Neurological diseases are devastating illnesses that affect over one billion people worldwide. Drosophila melanogaster provides a genetically tractable system in which to study gene function and the mechanisms of pathogenesis of neurological diseases. In this study I have investigated the function of survival motor neuron (SMN), the causative gene in the neuromuscular disease spinal muscular atrophy (SMA), in growth and differentiation in Drosophila. In addition, I have used the fruit fly to investigate a putative modifier of a previously characterised Drosophila model of Parkinson's disease. Spinal muscular atrophy is an autosomal recessive neurological disease that is characterised by motor neuron loss resulting in muscle weakness. The disease is caused by the deletion or mutation of the survival motor neuron (SMN) gene. In Drosophila, SMN was found to be highly expressed in dividing tissues and a reduction in SMN levels resulted in growth defects, stem cell defects and developmental delay. SMN was also shown to regulate chromosome morphology of the endocycling nurse cells of the female germline. Therefore it appears that SMN has a role in growth control and development in Drosophila. Parkinson's disease is a common disorder that results in widespread neurodegeneration with a predilection for dopaminergic neuron loss resulting in movement defects. A defining neuropathological feature of the disease is the presence of alpha-synuclein containing inclusions. Using a Drosophila model of PD, I have shown that specific alpha-synuclein-induced phenotypes in the fly can be suppressed by the overexpression of the E3 ubiquitin ligase, Nedd4.

Although death due to lethal cardiac arrhythmias is the leading cause of mortality in Western Society, many of the fundamental mechanisms underlying their onset, maintenance and termination, still remain poorly understood. In recent years, experimental techniques such as optical mapping have provided useful high-resolution recordings of cardiac electrical dynamics during complex arrhythmias and defibrillation episodes, which have been combined with detailed computer simulations to further our understanding of these phenomena. However, mechanistic enquiry is severely restricted as the optical mapping technique suffers from a number of distortion effects which compromise the fidelity of the experimental measurements, presenting difficulties in the comparison of experimental data with computational simulations. This Thesis presents a thorough investigation into the distortion effects encountered in optical mapping experiments, guided by the development of a coherent series of computational models. The models presented successfully characterise the specific mechanisms of fluorescent signal distortion due to photon scattering. Photon transport in cardiac tissue is modelled using both continuous (reaction-diffusion) and discrete stochastic (Monte Carlo) approaches to simulate the effects of photon scattering within the myocardium upon the recorded fluorescent signal, which include differing levels of detail and associated computational complexity. Specifically, these models are used to investigate the important role played by the complex ventricular structural anatomy, as well as the specifics of the experimental set-up itself. In addition, a tightly coupled electromechanical model of a contracting cardiac fibre is developed which provides an important first-step towards the development of a model to quantitatively assess the distortion observed when recording from a freely contracting cardiac preparation. Simulation of these distortion effects using the models allows discrimination to be made between those parts of the experimental signal which are due to underlying tissue electrophysiology and those due to artifact, facilitating a more accurate interpretation of experimentally-obtained data. The models presented succeed in two main respects. Firstly, they provide a ‘post-processing’ tool which can be added on to computational simulations of electrical activation, allowing for a more accurate and faithful comparison between simulations and experiments, helping to validate predictions made by electrical models. Secondly, they provide a higher degree of mechanistic insight into the fundemental ways in which optical signals are distorted, showing how this distortion can be maximised or controlled. The understanding and quantification of the fundemental mechanisms of optical mapping signal distortion, provided by this Thesis, therefore fulfils an important role in the study of arrhythmia mechanisms.

Thesis (M.D.S.) - Department of Dental Health, University of Adelaide, 1982. / Typescript (photocopy).

No

Parkinson's disease (PD) is an incurable neurodegenerative motor disorder caused by the inexorable loss of dopamine neurones from the substantia nigra pars compacta. Cell loss is characterised by the perturbation of multiple physiological processes (including mitochondrial function, autophagy and dopamine homeostasis) and much of this pathophysiology can be reproduced in vitro using the mitochondrial toxin MPP+ (1-methyl-4-phenylpyridinium). It was hypothesised that MPP+ toxicity could be modelled using protein-protein interaction networks (PPIN) in order to better understand the interplay of systems-level processes that result in eventual cell death in MPP+ models and PD. Initially, MPP+ toxicity was characterised in the human, dopamine-producing cell line BE(2)-M17 and it was confirmed that the neurotoxin resulted in time and dose dependent apoptosis. A radio-label pulse-chase assay was developed and demonstrated that MPP+ induced decreased autophagic flux preceded cell death. Autophagic dysfunction was consistent with lysosome deacidification due to cellular ATP depletion. Pertinent PPINs were sampled from publically available data using a seedlist of proteins with validated roles in MPP+ toxicity. These PPINs were subjected to a series of analyses to identify potential therapeutic targets. Two topological methods based on betweenness centrality were used to identify target proteins predicted to be critical for the crosstalk between mitochondrial dysfunction and autophagy in the context of MPP+ toxicity. Combined knockdown of a subset of target proteins potentiated MPP+ toxicity and the combined resulted in cellular rescue. Neither of these effects was observed following single knockdown/overexpression confirming the need for multiple interventions. Cellular rescue occurred via an autophagic mechanism; prominent autophagosomes were formed and it was hypothesised that these structures allowed for the sequestration of damaged proteins. This thesis demonstrates the value of PPINs as a model for Parkinson's disease, from network creation through target identification to phenotypic benefit.

Innate immune activation is thought to play a central role in IBD pathogenesis because genetic polymorphisms in NOD2 and NLRP3, cytosolic innate immune receptors belonging to the NLR family, have been associated with IBD susceptibility. However, the mechanisms through which NLR mutations predispose to IBD remain unclear. The aim of this project was to dissect the functional roles of different NLRs in intestinal inflammation. Using the well-established DSS-induced colitis model as well as experimental models of IBD based on infection with Helicobacter hepaticus, we found that Nod2 expression was significantly increased at the peak of intestinal inflammation, and remained elevated throughout the resolution process. This observation suggests a possible role for Nod2 in the resolution of inflammation. Conversely, upon infection with the acute intestinal pathogen Citrobacter rodentium, Nlrp3-/- mice suffered from increased bacterial colonization as early as 3 days post infection, resulting in exacerbated intestinal inflammation and severe weight loss. Analysis of irradiation bone marrow chimeras showed that the protection required Nlrp3 activation in the non-haematopoietic compartment. Furthermore, this protective mechanism was independent of the inflammasome-associated cytokines IL-1β or IL-18. Therefore, this study implicates Nlrp3 activation in intestinal tissue cells as having a crucial role in controlling pathogenic bacterial colonization, providing a potential mechanism by which NLRP3 polymorphisms could lead to increased susceptibility to IBD.

Sudden cardiac death represents one of the leading causes of death worldwide, with the majority of these deaths caused by arrhythmias derived from ischæmic events. However, the mechanisms leading from ischæmia to re-entry, arrhythmia and eventual death are poorly understood. Furthermore, variability in the action potential of cardiac tissue, while important in determining arrhythmic risk, is only recently being addressed in computational modelling, with little known about the causes and mechanisms underlying it, nor regarding its evolution in response to pathological conditions such as ischæmia. This dissertation investigates the causes of variability in the repolarisation of the action potential of the rabbit ventricular myocyte, and the response of this variability to ischæmia. The effect of variability in ion channel conductances is investigated by means of a complete search of the parameter space revealed by simultaneous variation in multiple parameters describing ion channel conductances in computational models of the rabbit ventricular action potential. Rabbit data and models are used in this thesis due to the similarities to human data, both in terms of electrophysiology generally, and the response to ischæmia specifically. The response of two different model frameworks is assessed to determine similarities and differences between model frameworks that are designed to reproduce the same system. Those models producing action potential durations that fall within an experimentally derived range at multiple pacing rates are used to define model populations that thus reproduce experimental variability in repolarisation. These model populations are used to investigate the effects of ischæmic conditions on population variability. Variability is measured not only for action potential duration, but also for other biomarkers commonly implicated in the development of re-entry. The work presented in this dissertation is significant for: (1) presenting a comprehensive study of the effect of simultaneous variation in ion channel conductances, with details regarding the interactions between conductances and how these interactions change depending on the pacing rate; (2) detailed examination of the differences between two models of the same system; (3) production of the largest extant populations reproducing experimentally observed variability in action potential duration; (4) the first time model populations have been used to investigate the effects of ischæmia on variability.

Thesis (Ph. D.)--Rutgers University, 2010. / "Graduate Program in Chemistry and Chemical Biology." Includes bibliographical references (p. 153-165).

An increasingly important research area within the field of cardiac modelling is the development and study of methods of model-based parameter estimation from clinical measurements of cardiac function. This provides a powerful approach for the quantification of cardiac function, with the potential to ultimately lead to the improved stratification and treatment of individuals with pathological myocardial mechanics. In particular, the diastolic function (i.e., blood filling) of left ventricle (LV) is affected by its capacity for relaxation, or the decay in residual active tension (AT) whose inhibition limits the relaxation of the LV chamber, which in turn affects its compliance (or its reciprocal, stiffness). The clinical determination of these two factors, corresponding to the diastolic residual AT and passive constitutive parameters (stiffness) in the cardiac mechanical model, is thus essential for assessing LV diastolic function. However these parameters are difficult to be assessed in vivo, and the traditional criterion to diagnose diastolic dysfunction is subject to many limitations and controversies. In this context, the objective of this study is to develop model-based applicable methodologies to estimate in vivo, from 4D imaging measurements and LV cavity pressure recordings, these clinically relevant parameters (passive stiffness and active diastolic residual tension) in computational cardiac mechanical models, which enable the quantification of key clinical indices characterising cardiac diastolic dysfunction. Firstly, a sequential data assimilation framework has been developed, covering various types of existing Kalman filters, outlined in chapter 3. Based on these developments, chapter 4 demonstrates that the novel reduced-order unscented Kalman filter can accurately retrieve the homogeneous and regionally varying constitutive parameters from the synthetic noisy motion measurements. This work has been published in Xi et al. 2011a. Secondly, this thesis has investigated the development of methods that can be applied to clinical practise, which has, in turn, introduced additional difficulties and opportunities. This thesis has presented the first study, to our best knowledge, in literature estimating human constitutive parameters using clinical data, and demonstrated, for the first time, that while an end-diastolic MR measurement does not constrain the mechanical parameters uniquely, it does provide a potentially robust indicator of myocardial stiffness. This work has been published in Xi et al. 2011b. However, an unresolved issue in patients with diastolic dysfunction is that the estimation of myocardial stiffness cannot be decoupled from diastolic residual AT because of the impaired ventricular relaxation during diastole. To further address this problem, chapter 6 presents the first study to estimate diastolic parameters of the left ventricle (LV) from cine and tagged MRI measurements and LV cavity pressure recordings, separating the passive myocardial constitutive properties and diastolic residual AT. We apply this framework to three clinical cases, and the results show that the estimated constitutive parameters and residual active tension appear to be a promising candidate to delineate healthy and pathological cases. This work has been published in Xi et al. 2012a. Nevertheless, the need to invasively acquire LV pressure measurement limits the wide application of this approach. Chapter 7 addresses this issue by analysing the feasibility of using two kinds of non-invasively available pressure measurements for the purpose of inverse parameter estimation. The work has been submitted for publication in Xi et al. 2012b.

Novel infectious diseases in humans are of great concern to public health authorities and researchers in epidemiology. Zoonotic pathogens in particular have the potential to cause epidemics without any or little warning. In this thesis, I investigate evolutionary and environmental conditions, and the interactions between both, which facilitate the zoonotic emergence of novel pathogens. I start with a list of the mechanisms and processes which might influence a zoonotic emergence, and identify some unsolved problems. I address these with multiple, theoretical models. First, I use a village-city model with different adaptation scenarios to examine the influence of spatial heterogeneity on the emergence process. I derive general analytical results for the statistical properties of emergence events, including the probability distribution of outbreak sizes. My results suggest that, for typical connection strengths between communities, spatial heterogeneity has only a weak effect on outbreak size distributions, and on the risk of emergence per introduction. Next, I extend the research on environmental conditions by looking at pathogen specialisation in multi-host systems. I derive threshold connectivities for which generalist pathogens, which infect multiple species and might therefore be more dangerous to cross into the human species, can sustain transmission and are not dominated by specialists, which can only cause sustained transmission chains in a single host species, but are able to cause emergences with little warning. My third research chapter is interested in the effect of the loss of biodiversity. I analytically derive expected prevalences for fast growing and slow growing species. If fast growing species tend to perform better in degraded environments, my analytical results suggest that the overall prevalence level of infectious diseases will rise as environments degrade, which facilitates the chance of zoonotic jumps. In my last research chapter, I examine the actual impact of a novel, emerging infectious disease. I use data from the recent `Swine flu' epidemic in England to estimate epidemiological parameters of the infectious agent. My results suggest that the majority of infected cases showed no or only mild symptoms. This reveals that more data than just the estimated number of cases are necessary to fully evaluate the danger of a possible zoonotic, emerging infectious disease. I conclude by discussing my results and the implications which these might have.