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The theory and the practical application of the simulation of root infection of winter wheat by the take-all fungus, <i>Gaeumannomyces graminis</i> var. <i>tritici</i>, are critically evaluated with respect to field epidemics and to infection of seedlings within controlled environments. Several simple models for disease progress in field epidemics are evaluated with respect to field data, including a generalized logistic equation and systems of simple non-linear differential equations, with and without algebraic solutions. An investigation is made of disease heterogeneity in the field and transect data derived from sampling 11,000 plants are analysed for the presence of significant pattern. The effect of the observed spatial heterogeneity on the precision of field data is also empirically investigated. The use of a controlled-environment experiment to model the effect of volunteer infestation on inoculum survival in the field is demonstrated, and a simple model is used to quantitatively estimate the effect of volunteer infestation on inoculum multiplication. Data for a seedling disease epidemic are simulated by three mathematically and computationally diverse simulators derived from a single underlying theoretical model. The first is a complex simulator written in FORTRAN and run on a mainframe computer which resolves the infection process into a number of detailed submodels. The second simulator is written in BBCBASIC and 6502 machine code and makes use of a discrete root map to hold information on host growth and infection. In the third simulator the model is expressed as a series of rate equations and is run on a simulation package on the BBC microcomputer. The simulation techniques used are discussed and evaluated with respect to model development and the descriptive accuracy of the simulators. In conclusion a strategy is proposed for the development of a comprehensive model for field epidemics of take-all by means of controlled-environment experimentation.

While there has been an opulence of data and studies surrounding the study of the developing pancreas in mammals and other vertebrates, the focus has largely been in mice. The paucity of research in the development of the human pancreas has led to diminished knowledge in the area, compared to other species. Recent discoveries provide growing evidence for discrepancies between mouse and human pancreatic development and diseases and highlight the fact that developmental studies of the pancreas in humans are imperative. The need to develop therapies for diabetes, a growing and one of the leading health problems worldwide, further compels more exploration in this area to deepen our understanding in the different aspects of diabetes in humans and its underlying causes. Research involving modelling human diseases in vitro enables the investigation of the cellular and molecular mechanisms underlying these diseases as well as the development of therapies for treating them. The availability of hPSCs brings with it the advantage of overcoming the limitations of animal models for certain disorders such as pancreatic agenesis, the focus of my project. The use of site-specific nucleases such as TALENs for such a purpose represents a paradigm shift in disease modelling, where TALENs are capable of directly correcting disease-causing mutations, therefore permanently eliminating the symptoms with precise genome modifications. Alternatively, TALENs can also be used to inactivate specific genes by inducing site-specific mutations. Using these tools, I found that GATA6 is required for the formation of the definitive endoderm (DE) and pancreas in humans; hPSCs harbouring homozygous GATA6 mutations fail to form the definitive endoderm, and consequently the pancreas, whereas hPSCs harbouring heterozygous GATA6 mutations exhibited impairment in definitive endoderm development, although it remains unclear if this is a protocol dependent defect. At the pancreatic stage, heterozygous GATA6 mutations consistently compromised pancreas formation regardless of protocol used. I also found that GATA6 transcriptionally activates the development of the definitive endoderm and pancreatic endoderm, and possibly represses the development of mesoderm. Furthermore, I also established that GATA6 directly interacts with key definitive endoderm markers CXCR4 and SOX17, and pancreatic marker PDX1. Taken together, the work herein demonstrates the successful use of hPSCs coupled with the TALEN genome editing technology as a unique in vitro system for disease modelling. These findings also establish two developmental windows, the DE and pancreatic progenitor stages, where GATA6 haploinsufficiency can result in the impairment of pancreatic development leading to pancreatic hypoplasia observed in human GATA6 heterozygous patients. Lastly, my work also provides the molecular mechanism by which GATA6 regulates pancreatic development. Overall, this study provided new insights in the role of GATA6 during development of the human pancreas. These results will be important in developing new methods of differentiation for hPSCs and understanding the interconnection between early organogenesis and late onset of diabetes.

There exists a need to estimate the potential financial, epidemiological and societal impact that diseases, and the treatment thereof, can have on society. Markov processes are often used to model diseases to estimate these quantities of interest and have an advantage over standard survival analysis techniques in that multiple events can be studied simultaneously. The theory of Markov processes is well established for processes for which the process parameters are known but not as much of the literature has focussed on the estimation of these transition parameters. This dissertation investigates and implements maximum likelihood estimators for Markov processes based on longitudinal data. The methods are described based on processes that are observed such that all transitions are recorded exactly, processes of which the state of the process is recorded at equidistant time points, at irregular time points and processes for which each process is observed at a possibly different irregular time point. Methods for handling right censoring and estimating the effect of covariates on parameters are described. The estimation methods are implemented by simulating Markov processes and estimating the parameters based on the simulated data so that the accuracy of the estimators can be investigated. We show that the estimators can provide accurate estimates of state prevalence if the process is stationary, even with relatively small sample sizes. Furthermore, we indicate that the estimators lack good accuracy in estimating the effect of covariates on parameters unless state transitions are recorded exactly. The methods are discussed with reference to the msm package for R which is freely available and a popular tool for estimating and implementing Markov processes in disease modelling. Methods are mentioned for the treatment of aggregate data, diseases where the state of patients are not known with complete certainty at every observation and diseases where patient interaction plays a role. / Dissertation (MSc)--University of Pretoria, 2010. / Statistics / unrestricted

The ability to generate cardiomyocytes from either embryonic stem cells or induced pluripotent stem cells provides an unprecedented opportunity to establish human in vitro models of cardiovascular disease as well as to develop platforms for the testing of novel cardiac therapeutics. We designed two different platforms, a biowire platform and post deflection platform, to generate engineered heart tissues (EHTs) to study a fundamental process in cardiomyocytes: hypertrophy. Both pathological and physiological hypertrophy was studied in order to garner a better understanding of each process. Physiological hypertrophy characteristics were observed using the biowire platform seen in improved myofibril alignment and downregulation of fetal genes. When electrical stimulation was added, a rate dependent effect on sarcomere maturation was observed by the increased frequency of I-bands and H-zones. Certain hallmark features of pathological hypertrophy, such as upregulation of brain natriuretic peptide and sarcomere structure breakdown, were recapitulated when EHTs were treated with isoproterenol.

The ability to generate cardiomyocytes from either embryonic stem cells or induced pluripotent stem cells provides an unprecedented opportunity to establish human in vitro models of cardiovascular disease as well as to develop platforms for the testing of novel cardiac therapeutics. We designed two different platforms, a biowire platform and post deflection platform, to generate engineered heart tissues (EHTs) to study a fundamental process in cardiomyocytes: hypertrophy. Both pathological and physiological hypertrophy was studied in order to garner a better understanding of each process. Physiological hypertrophy characteristics were observed using the biowire platform seen in improved myofibril alignment and downregulation of fetal genes. When electrical stimulation was added, a rate dependent effect on sarcomere maturation was observed by the increased frequency of I-bands and H-zones. Certain hallmark features of pathological hypertrophy, such as upregulation of brain natriuretic peptide and sarcomere structure breakdown, were recapitulated when EHTs were treated with isoproterenol.

The informal settlement of Kibera in Nairobi has a significantly higher prevalence of HIV than the Kenyan average. Sex workers and their male clients from Kibera were recently surveyed; their responses indicate a population that is well mixed with large variance in individual number of sexual contacts. Hence, a multigroup two-sex model is created to study HIV spread in the sex working population of Kibera. This model is parameterized to the Kibera data, and the e ffects of various parameters on the prevalence of HIV and the risk of infection to individuals are studied by an elasticity analysis. The probability of infection from males to females per sexual act has the greatest implications for HIV control. A simplified model is presented and a theoretical analysis gives the reproduction number and proves global stability of the endemic equilibrium. / Graduate

Cardiovascular disease, including coronary artery disease and aortic valve stenosis, impacts tens of millions of people annually and carries a massive global economic burden. Advances in medical imaging, hardware and software are leading to an increased interest in the field of cardiovascular computational modelling to help combat the devastating impact of cardiovascular disease. Lumped parameter modelling (a branch of computational modelling) holds the potential of aiding in the early diagnosis of these diseases, assisting clinicians in determining personalized and optimal treatments and offering a unique in-silico setting to study cardiac and circulatory diseases due to its rapid computation time, ease of automation and relative simplicity. In this thesis, cardiovascular lumped parameter modelling is presented in detail and a patient-specific framework capable of simulating blood flow waveforms and hemodynamic data in the heart and coronary arteries was developed. The framework used only non-invasive clinical data and images (Computed Tomography images, echocardiography data and cuff blood pressure) as inputs. The novel model was then applied to 19 patients with aortic stenosis who underwent transcatheter aortic valve replacement. The diastolic coronary flow waveforms in the left anterior descending artery, left circumflex artery and right coronary artery were validated against a previously developed patient-specific 3D fluid-structure interaction model for all 19 subjects (pre and post intervention). There were strong qualitative and quantitative agreements between the two models. After the procedure, aortic valve area and net pressure gradient across the aortic valve improved for almost all the subjects. As for the hemodynamic data, according to the model, there was substantial variability in terms of the increase or decrease post intervention. On average, left ventricle workload and maximum left ventricle pressure decreased by 4.5% and 13.0% while cardiac output, mean arterial pressure and resting heart rate increased by 9.9%, 6.9% and 1.9% respectively. There were also subject specific changes in coronary blood flow (37% had increased flow in all three coronary arteries, 32% had decreased flow in all coronary arteries, and 31% had both increased and decreased flow in different coronary arteries). All in all, a proof-of-concept cardiac and coronary lumped parameter framework was developed, validated, and applied in this thesis. / Thesis / Master of Applied Science (MASc) / The heart is a vital part of the cardiovascular system, which helps deliver and regulate blood flow through the entire human body. The coronary arteries are a crucial part of this system since they deliver blood directly to heart muscles. For numerous reasons, the cardiovascular system can become diseased over time and require clinical treatment. Coronary artery disease and aortic valve stenosis are among the most prevalent cardiovascular diseases globally. While medical imaging on its own is a crucial part of the disease management and treatment process, advanced computational models can further enhance the process and provide clinics with data and predictions they might otherwise miss. In this thesis, a patient specific computational framework capable of simulating blood flow waveforms and cardiovascular data in the heart and coronary arteries using only non-invasive clinical data and images was developed and validated. The novel model was applied to a series of patients with aortic stenosis who underwent heart valve replacement with the aim of studying the impact on coronary blood flow and global cardiovascular metrics.

O Zika vírus (ZIKV) é um flavivírus transmitido pelo mosquito Aedes aegypti e que se espalhou rapidamente pelas Américas, causando uma epidemia no Brasil em 2015 . Um número crescente nos casos de infecções veio acompanhado de um aumento no número de fetos e bebês nascidos com microcefalia, levando a um chamado de emergência mundial de saúde. Históricamente, o ZIKV não havia causado infecções de destaque em humanos e a reermegência dessa ameça viral associada à defeitos do nascimento foi logo relacionada à evolução e consequente distinção entre os genótipos virais, o original Zika africano e seu descendente Zika asiático, que chegou ao Brasil. A hipótese da cepa brasileira do ZIKV ser a causadora de microcefalia e de outros defeitos do nascimento ganhou mais respaldo após a identificação do vírus em amostras de tecido cerebral e líquido amniótico de fetos. Posteriormente, a associação direta entre microcefalia e a síndrome congênita com o ZIKV foi confirmada por meio da aplicação de modelos biológicos experimentais que se revelaram susceptíveis à infecção viral, como células do sistema nervoso central em sistemas 2D e 3D in vitro e camundongos prenhês. Esse trabalho teve como objetivo investigar a infecção da cepa brasileira do ZIKV (ZIKVBR) em diferentes células humanas neurais in vitro diferenciadas a partir de células-tronco pluripotentes induzidas, além de criar uma plataforma para teste de fármacos in vitro contra o vírus. Nossos resultados comprovaram a susceptibilidade e permissividade celular à infecção do ZIKVBR em células neuronais e, em especial, progenitoras neurais, causando morte celular por apoptose. Além disto, quando células progenitoras neurais foram cultivadas em suspensão, formando neuroesferas, o ZIKVBR foi capaz de causar uma redução na população de células, gerando uma anormalidade morfológica semelhante à microcefalia. Além do mais, quando células progenitoras neurais infectadas com ZIKVBR foram diferenciadas em neurônios maduros, a análise da sinaptogênese revelou que esses neurônios apresentavam uma menor densidade de puncta sináptica, indicando um comprometimento no funcionamento das sinapses que pode estar contribuindo para os problemas associados com a síndrome congênita do ZIKV. Por fim, o tratamento dessas células com a droga Sofosbuvir, um inibidor de RNA polimerase dependente de RNA aprovado para uso clínico, foi capaz de resgatar NPCs e neurônios apoptóticos. Em suma, nossos dados indicam que o ZIKVBR infecta preferencialmente células progenitoras neurais, replicando-se eficientemente e causando morte por apoptose nessas células e neurônios maduros diferenciados de células progenitoras neurais infectadas apresentam uma menor desidade de puncta sináptica. Finalmente, a reutilização de compostos farmacêuticos já aprovados para uso clínico pode acelerar o tratamento para indivíduos infectados pelo ZIKV onde a prevenção já não é mais opção, como no caso de mulheres grávidas. / Zika virus (ZIKV) is a mosquito-borne flavivirus transmitted by Aedes aegypti that has rapidly spread through the Americas, causing a widespread epidemic in Brazil in 2015. A increasing number of infection cases was followed by a rise in the number of fetuses and babies born with microcephaly, leading to a global health emergency call. Up to then, ZIKV had not caused meaningful infections in humans and the reemergency of this viral threat associated with birth defects was soon related to viral genotype mutations and its consequent distinction from the original african Zika strain to its descendent asian Zika strain, which reached Brazil. The hypotesis of the brazilian ZIKV strain being responsible for microcephaly and other birth defects gained support after the isolation and identification of the virus in samples of cerebral tissue and amniotic fluid of fetuses. Subsequently, the direct association between microcephaly and congenital syndrome with ZIKV was confirmed through the application of biologic experimental models which proved susceptible to viral infection, as for cells from the central nervous system cultured in 2D and 3D models as well as pregnant mice. The aim of this study was to investigate the brazilian ZIKV strain (ZIKVBR) infection in different human neural cells in vitro differentiated from induced pluripotent stem cells, as well as creating a platform for in vitro drug testing with antiviral capabilities. Our results showed cellular infection susceptibility and permissiveness to ZIKVBR in neurons and, specially, neural progenitor cells, displaying cell death by apoptosis. Futhermore, when neuronal progenitor cells cultured in suspension, forming neurospheres, were infected with ZIKVBR, it caused a reduction in cell population, displayed by evident morphological abnormalities resembling to microcephaly. Additionally, when neural progenitor cells infected with ZIKVBR were diferentiated further into mature neurons, synaptogenesis analysis revealed these neurons displayed fewer synaptic puncta density, indicating a compromise in synapse functioning that may be contributing to problems associated with ZIKV congenital syndrome. Moreover, cell treatment with Sofosbuvir, a RNA polymerase RNAdependent inhibitor approved for clinical use, was able to rescue apoptotic NPCs and neurons. In summary, our results reveal that ZIKVBR preferentially infects neural progenitor cells, efficiently replicating itself and causing death by apoptosis in these cells and mature neurons differentiated from infected neural progenitor cells display reduced synaptic puncta density. Lastly, the repurpose of FDA approved compounds may aid in accelerating treatment for infected individuals whose prevention is no longer an option, as it is for pregnant women.

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