds. Our laboratory has previously demonstrated that PDI overexpression is protective against mutant SOD1, TDP-43 and FUS in neuronal cell cultures. Hence, here we examined that PDI overexpression is also protective against ALS mechanisms of pathogenesis triggered by mutant forms of novel ALS protein, Cyclin F. Previous studies have also identified that PDI's disulphide interchange activity is fundamental for its protective activity. Therefore, here we further investigated the properties of PDI which are important in mediating this activity. Results obtained suggest that PDI's a domain is essential for PDI's protective function, as well as its oxidase activity (capability to form disulphide bonds). These properties were verified in cell culture models expressing mutant FUS. Ultimately, there is a need for more effective therapeutics in ALS, thus, peptides mimicking PDI's a domain and oxidase activity were developed and analysed here for their protective effect in ALS cell models.

1 online resource (xxii, 607 pages) colour illustrations

Empirical thesis.

Bibliography: pages 194-221.

Chapter 1. Introduction  – Chapter 2. Interferon signalling is frequently downregulated in melanoma  – Chapter 3. TNFα effects on immune marker expression in melanoma cells  – Chapter 4. Immunotherapy resistance mechanisms in melanoma cells  – Chapter 5. Differential regulation of PD-L1 and PD-L2 expression in melanoma cells  – Chapter 6. Conclusion  – References.

Immunotherapy in the form of immune checkpoint inhibitors has significantly improved the survival of patients with advanced melanoma. However, immune checkpoint blockade is only effective in 15-40% of melanoma patients and failure to respond to immune checkpoint blockade may occur via several tumour intrinsic or extrinsic mechanisms, including overexpression of immune inhibitory ligands and receptors, alterations in antigen processing and presentation, and defects in the interferon gamma (IFNγ) signalling pathway.

In this PhD project, we first examined IFNγ responses in a large panel of immunotherapy-naïve melanoma cell lines with defined genetic drivers; BRAF-mutant, NRAS-mutant, BRAF/NRAS wild type cutaneous melanoma, and GNAQ/GNA11-mutant uveal melanomas (Chapter 2). We investigated the basal and IFNγ-induced expression of immune inhibitory ligands PD-L1 and PD-L2, antigen presenting molecules HLA-ABC and HLA-DR, and nerve growth factor receptor (NGFR) on the surface of these immunotherapy-naïve cell lines to determine the influence of melanoma driver oncogenes on IFNγ signalling. In Chapter 3, we compared tumour necrosis factor (TNFα) response in the same panel of cell lines. We found that melanoma response to IFNγ and TNFα are heterogeneous, and that IFNγ induced PD-L1, PD-L2, HLA-DR and HLA-ABC expression more potently, whereas TNFα preferentially up regulated NGFR expression. We further identified two well-recognised mechanisms of immunotherapy resistance, including the loss of β-2-microglobulin (β2M) and interferon gamma receptor 1 (IFNGR1) expression in these immunotherapy-naïve cells.

In Chapter 4, we extended our characterization of resistance mechanisms to a panel of 16 short-term melanoma cell lines (PD-1 PROG cell lines) derived from patients who progressed on anti-PD-1 or combination of anti-PD-1 and anti-CTLA-4 based immunotherapy. We assessed expression of IFNGR1, PD-L1, PD-L2, HLA-ABC, HLA-DR and B2M in these cells. We additionally analysed expression of transcription factors, melanoma pigment antigens and markers of de-differentiation, including SOX10, MLANA, AXL, MITF and NGFR. We identified several potential mechanisms of immunotherapy resistance in this panel of PD-1 PROG cell lines including loss of β2M expression, increased expression of immune inhibitory molecules, diminished response to IFNγ stimulation and melanoma de-differentiation.

Finally, in Chapter 5, we examined the regulation of PD-L1 and PD-L2 in the melanoma cell lines. Specifically, we assessed the temporal accumulation and stability of total and cell surface-specific expression of PD-L1 and…

Thesis by publication.

Cotutelle thesis with Universität Bern (the University of Bern (Switzerland)).

1. Introduction  – 2. Publication and manuscripts  – 3. Discussion  – Appendices.

Malaria is a parasitic disease which puts almost half of the world's population at risk of infection. It is caused by Plasmodium, a parasite transmitted by Anopheles mosquitoes. During its life cycle in the vertebrate host, the malaria parasite invades and multiplies in hepatocytes and in red blood cells. During invasion of sporozoites and merozoites, the host cell membrane invaginates around the parasite to form the parasitophorous vacuole membrane (PVM). The membrane is extensively remodeled by the parasite: host cell membrane proteins are removed and parasite proteins are incorporated. The PVM shields the parasite from a direct attack by cytosolic host cell immune responses and provides the host-parasite interface. Important roles of the PVM are nutrient acquisition, excretion of waste products and export of host-targeted proteins which add to parasite fitness and virulence. Finally, egress is critical for parasite release from its host cell and the first step is PVM disintegration. PVM biology and remodeling is of great interest, but despite all of these important tasks fulfilled by proteins in the PVM, the composition is not yet clear. Here, a proximity labeling technique (BioID) was used to identify PVM or PVM-interacting proteins in P. berghei blood stage parasites. Using this technique, many of the already known PVM proteins were found. Apart from known resident PVM proteins, I detected several proteins that were that were hitherto unknown as PVM proteins. Uncharacterized protein candidates were endogenously GFP-tagged and analyzed using live- and fixed-cell microscopy during the liver and the blood stage. Using this approach, I was able to identify several novel PVM proteins, and proteins that come in close proximity to the PVM. During the intraerythrocytic symptomatic blood stage, Plasmodium digests up to 70% of host hemoglobin. Heme is an essential component of a number of proteins including hemoglobin and is essential for life. It is synthesized by a pathway involving at least eight enzymatic steps, and deficiency of any of these result in porphyria. Despite the abundance of hemoglobin and heme during the blood stage, the parasite expresses all enzymes of its canonical heme pathway, which can facilitate the de novo heme synthesis. Previous research has indicated that the parasite heme pathway is non-essential during the blood stage, and that the deficiency of host heme enzymes can influence parasite growth. That Plasmodium can be greatly influenced by the host background is well established. Here we hypothesized that host porphobilinogen deaminase (PBGD) and coproporphyrinogen oxidase (CPOX), two additional heme pathway enzymes, play a role during the blood stage. To facilitate this goal, in-depth studies by using rodent malaria species were utilized to infect mice deficient in PBGD and CPOX. Blood from…

Empirical thesis.

Bibliography: pages 197-237.

1. General introduction  – 2. Materials and methods  – 3. C9ORF72 regulates actin dynamics  – 4. Dysregulation of actin dynamics in ALS  – 5. Mechanisms involved in dysregulation of actin dynamics in ALS  – 6. General discussion  – 7. References  – 8. Appendices.

Mutations in the C9ORF72 (chromosome 9 open reading frame 72) gene account for 40% of familial cases of Amyotrophic lateral sclerosis (ALS), and pathological forms of TDP-43 in motor neurons are present in almost all cases of ALS. Currently, there is no effective treatment for this disorder. Therefore, given their importance in ALS, understanding the pathological roles of C9ORF72 and TDP-43 is crucial for developing effective therapeutic strategies.

The mechanisms underlying neurodegeneration in ALS are still not fully understood. Whilst defects in cytoskeletal organisation and cytoskeletal proteins have been previously associated with ALS, the role of actin filaments and actin binding proteins in ALS has not been previously examined. The overall aim of the studies described in this thesis was to examine the regulation of actin dynamics and actin binding proteins in C9ORF72 and TDP-43 related ALS. Firstly, the normal cellular function of C9ORF72 was examined in Chapter 3. Here it was demonstrated that C9ORF72 is an actin binding protein that regulates actin dynamics. Furthermore, the uDENN domain of C9ORF72 is required for this activity via cofilin-mediated signalling mechanisms. These studies therefore provide novel insights into the normal cellular function of C9ORF72. Secondly, in Chapter 4, it was demonstrated that actin dynamics is disturbed by pathological forms of TDP-43 in cells expressing cytoplasmic TDP-43, in the TDP-43 rNLS mice model and in sporadic ALS (SALS) patient tissues. These studies therefore demonstrate that TDP-43 pathology is associated with increased actin polymerisation, possibly mediated by a direct interaction between actin and TDP-43. Moreover, actin polymerisation causes both mis-localisation of TDP-43 to the cytoplasm and stress granule formation, implying that actin polymerisation induces pathological events relevant to ALS. Finally, the regulation of actin-binding and regulatory proteins in the TDP-43 rNLS mice model and in SALS patients was examined in Chapter 5. Cofilin phosphorylation and profilin-1 expression was enhanced in SALS patients. Furthermore, increased levels of Rac1/cdc42 and Limk1 phosphorylation were also detected, thus providing mechanistic insights into these observations. Similarly, increased levels of profilin-1 and cofilin phosphorylation correlated with disease course pathologically and phenotypically in TDP-43 rNLS mice, consistent with the findings obtained in SALS patients. While actin related proteins; Arp2, Arp3 and ARPC3 were decreased at a later stage in TDP-43 rNLS mice. In conclusion, this thesis provides novel insights into the mechanisms of neurodegeneration in ALS. Importantly, it identifies dysregulation of actin dynamics and…

Thesis by publication.

Bibliography: pages 333-382.

Chapter I. Literature review  – Chapter II. Materials and methods  – Chapter III. The functional influence of breast implant outer shell morphology on bacterial attachment and growth  – Chapter IV. The influence of implant surface on biofilm formation in an in vivo porcine model  – Chapter V. Analysis of bacterial biofilm and host response in new cases of breast Implant-associated anaplastic large-cell lymphoma  – Chapter VI. Differential mitogenic response of breast implant-associated anaplastic large-cell lymphoma to gram-negative lipopolysaccharide  – Chapter VII. Differential mitogenic response of breast implant-associated anaplastic large-cell lymphoma to staphylococcal superantigens  – Chapter VIII. The development of a co-culture system of mammalian cells and biofilm composed of different bacterial species  – Chapter IX. Effect of TLR4 on LPS stimulation of BIA-ALCL tumour cells  – Chapter X. General discussion  – Appendices  – References.

Breast implant-associated anaplastic large-cell lymphoma (BIA-ALCL) is a recently diagnosed, rare non-Hodgkin T-cell lymphoma in tissue around a breast implant. Since 2000, its detection and incidence has risen worldwide due to the increase use of breast implants in breast surgery. Although the aetiopathogenesis is unclear, it is postulated that the cancer results from chronic bacterial antigen stimulation and sustained T-cell proliferation that potentially leads to malignant transformation. This is in conjunction with implant properties, implant exposure time and host predisposition or genetic factors. The experiments described in this thesis explore the influence of implant surface texture, bacterial load and host response in patient specimens, and initiating and potentiating factors to malignancy.

The majority of BIA-ALCL cases have occurred in patients with textured implants, which have been shown to support a higher bacterial load. The work described in Chapter III of this thesis describes the development of an in vitro bacterial attachment assay to further characterise the surface texture of implants and their capacity to support bacterial growth in vitro. We describe a significant relationship between the measurement of available surface area, surface roughness and potentiation of bacterial growth for both Gram-positive and Gram-negative bacteria. In Chapter IV, we examine the influence of implant texture in vivo using a well-established porcine model. We describe the association between textured implant surfaces with bacterial attachment, biofilm formation, development of capsular contracture and host response following artificial bacterial contamination of breast implants in pigs.

The role of bacteria in BIA-ALCL has recently been supported by the discovery of high levels of bacterial contamination within BIA-ALCL specimens. In Chapter V, we compare the bacterial load and host response in fresh implants and capsules from new cases of BIA-ALCL to non-tumour specimens.

In Chapter VI, we utilise…

Thesis by publication.

Bibliography: pages 222-238.

1. Introduction  – 2. Literature review  – 3. Optimisation of mechanical stretch of endothelial cells for protein and RNA quantification  – 4. Cyclic stretch as a novel modulator of APP and associated inflammatory markers in human cerebral endothelial cells  – 5. Signalling pathways that mediate cerebral endothelial responses to cyclic stretch  – 6. Effect of endothelial cell passaging and cyclic stretch on APP expression, amyloid secretion, and NO signalling  – 7. Effect of cyclic stretch in cerebral endothelial cells pre-exposed to Aβ  – 8. Efflect of cyclic stretch and glycosphingolipid inhibition on cerebral endothelial cells  – 9. A preliminary study of the effect of a high salt diet on APP processing, eNOS signalling and aortic stiffness  – 10. Conclusions, limitations and future directions  – Appendices.

Alzheimer's disease (AD) is characterised by amyloid-β (Aβ) plaques arising from amyloid precursor protein (APP) processed by β secretase-1 (BACE-1). Increasing evidence suggests a role of vascular factors in AD, namely hypertension, elevated pulse pressure and arterial stiffness, all associated with increased vascular pulsatility imposing mechanical stretch on the endothelium. This thesis addresses the role of cyclic stretch on human cerebral microvascular endothelial cells (HCMECs) in expression and processing of APP. It also investigates effect of high salt diet on APP processing in rat brains, given associations of high salt diet and cognitive impairment. In vitro studies involved cultured HCMECs subjected to 0%, 5%, 10% or 15% stretch (18 hours, 1 Hz) and analysis of protein and RNA expression, nitric oxide and Aβ levels. In vivo study included treatment of rats with high (8% NaCl, HS) or a low (0.26% NaCl, control) for 10-13 weeks and examination of brain tissue. Established for the first time was that APP expression and Aβ secretion are altered in response to HCMECs stretch, and that this response is differentially mediated in early and late passage HCMECs. In late passage HCMECs, APP and BACE-1 expression increased 2-3-fold with 10 and 15% stretch compared to 0%, with proportional increases in Aβ42/Aβ40 with % stretch (R2=0.21). In early passage HCMECs stretched at 15%, APP expression, BACE-1, Aβ42 levels were decreased 2-3-fold compared to late passage HCMECs. Glycosphingolipid inhibition prior to cyclic stretching at 15% increased APP expression and Aβ42 secretion 1-fold. In vivo findings provide preliminary evidence of altered APP processing in HS rats compared to controls parallel with increases in markers of arterial stiffness. Overall results suggest a role of arterial stiffness and vascular pulsatility strengthening the evidence of vascular contributions to AD. Future studies identifying associated molecular mechanisms will provide novel therapeutic targets for AD.

1 online resource (xx, 238 pages : illustrations)

Empirical thesis.

Bibliography: pages 140-150.

Chapter 1. Introduction  – Chapter 2. Protocol development and optimization  – Chapter 3. A versatile upconversion surface evaluation platform for bio-nano surface selection for the nervous system  – Chapter 4. Evaluation of the effect of shape upon endocytosis of transferrin-coated UCNPs, and their ability to cross the blood-brain barrier  – Chapter 5. The effect of modifying the aspect ratio of PEGylated UCNPs upon their cellular uptake  – Chapter 6. Summary and future scope.

Brain diseases including Alzheimer’s disease, Huntington’s disease and amyotrophic lateral sclerosis (ALS) are fatal diseases without effective treatments. In past decades, some potentially effective therapeutics have been developed and demonstrated promising effects for brain diseases in pre-clinical cell culture and animal model evaluation. However, most of these developed drugs cannot reach the brain for therapy due to their inability to cross the blood-brain barrier (BBB). To overcome the BBB, nanotechnology is emerging as a promising approach to mediate and increase BBB penetration of drugs to the specific site of the brain. However, the efficiency of nanoparticle-based BBB penetration is still very low (<1%), with much to learn about how the biophysical properties of nanoparticles such as which size, surface, shape of nanoparticles facilitate BBB penetration. This project takes advantage of the specific properties of upconversion nanoparticles (UCNPs) including low auto-fluorescence, non-photobleaching, deep tissue penetration and adjustable size and shape, to develop a versatile platform to systematically evaluate the effects of nanoparticle surface, shape on BBB crossing in vitro and in vivo for future construction of multifunctional nano-carrier for theranostic applications in neurodegenerative diseases.

In the first chapter, this thesis introduced the BBB and currently understood mechanisms of BBB penetration, and reviewed recent advances in nanoparticle-based BBB penetration strategies and approaches for brain disease therapy.

In the second chapter, I described the key experimental methods that were developed and optimized for use in this project. Typically, the UCNPs employed in this project were fabricated via modified and optimized standard protocols for specific application as required. Importantly, the approaches of zebrafish imaging and microinjection were developed brand new in this project.

In the third chapter, a UCNPs-based evaluation platform was synthesized for bio-nano surface selection in vitro and in vivo to systemically evaluate the suitability of various surface modifications for theranostic applications in neurodegenerative diseases. First, high lanthanide-doped UCNPs was designed, which provide strong tissue penetrable emission at 800nm. Then, these as-prepared UCNPs were further modified with four popular surfaces (OA-free, DNA-modified, silica coated and PEG-COOH capped) for comparison. The result showed that PEG-COOH performed superior cell…

Empirical thesis.

Bibliography: pages 197-253.

Chapter 1. Literature review  – Chapter 2. Methodology  – Chapter 3. Mesenteric artery remodelling in CKD  – Chapter 4. AT1 receptor blockade and resistance arteries in CKD  – Chapter 5. CCB treatment and resistance arteries in CKD  – Chapter 6. Resistance artery endothelial dysfunction in CKD  – Chapter 7. Final discussion  – Chapter 8. References.

Hypertension is a significant complication among chronic kidney disease (CKD) patients, resulting in higher mortality rates in these patients than renal disease itself. The process of cardiovascular damage starts very early during progression in well-defined CKD, long before the renal treatment stage is reached (Vanholder et al., 2005), thus making kidney disease the most common “secondary” form of hypertension (Cohen and Townsend, 2009, Ross and Banerjee, 2013). Resistance arteries play a particularly important role in the maintenance of hypertension in these CKD patients (Li et al., 1997, Paisley et al., 2009), as they are the main contributors to total peripheral resistance (TPR) (Beevers et al., 2001, Mulvany et al., 1978, Mulvany and Halpern, 1977). Although it is well-established that alterations in resistance arteries are likely to contribute to increased cardiovascular risk and act as a substrate for end-organ damage (Schiffrin, 2012), resistance artery alterations are still poorly understood in the CKD-mediated hypertensive state, and hence were the focus of this thesis.

To investigate the effects of CKD on the resistance vasculature as part of the overall cardiovascular disease (CVD) state, we employed an established rat model of CKD-mediated hypertension, the Lewis polycystic kidney (LPK) rat. In a series of four studies, we sought to examine alterations in resistance artery structure, function and biomechanical properties, in association with disease progression. In addition, we investigated the effects of treatment (AT1 receptor antagonism and calcium channel blockade) on the resistance vasculature.

In study 1, we explored temporal changes in LPK resistance artery structure, function and biomechanical properties, in association with hypertension and renal disease progression. We investigated three time-points: 6 weeks of age, where LPKs are hypertensive and have gross derangement of the kidney cortex and medulla; 12 weeks of age, where LPKs demonstrate signs of renal dysfunction in addition to hypertension and increased sympathetic nerve activity (SNA); and 18 weeks, where LPK have marked renal disease and still present with hypertension and elevated SNA (Phillips et al., 2007). These experiments revealed that alterations in the vasculature tended to emerge after the 6 week time-point in LPK, and that older age was associated with negative outcomes. Older LPK resistance arteries underwent structural alterations which were characterised by eutrophic and hypertrophic inward remodelling at established and severe renal disease time points, respectively, and these structural alterations were…

Empirical thesis.

Bibliography: pages 138-167.

Chapter 1. Cerebrovascular diseases : an introduction  – Chapter 2. Role of Computational Fluid Dynamics (CFD) in the management of cerebrovascular diseases  – Chapter 3. The influence of flow diverter’s angle of curvature across the aneurysm neck on its hemodynamics  – Chapter 4. Identification of a hemodynamic parameter for assessing treatment outcome of EDAS in Moyamoya disease  – Chapter 5. Assessing surgical treatment outcome following superficial temporal artery to middle cerebral artery bypass based on Computational Hemodynamic Analysis  – Chapter 6. Hemodynamic assessment of surgical treatment outcome on Moyamoya disease patients with complete circle of Willis following revascularization surgery  – Chapter 7. Discussion and conclusion.

The vasculature that supplies blood to the brain is tightly regulated and any abnormalities in its structure; aneurysms and Moyamoya disease (MMD) cause a multitude of complications including but not limited to Sub-arachnoid hemorrhage (SAH), transient ischemic attacks(TIA), stenosis etc. The blood flow dynamics of these vascular disorders plays a critical role in the origin and development of the disease. Computational Fluid Dynamic (CFD) techniques help us understand the underlying mechanism that promotes disease prognosis. In this study we aim to computationally analyze the hemodynamic parameters that will help assess the treatment outcome of cerebrovascular diseases such as intracranial aneurysms and MMD.

In our initial study we attempted to understand the relationship between the free segment of Flow Diverter (FD)’s angle and the Metal Coverage Rate (MCR) across the aneurysm neck and its influence on flow reduction and Energy Loss (EL). Our results from two patient specific aneurysms; A (Aspect Ratio=3.1) and B (Aspect Ratio=2.9) with three FD configurations each (0°, 10° and 25°), suggested that an optimal MCR in the range of 50-60% across the aneurysm neck brings about maximum flow reduction inside the aneurysm facilitating its occlusion. It also yielded higher percentage reduction in Energy Loss when compared to the no-stent case indicating a lower risk of rupture compared to other stent configurations.

The second part of the dissertation deals with the application of CFD principles to understand the hemodynamics of scarcely known Moyamoya disease, which is caused due to progressive occlusion of the Internal Carotid Arteries (ICA). A total of 34 adult MMD patients; 8-treated bilaterally with indirect EDAS revascularization, 26-treated unilaterally with combined direct STA-MCA bypass and indirect EDMS revascularization (18-incomplete Circle of Willis, 8-complete Circle of Willis), were computationally analyzed to identify a novel hemodynamic parameter, Pressure Drop Index (PDI) which correlated with Matsushima’s angiographic grading (A-Significant improvement; B-Limited Improvement; C-No Improvement) across all patients. We also sought to evaluate the characteristic remodeling of ICA post-surgery in the18…

Empirical thesis.

Bibliography: pages 185-232.

Chapter 1. Literature review  – Chapter 2. Methods of biofilm studies  – Chapter 3. Biofilms harbouring multi-drug resistance organisms in the Intensive Care Unit  – Chapter 4. Contamination of surgical bed by shoe covers  – Chapter 5. Development of a dry surface biofilm model  – Chapter 6. The efficiency of chlorine for biofilm destruction  – Chapter 7. Heat treatment efficacy to inactivate biofilm  – Chapter 8. General discussion and conclusion.

There is incontrovertible evidence that nosocomial pathogens contaminate inanimate items in the hospital environment, such as surfaces and medical equipment. Transmission of infectious agents from contaminated fomites to patients is a known infectious route, although the contribution to overall hospital acquired infections (HAI) is unknown. However, the risk of developing HAI, has been shown to be increased 73% if the patient previously occupying the room had a multi-antibiotic resistant organism (MRO). Acinetobacter baumannii, vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus are the main non-sporing infectious agents that have been clearly demonstrated to survive in environmental reservoirs.

Bacteria readily adhere to surfaces. Once adhered they secrete a slimy matrix principally composed of protein, carbohydrate and DNA which surrounds them protecting them from the external environment. Bacterial reproduction and recruitment, leads to the development of a mature biofilm.

The environmental conditions associated with hospital surfaces, especially in Intensive Care Units (ICU), are conducive to development of biofilm, which could have adverse consequences in these seriously ill patients. Up to 20% of intensive care patients become colonised by MROs increasing their risk of developing a HAI. Dry surfaces biofilms development increases the MDRO persistence rate in the hospital environment, and thereby likely increases the risk of hospital acquired infections and outbreaks. Hence, one cannot emphasise enough the importance of thoroughly cleaning and disinfecting hospital surfaces.

The ultimate aim of this project was to determine if persistence of antibiotic resistant human pathogens in the hospital environment was due to MRO being incorporated into biofilms contaminating dry hospital surfaces. If incorporated into biofilms it was hypothetised that they would be protected from environmental desiccation, cleaning and disinfectants agents.

The findings of this study revealed that bacteria encased in biofilms, including those causing serious infections, such as S. aureus, were present on over 90% of ICU surfaces. We determined that the majority of clinical biofilms incorporated Staphylococcus species and therefore developed an in vitro model representative of clinical dry-surface biofilms. The thickness of the biofilms and the number of cells in the biofilms in this model was reproducible between repetitions of the experiments, at least for one strain of S. aureus. We then…

Theoretical thesis.

Bibliography: pages 239-279.

General introduction  – Chapter 2. Methods and materials  – Chapter 3. PDI is protective against major familial ALS causing proteins  – Chapter 4. Mechanism of action of PDI against mutant SOD1, TDP-43 and FUS in neuronal cells  – Chapter 5. Examining the role of PDI family members ERp57 and ERp72 in ALS  – Chapter 6. Cytoplasmic PDI is protective against mutant SOD1, TDP-43 and FUS in neuronal cell cultures  – Chapter 7. General discussion.

Superoxide dismutase (SOD1), Tar-DNA binding protein-43 (TDP-43) and Fused in Sarcoma (FUS) are major proteins linked to Amyotrophic Lateral Sclerosis (ALS) pathology. Whilst neurodegenerative mechanisms are not fully defined in ALS, dysfunction to the Endoplasmic Reticulum (ER) is increasingly implicated in the pathology of the disease. Protein disulphide isomerase (PDI) is an ER chaperone which also functions as an isomerase and aids in the formation and reduction of protein disulphide bonds. It is primarily located in the ER but it is also found in other cellular locations. Our laboratory previously demonstrated that over-expression of PDI is protective against mutant SOD1 in neuronal cultures. PDI has also been shown to co-localise with FUS and TDP-43 positive inclusions in ALS patients. Here we examined whether over-expression of PDI is protective against mutant TDP-43 and FUS induced ER stress and ER-Golgi transport defects and mislocalisation into cytoplasm in cellular models. Furthermore, we examined the mechanism by which PDI is protective and suggests that the disulphide interchange activity is important for its protective function. Also PDI in the cytoplasm further accentuates its protective ability. Importantly, PDI was examined in vivo and it was demonstrated that a small molecule mimic of the PDI active site -1,2 bis (mercaptoacetamido) cyclohexane BMC reduced the loss of motor neuron in SOD1G93A mice signifying the relevance of PDI in disease pathology. The results in this study demonstrate that PDI is protective against the major misfolded proteins linked to ALS; therefore the molecular mimic may be a novel therapeutic target in multiple forms of ALS.

1 online resource (xvii, 309, [4] pages) illustrations (some colour)

Empirical thesis.

Bibliography: pages 263-303.

1. General introduction and outline  – 2. Introduction  – 3. Experimental methodology  – 4. Desensitisation and kinase modulators  – 5. Regulation of N-terminal SNPs of hMOPr  – 6. Regulation of TM1 and ICL2 SNPs of hMOPr  – 7. The ICL3 : hMOPr SNPs and phosphosite mutants  – 8. Regulation of C-terminal phosphosite mutant of hMOPr  – 9. Summary and prospects.

Opioid drugs are highly effective for the treatment of moderate to severe nociceptive pain. They exert their analgesic and rewarding effects primarily by signalling through the μ-opioid receptor (MOPr). The focus of this project was to better understand MOPr signalling regulation by investigating natural variants of MOPr and MOPr phosphosite mutants.

Isogenic, stably transfected mouse pituitary adenoma (AtT20) cell lines expressing eight naturally occurring human MOPr variants and four phosphomutants were created. Opioid-stimulated changes in membrane potential were measured using a membrane potential-sensitive dye, while receptor phosphorylation of Ser 377 residue was determined by Western Blot and whole-cell ELISA was used to obtain the receptor surface loss dynamics.

The N-terminal MOPr variants, A6V and N40D, are the most common single-nucleotide polymorphisms found worldwide. Their signalling regulation was quite similar to the wild-type MOPr in each assay, where buprenorphine was the opiod with the most variance observed. In AtT20-hMOPr-L85I cells morphine mediated internalisation was not as substantial as previously reported, while the second intracellular loop (ICL2) polymorphism R181C dramatically impacted receptor ability to signal by affecting opioid affinity and probably G protein binding. The majority of the third intracellular loop (ICL3) variants had detrimental effect in receptor signalling and regulation which indicates the important role this region plays in G protein activation. In addition the multiple phosphorylation mutants also affected membrane expression which was related to endoplasmic reticulum sequestration and possible changes in receptor stability. Finally, deleting all the putative phosphorylation sites in the human MOPr C-terminal domain did not greatly influence homologous desensitisation of the membrane potential signal, yet completely abolished internalisation as expected. In contrast heterologous desensitisation was deeply compromised in some mutants of the ICL3 while total phosphorylation deletion of the C-terminal was the only variant to increase desensitisation of somatostatin signalling. Interestingly buprenorphine induced signalling had a quite different profile across the variants, and morphine and methodone signalling were more affected by the ICL3 changes when compared to opioid peptides and buprenorphine.

Overall, these results support the hypothesis of multiple mechanisms involved in regulation of MOPr, where ICL2 and ICL3 are crucial for G protein signalling, and receptor phosphorylation is not necessary for receptor desensitisation. In addition,…

Empirical thesis.

Bibliography: pages 144-188.

1. Introduction  – 2. Recording, labelling and transfection of single neurons in deep brain structures  – 3. Somatostatin 2a receptors are not expressed on functionally identified respiratory neurons in the ventral respiratory column of the rat  – 4. The connectome of rostral ventrolateral medulla C1 neurons  – 5. General discussion  – References  – Appendices.

The autonomic nervous system governs basic homeostatic functions such as regulating cardiac output, blood pressure, body temperature, and the generation of respiratory rhythm. Although traditionally conceptualised as independent entities, the neural networks that underlie respiratory rhythm and vasomotor tone are intimately connected, with each component influencing the activity of the other. This respiratory-sympathetic coupling contributes to cardiac sinus arrhythmia and underlies the strong respiratory modulation of vasomotor sympathetic nerve activity, which may play an important role in initiation and development of neurogenic hypertension. The experiments described in this thesis explore the neurochemical profiles, functional properties, and circuit architecture of medullary neurons that control breathing and circulation.

Genetically modifying a single functionally identified neuron in an intact neural network would provide the ultimate way to bridge neuronal circuit structure and function. The work described in Chapter 2 of this thesis describes a novel method that combines extracellular recording with the labelling of neurons recorded in the ventral lateral medulla with either dyes or plasmid DNA, permitting conventional cell labelling or gene delivery. We demonstrate that our approach has several advantages over traditional single-cell labelling approaches, and therefore provides a useful tool for correlation of functional properties of single neurons with their neurochemical phenotypes that can also be used for targeted single cell gene delivery under blind recording conditions.

We then apply this technique to address a controversial topic in the field of respiratory neuroscience; the neurochemical signature of the neurons responsible for generating respiratory rhythm. Previous investigations have suggested that the neuropeptide somatostatin plays a critical role in respiratory rhythmogenesis and is a marker for excitatory respiratory interneurons in the pre-Bötzinger complex (preBötC). Somatostatin receptor type 2a (sst₂ₐ) has been postulated as a mediator of such effects, and has also been proposed as a marker for respiratory neurons. In Chapter 3 we systematically examine the expression pattern of sst₂ₐ with regards to the functionally distinguished subcomponents of the ventral respiratory column. We further examine the feasibility of sst₂ₐ as a reliable marker of respiratory function at the single cell level.

In Chapter 4 we examine the anatomical substrate responsible for linking the respiratory and sympathetic circuits using a modified trans-synaptic tracing tool. We first…

Thesis by publication.

Bibliography:

Chapter 1. Thesis outline  – Chapter 2. Photoluminescent nanoparticles for cancer theranostics  – Chapter 3. Synthesis, design and applications of UCNPs in theranostics  – Chapter 4. Deep-penetrating photodynamic therapy with KillerRed mediated by upconversion nanoparticles  – Chapter 5. Facile assembly of functional upconversion nanoparticles for targeted cancer imaging and photodynamic therapy  – Chapter 6. The effect of surface characteristics of upconversion nanoparticle on corona formation and cell interaction  – Chapter 7. Systematic assessment of blood circulation time of functionalized upconversion nanoparticles in the chick embryo  – Chapter 8. Summary and future scope  – Appendices.

Development of new approaches for diagnosis and therapy of tumours, termed theranostics, is one of the most dynamic areas of the life sciences, where new nanomaterials afford new opportunities. The nanomaterial merits include programmability of their physical and chemical properties; abundance of reactive functional groups on their surface; large effective surface area and optimal size, which determines preferential accumulation of nanoparticles (NPs) in tumour tissue.Photoluminescent nanomaterials add to the list of merits their high optical contrast achievable on the crowded background of biological cells, tissues and whole organisms.In order to fully harness the potential of photoluminescent NPs, they need to be coupled with functional biological molecules, accomplished by procedures of NP surface redressing and bioconjugation, to enable controlled targeting and therapeutic action in the delicate environment of a biological system. Interactions of these biofunctional hybrid assemblies with live cells and organisms, however, are overly complex, poorly understood and controlled. It is increasingly being accepted that NPs encountering biological medium are swiftly coated by a biomolecular adsorption layer called “protein corona”. Consequently, the molecular machinery of a living cell or organism will interact with the corona rather than biofunctional hybrid assemblies, making it a key determinant of the biological response of NP exposure. In this PhD thesis, the design and characterisation of photoluminescent biofunctional nanoparticles and their applications for targeted delivery and photodynamic therapy of cancer cells are addressed, in addition to the systematic investigation of protein corona formation on polymer-coated nanoparticles.

My thesis research was centred at a new-generation of biofunctional photoluminescent nanoparticles with unique optical properties termed upconversion nanoparticles (UCNPs). UCNPs are photoexcited by near-infrared light (NIR) at 980 nm capable for deep penetration (up to 1 cm) in biological tissue. This excitation confers another key advantage of background-free imaging in cells and tissues due to very little excitation of autofluorescence. UCNP emission is tuneable by design from ultraviolet to visible or even NIR spectral bands, which…

Thesis by publication.

CHapter 1. Introduction  – Chapter 2. Methods  – Chapter 3. Study I: Overexpression of αvβ6 integrin alters the colorectal cancer cell proteome in favor of elevated proliferation and a switching in cellular adhesion that increases invasion  – Chapter 4. Anti-sense suppression of the β6 subunit attenuates metastatic phenotypes  – Chapter 5. Study IV: A novel multiplexed immunoassay identifies CEA, IL-8 and prolactin as prospective markers for Dukes’ stages A-D colorectal cancers  – Chapter 6. General discussion, conclusion and future directions  – Chapter 7. Appendices.

Globally, colorectal cancer (CRC) afflicts millions of men and women each year. The progression of an abnormal epithelial cell to a benign tumour and then a malignant cancer exploits several mutated genes and perturbed signalling and biochemical pathways. The expression of proteins such as the β6 integrin subunit, uPAR and TGFβ have been extensively associated with CRC progression.

The primary aim of this thesis was to contribute new knowledge regarding the role of integrin αvβ6 in CRC by investigating the proposed uPAR•αvβ6•TGFβ metastasome. This was achieved by employing five CRC cell lines as model cellular systems, in which β6 had been artificially overexpressed, suppressed or truncated (SW480Mock, SW480β6OE, SW480β6DEL, HT29Mock, HT29β6AS). The consequences of β6 expression following latent TGFβ or plasminogen treatment was assessed using a variety of phenotypic, cellsignalling (i.e., AlphaScreen® SureFire® Assay) and state-of-the-art proteomic technologies. β6-expressing cells were significantly more proliferative and invasive with increased ERK1/2 activity, which was further promoted following ligand (latent TGFβand/or plasminogen) treatment. Integrin β6 expression was found to translate subphysiological levels of these zymogens into significantly enhanced metastatic behaviours, through a mechanism that could be disrupted either by antagonising TGFβ receptor signalling, plasmin activity or the uPAR•αvβ6 interaction. β6 overexpression and suppression induced extensive proteomic changes, altering pivotal biomolecular pathways that can be associated with CRC and provided insights into the role and central function of β6 within the uPAR•αvβ6•TGFβ metastasome.

Furthermore, an Olink Proseek multiplexed study, interrogated Dukes’ stage A-D CRC patient plasma samples and identified CEA, IL-8 and prolactin as differential markers of early stage Dukes’ A/ B and later (malignant) stage Dukes’ C/D plasmas from those obtained from unaffected healthy controls.

This thesis demonstrates the power of modern proteomic and multiplexing technologies to elucidate β6-promoted CRC pathenogenesis at the molecular level whilst identifying potential avenues for future biomarker exploration.

1 online resource (xiv, 410 pages)

Thesis by publication.

Bibliography: pages 177-207.

Chapter I. General introduction  – Chapter II. Study I. In vitro determination of the efficacy of illicit synthetic cannabinoids at CB1 receptors  – Chapter III. Study II. Differential activation of G-protein-mediated signalling by synthetic cannabinoid receptor agonists  – Chapter IV. Study III. Brodifacoum does not modulate human cannabinoid receptor-mediated hyperpolarization of AtT20 cells or inhibition of adenylyl cyclase in HEK 293 cells  – Chapter V. Study IV. Absence of Entourage: Terpenoids Commonly Found in Cannabis sativa Do Not Modulate the Functional Activity of Δ9-THC at Human CB1 and CB2 Receptors  – Chapter VI. Study V. Investigating the specificity of cannabidiol signalling  – Chapter VII. General discussion  – Appendices  – References.

Cannabinoids from plants are some of the oldest human medicine, while synthetic cannabinoid receptor agonists (SCRAs) new psychoactive substances have been responsible for hundreds of deaths all over the world. The studies presented in this thesis were undertaken to further the understanding of the molecular pharmacology of cannabinoids, and how structurally diverse cannabinoids have greatly varied outcomes when acting via the same target. The characterisation of cannabinoids at activating cannabinoid receptors (CB1 and CB2) combined with operational model, functional selectivity and allosteric modulation together form the basis of the current thesis to better understand the molecular contributions to the toxic effects of SCRAs and therapeutic effects of phytocannabinoids. The first major finding of this thesis was the quantitative determination of the efficacy of SCRAs downstream of CB1 using the operational model of pharmacological agonism. A panel of 17 cannabinoids were compared for their ability to induce response after the pharmacological knockdown of CB1 receptor using AM6544 (irreversible CB1 antagonist). The operational efficacy of cannabinoids ranged from 233 (5F-MDMB-PICA) to 0.9 (THC), with CP55940 in the middle of the efficacy rank order. SCRAs generally demonstrated substantially higher efficacy at activating CB1 receptors than THC. This work is the first of its kind to provide a framework to quantify the efficacy of chemically diverse cannabinoids and help identify some of the potential underlying molecular mechanisms regarding the SCRAs-related adverse effect on CB1 activation. The functional activity of the same panel of cannabinoids was also characterised in two signalling endpoints - Gαi/o (inhibition) and Gαs (stimulation) of cAMP signalling. The rank order of potency of the cannabinoids to stimulate Gαs-like signalling compared to Gαi/o signalling was significantly different. This suggests the differing ability of cannabinoids to preferentially induce CB1-dependent cAMP inhibition over stimulation of cAMP (functional selectivity). Cannabinoids showed diverse signalling profile (wide range of EMAX) at Gαs-like pathway than their activity at canonical Gi-mediated signalling pathway.…

Empirical thesis.

Bibliography: pages 131-166.

Chapter 1. Introduction  – Chapter 2. Recording, labelling and transfection of single neurons in deep brain structures  – Chapter 3. Mapping sources of pre-synaptic input to bulbospinal RVLM neurons  – Chapter 4. Direct projections from the midbrain colliculi innervate putative pre-motor sympathetic, respiratory and somatomotor populations in the ventral medulla  – Chapter 5. Synthesis and future directions.

The basal and evoked activity of sympathetic nerves, and therefore the physiological consequences of sympathetic nerve activity (e.g. cardiovascular homeostasis), are directly dependent on synaptic drive arising from groups of sympathetic premotor neurons in the medulla and hypothalamus. The central theme of this thesis is the employment of genetic tools, primarily viral based tracing strategies, to map the locations of neurons distributed throughout out the brain that provide synaptic drive to sympathetic premotor neurons in the ventral medulla.

Genetic tools grant researchers the ability to selectively manipulate neuronal populations based on either anatomical criteria or the constitutive expression of specific promoters. In Chapter 2 of this thesis we developed a novel technique that permits the genetic manipulation of single neurons in the rat, based on functional (electrophysiological) criteria. The work described in this chapter is foundational for future experiments that intend to map functionally relevant circuits in combination with tran-synaptic viral tracing strategies.

Sympathetic premotor neurons within the rostral ventrolateral medulla (RVLM) are essential for the generation of vasomotor tone. However, key questions regarding the architecture of the circuits that control their activity remain. In Chapter 3 we address this topic by employing a trans-synaptic viral tracing strategy to identify neurons monosynaptically connected to bulbospinal RVLM neurons. The anatomical data acquired using this approach has allowed us to generate a brainwide map of the neurons that provide monosynaptic input to bulbospinal RVLM neurons, and therefore determine major sources of synaptic drive likely to underlie the generation of SNA. We describe an arrangement for afferent input to RVLM bulbospinal neurons that emphasises input from hitherto unappreciated local medullary sources, but is overall qualitatively similar to currently held circuit schemes.

In Chapter 4 we examine the anatomical substrates that underlie the recruitment of autonomic and motor responses to alerting stimuli. Using an anterograde viral tracing strategy we identify a previously undescribed direct efferent projection from the superior and inferior colliculi to the ventral brainstem. We describe termination of axons originating from the colliculi to regions of the brainstem previously associated with sympathetic, respiratory, and motor functions, and observe putative synaptic contacts in close apposition to bulbospinal neurons in the rostral ventromedial medulla and medullary…

Empirical thesis.

Thesis carried out as part of a co-tutelle arrangement with Tohoku University.

Bibliography: pages 125-136.

Chapter 1. Introduction  – Chapter 2. Materials and methods  – Chapter 3. Structural optimisation of flow-diverting stent  – Chapter 4. Haemodynamic simulation of flow-diversion treatments with stent compaction applied  – Chapter 5. Haemodynamic simulation of flow-diversion treatment with multiple flow-diverting stents  – Chapter 6. Investigation of incomplete stent expansion effects on aneurysmal haemodynamics  – Chapter 7. Applying computational simulation to the design of flow-diversion Tteatment : a showcase  – Chapter 8. Conclusion and outlook.

Despite various complications reported, flow-diverting (FD) stent implantation has become a major mode of treatment for intracranial aneurysms. Review of literature suggests that the treatment outcomes are closely associated with the aneurysmal haemodynamics changed by the implanted stent. In this thesis, I sought to examine the haemodynamic changes following different strategies of FD treatment and provide practical solutions to improve the stent's flow-diversion efficacy.

As the first accomplishment, I developed in Chapter 3 an automated optimisation method to be used to accommodate stent configuration to a specific aneurysm geometry. Adopting this method, wire structure of a stent can be modified to alter the local haemodynamics towards maximal reduction of the aneurysmal inflow. Furthermore, employing a virtual stent deployment technique and computational fluid dynamics analysis, I systematically investigated in Chapter 4 and 5 the aneurysmal haemodynamics following the flow-diversion treatment with a stent compaction technique applied or with dual-FD stents deployed - two of the most commonly adopted treatment strategies in the current interventional practice. These studies provided quantitative results illustrating stent wire characteristics and the subsequent aneurysmal haemodynamic changes in various flow-diversion scenarios. Finally, I examined in Chapter 6 the aneurysmal haemodynamics affected by incomplete stent expansion (IncSE) - a condition suspected to cause delayed aneurysm occlusion, for which various severities of IncSE occurring at different segments of the parent artery were modelled and examined. Results suggest that the effects of IncSE vary greatly with respect to the location where it occurs.

Based upon this series of studies, a workflow was put forward to individualise the treatment plan corresponding to the haemodynamic characteristics of a specific patient, from the patient aneurysm model reconstruction, to computer-aided stent deployment rehearsal, and then to the haemodynamic outcome analysis to determine the most effective treatment plan prior to the real treatment. An example of implementing this workflow to predict the post-treatment haemodynamics thereby determining the most favourable treatment strategy was illustrated in detail in Chapter 7.

Apart from this useful application,…

Thesis by publication.

Bibliography: pages 205-294.

1. Introduction  – 2. Increased excitatory regulation of the hypothalamic paraventricular nucleus circulating vasopressin results in the hypertension observed in polycystic kidney disease  – 3. Angiotension II differentially regulates blood pressure and sympathetic nerve activity in polycystic kidney disease  – 4. The subfornical organ drives hypertension in polycystic kidney disease via the hypthalamic paraventricular nucleus  – 5. High water intake provides renal and cardiac autonomic benefits in a rat model of polycystic kidney disease  – 6. General discussion  – References  – Appendices.

Polycystic kidney disease (PKD) is characterised by the progressive accumulation of multiple bilateral renal cysts that threaten body-fluid homeostasis and glomerular filtration. In PKD patients, the development of hypertension and baroreflex dysfunction both contribute to a high risk of cardiovascular mortality, but the underlying mechanisms are poorly understood. This thesis examined the hypothesis that altered signalling within the hypothalamus and elevations in extracellular fluid osmolality contribute to hypertension and baroreflex dysfunction in a rat model of PKD, the Lewis Polycystic Kidney (LPK) rat.

The first goal of this thesis was to determine whether the ongoing activity of neurons in the hypothalamic paraventricular nucleus (PVN), a structure that regulates arterial pressure through multiple neural and humoral outputs, contributes to the maintenance of hypertension and baroreflex dysfunction in LPK rats in early and advanced stages of the disease. Acute pharmacological experiments in anaesthetised animals showed that an early increase in the glutamatergic excitation of PVN neurons maintains hypertension, but not baroreflex dysfunction, in LPK animals. This study also suggested that vasopressin release maintains a component of the hypertension in anaesthetised LPK rats but is independent of PVN neuronal activity and might therefore involve the supraoptic nucleus. However, additional work showed that while supraoptic nucleus vasopressin neurons are more active in LPK rats, chronic systemic inhibition of V1A receptors is not anti-hypertensive.

Further studies examined the source of elevated PVN glutamatergic tone in LPK rats. The first hypothesis examined was that the local signalling of angiotensin II, a critical regulator of PVN glutamatergic tone, is enhanced in LPK rats. It was shown that the sensitivity but not tonicity of the angiotensin II type 1 receptor (AT1R) is enhanced in LPK rats and that its activation preferentially drives vasopressin release rather than sympathetic outflow via an indirect interaction with PVN astrocytes. Subsequent work determined whether heightened PVN glutamatergic tone is produced by a greater ongoing discharge of glutamatergic afferents in LPK rats. It was demonstrated that the subfornical organ (SFO), a sensory structure that detects plasma angiotensin II and osmolality levels, contains more activated…

Theoretical thesis.

Bibliography: pages 160-199.

Chapter 1. Introduction  – Chapter 2. Mitogen-activated protein kinase dependency in BRAF/RAS wild type melanoma: a rationale for combination inhibitors  – Chapter 3. Influence of p53 on melanoma responses to trametinib  – Chapter 4. Multiple signaling pathways are active in BRAF/RAS wild type melanoma  – Chapter 5. Conclusion.

Selective inhibition of the mitogen-activated protein kinase (MAPK) pathway has significantly improved the survival of patients with BRAF V600 - mutant advanced melanoma. However, BRAF / RAS wild type (WT) melanomas have no known actionable mutations, and immune checkpoint inhibitors remain the only effective therapy for patients with this melanoma subtype. In this PhD project, we explored the signalling activity and response of BRAF / RAS WT melanomas to combination small molecule inhibitors. In Chapter 2, we investigated MAPK dependency in 23 melanoma cell lines, including 10 BRAF V600 - mutant and 13 BRAF / RAS WT (seven NF1 - mutant and six triple WT) melanomas. Melanoma cell lines were treated with the MEK inhibitor trametinib, and the impact of MEK inhibition on cell survival and proliferation were examined. We showed that BRAF / RAS WT melanomas had variable responses to MEK inhibition; 23% were highly sensitive, indicating dependency on MAPK signalling for survival and proliferation, whereas 38% were resistant, and this was commonly associated with high mutation burden and loss - of - function mutation s in NF1. We demonstrated that NF1 loss conferred MEK inhibitor resistance in BRAF V600 - mutant cells but not in BRAF / RAS WT melanomas. The mutational profiles of BRAF / RAS WT melanomas revealed concurrent mutations in RASopathy genes, and enrichment of TP53 mutations. In Chapter 3, we explored the precise contribution of p53 loss to MEK inhibitor resistance in our panel of melanoma cells. We also examined the efficacy of a p53 activator in combination with MEK inhibition on suppressing melanoma proliferation. Finally, in Chapter 4, the activity of oncogenic signalling pathways in BRAF/RAS WT melanoma cell lines was examined and the activity of combination inhibitors targeting activated cascades w as tested .

1 online resource (xvii, 199 pages) illustrations

Thesis by publication.

Contains bibliographical references.

Chapter 1: KP and MND  – Chapter 2: Case study  – Chapter 3: BMAA & Neurotoxicity  – Chapter 4: Other publications arising from the candidature.

Motor Neuron Disease (MND) or Amyotrophic Lateral Sclerosis (ALS) is a devastating neurological disease with no biological diagnostic markers, no effective treatment, and no cure. We investigate the immune related Kynurenine Pathway (KP) for a role in ALS. The production of neuroactive metabolites during the KP indicate that there is an overlap with the mechanisms of ALS, particularly with the neurotoxin quinolinic acid. Subsequently, we investigate the KP metabolome, analysing 10 metabolites using biochemical analyses including High Performance Liquid Chromatography and Gas Chromatography/Mass Spectrometry. Using serum from a longitudinal cohort of 66 ALS patients, we establish a potential for KP metabolomics to be used a biomarker for ALS. To increase specificity and reliability of these results, in collaboration with Macquarie University Neurology, we established a Neurodegenerative Diseases Biobank to collect patient biological samples. These samples would facilitate future investigations into the mechanisms, genetics, biomarkers, and to detect the presence of toxic compounds such as metals, or β-methylamino-L-alanine (BMAA). We describe the establishment of the biobank as a case study for future references. BMAA is known to be neurotoxic, and we investigate its role ALS. We reveal its role in promoting axonal degeneration and neuronal death, and show for the first time, its ability to spread transcellularly  – abstract.

1 online resource (xvi, 242 pages) illustrations

Thesis by publication.

1. Human proteome project  – 2. Mass spectrometry based proteomics  – 3. Role of uPAR in colorectal cancer  – 4. Antagonism of metastasis in late-stage colorectal cancer (CRC)  – 5. Thesis discussion and future directions.

Accurate management of any human disease requires a thorough understanding of the molecular underpinnings (i.e., genome + epigenome + transcriptome + proteome + peptidome + protein post-translational modifications + metabolome + microbiome) driving the biology of that specific disease. Given the intricate and expanding roles played by proteins in human health and disease, these have been studied extensively to uncover disease mechanisms, define diagnostic, prognostic and theranostic markers and identify novel therapeutic targets. Over the past decade, high throughput mass spectrometry (MS)-based proteomics with subsequent bioinformatic analysis have emerged as one major technological driving force in our attempts to expand human proteomics so that it has a noticeable impact on medicine, human health and the life sciences alike. The Human Proteome Project (HPP) provides a framework for communal proteomics research. It specifically adds value to the task of 'knowing thyself' in strictly molecular terms by mapping the ~20,000 proteins encoded by the human genome. It aims to do so as a corollary to the human genome using measurements at the highest possible accuracy and stringency. The HPP states that it has three initial primary aims, namely to; "i. complete the protein 'parts list' of Homo sapiens by identifying and characterizing at least one protein product and as many posttranslational modifications, single amino acid polymorphisms and splice variant isoforms as possible for each protein-coding gene;ii. transform proteomics so it becomes complementary to genomics across clinical, biomedical and life sciences through technological advances2iii. create knowledgebases for the identification, quantitation and characterization of the functionally networked human proteome." 8This thesis contributes to two major elements of the HPP (C-HPP, Chromosome-Centric-HPP and B/D-HPP, Biology/Disease-HPP) through the use of informatics and proteomics approaches. Expanding previous research efforts by our HPP team at Macquarie University, Chapter 1 aims to advance community-centric resources to accelerate the identification of missing proteins (MPs). This chapter provides a plausible explanation for the observed paucity in identifications of certain missing protein family groups that have failed to be identified by MS over the last decade - namely the olfactory receptors (ORs). Analysis of OR hydrophobicity, topological distribution, tryptic cleavage site and frequency, and ability to predict in silico uniquely-mapping, non-nested tryptic peptides of a communally-required length (9 amino acids or longer) indicated that multiple ORs are unable to generate peptides as per requirements set by the HPP to be called protein existence 1 (PE1 for short) proteins. These ORs may not be…

Theoretical thesis.

Bibliography: pages 109-118.

Chapter 1. Introduction  – Chapter 2. Materials and methods  – Chapter 3. Haemodynamics in a patient-specific intracranial aneurysm obtained from experimental and numerical approaches: a comparison between PIV, CFD, and PC MRI  – Chapter 4. Validation of CFD model results against PIV observations of haemodynamics in intracranial aneurysms treated with a flow-diverting stent  – Chapter 5. Sensitivity of aneurysmal haemodynamics to porous medium stent modelling setting  – Chapter 6. Effect of calibrated porous medium flow-diverting stent models on aneurysmal haemodynamic modifications  – Chapter 7. Conclusions and outlook.

Haemodynamic information is believed to be one of the most crucial factors affecting the initiation, development, growth, and rupture of intracranial aneurysms (IAs). Many studies of haemodynamic simulation contribute to the understanding of aneurysmal flow dynamics; however, clarification and justification of the validity of simulation results inconclusive remains. Besides, for most simulations of flow-diversion effect using a model flow-diverting (FD) stent, the properties of model FD stents failed to be calibrated to match the represented device, which would lead to inaccurate predictions of flow-diversion efficacy. Thus, this study aims to discover the factors that contribute to a more accurate and comprehensive simulation of aneurysmal haemodynamics and its flow-diversion treatment. Two main aspects were investigated in this thesis : 1) evaluation of the accuracy of computational fluid dynamics (CFD) predictions of aneurysmal haemodynamics before and after FD stent treatment, by comparing to in vivo and in vitro observation s; and 2 ) investigation of the practicality of using porous medium (PM) stent models, with calibrati on of stent model parameters to represent the commercially available FD stents, by derivation of parameters like permeability (k) that account for the flow resistance induced by the model stent.

To meet these needs, haemodynamic investigations of IAs and their flow-diversion treatments have been performed using different approaches. In Chapter 3 and 4, the validity of CFD predictions of aneurysmal haemodynamics was evaluated, by comparing the resolved velocity field in patient-specific aneurysms with experimental methods, such as particle image velocimetry (PIV) and phase-contrast magnetic resonance imaging (PCMRI). By comparing against a physical stent model, the PM model stent was found to be a practical tool to assist flow-iversion simulation. In Chapter 5 and 6, the PM model thickness range that would help to retain the simulation benefits without compromising the accuracy was first confirmed; then PM model FD stents were respectively calibrated to reflect the flow resistance created by several treatment modes using commercially available FD stents. Overall, CFD prediction is proved to be able to accurately resolve the aneurysmal flow dynamics , and the investigation of calibrated PM stent modelling provides…