Tropical – software for quantitative analysis of FRAP experiments : identifying the dynamics of linker histone H1°

Fluorescence recovery after photobleaching (FRAP) experiments using laser scanning microscopes to follow the in vivo dynamics of proteins tagged to fluorescent markers like the green fluorescent protein (GFP) has become a standard method in cell biology. FRAP perturbs the fluorescence distribution by photobleaching GFPtagged proteins in a specific area of a cell and monitors the fluorescence redistribution. Adequate methods to quantify the results of FRAP experiments have recently became available. Those methods allow the extraction of diffusion coefficients and dissociation constants of proteins diffusing inside distinct cellular compartments and undergoing dynamic binding and dissociation with immobile or mobile binding sites. However, software incorporating such methods was not available until now. Therefore I developed Tropical, a software for simulation and parameter estimation of reaction–diffusion models. Based on spatio-temporal microscopy images, Tropical estimates reaction and diffusion coefficients for user-defined models. Tropical allows the investigation of systems with an inhomogeneous distribution of molecules, making it well suited for quantitative analyses of microscopy experiments such as FRAP. Tropical was used in this thesis to analyze the dynamic behavior of linker histone H1°, which plays a crucial role in the dynamic organization of chromatin by stabilizing the nucleosome, a structure involved in DNA packing in eucaryotic cells. FRAP experiments were performed using three forms of linker histone H1°, the wild type, and two forms with mutated sites, that are likely to play a major role in DNA binding. Diffusion coefficients on the three forms were estimated with Tropical by fitting a pure diffusion model, assuming binding to happen instantaneously. The model showed a very good fit to the experimental data. It could be shown that lysine 52 significantly influences the DNA binding properties of H1° and its mutation resulted in a 3-fold enhanced diffusion coefficient. The H1° form containing six point mutations however showed an even higher diffusion coefficient, about 15 times higher than the one of the wild type histone, revealing a much larger contribution to DNA binding of these six mutated sites. Using Tropical to estimate the diffusion coefficients of linker histone H1° was another proof for the power and functionality of Tropical, besides the recently published one (Ulrich et al. 2006). Tropicals’ main advantages are (1) that it directly operates on microscopy images, (2) an inhomogeneous distribution of binding partners can be considered and (3) the obtained result can directly be verified. This thesis will first line out the current knowledge of eucaryotic chromatin organization, to clarify the role of linker histone H1. I will then give an overview of microscopy techniques available to reveal protein dynamics and their quantitative analysis using mathematical models. The next part will explain Tropical and its implemented methods in detail. Finally I will present the results obtained on the dynamics of H1° and critically discuss the applicability of Tropical to analyze FRAP data and FRAP as a method to reveal protein dynamics

A stochastic model of malaria transmission

Malaria models have evolved since Ross and Macdonald. By using an agent-based stochastic model we have looked into di erent aspects of disease transmission: 1. Gametocytemia phase transition between epidemic stability and disease elimination, and the potential bene t of combining gametocidal agents and ivermectin. 2. Heterogeneity promotes disease spreading. 3. Disease supression from the combined use of ivermectin and primaquine. 4. Utility of Hurst exponent and Shannon entropy in malaria forecasting. Results and conclusion: Malaria transmission was simulated with a computational agent-based model assuming a small African village. We have con rmed gametocytemia as a critical factor in disease transmission, revealing an abrupt phase transition between epidemic stability and disease elimination [326]. We have also found that synergism between gametocidal agents (primaquine) and ivermectin (a selective Anophelocide drug a ecting parasite maturation after mosquito infection) could e ectively suppress human-to-mosquito disease transmission [326]. We have found that heterogeneity ampli es disease transmission (roughly three times in our model). Different aspects of heterogeneity were analyzed such as human migration, mosquito density, and rainfall [327]. We have con rmed the potential bene t of suppressing heterogeneity-induced disease transmission with the use of gametocidal agents and ivermectin. Hurst exponent has been used in hydrology and in the stock market. No previous evidence of its application to infectious theory has been found. Yet, our data suggests that Hurst exponent and information entropy could be useful in malaria forecasting [328]. Our results support the combined use of gametocidal agents (primaquine or methylene blue) and ivermectin as part of an integrated approach to malaria.Os modelos de malária são úteis desde Ross e Macdonald. Através de um modelo estocástico de agente, foram analisados vários aspectos da transmissão da malária: 1. A existência de uma transição de fase entre estabilidade e eliminação da doença em função da gametocitemia. 2. O uso combinado de fármacos gametocidas e ivermectina na redução da transmissão. 3. O papel da heterogeneidadena propagação da malária. 4. A utilidade do expoente de Hurst e da entropia de Shannon na previão da malária. Resultados e conclusões: Foi utilizado um modelo computacional de agente com simulação da transmissão de malária numa pequena aldeia africana. Confirmámos a gametocitemia como um factor crítico na propagação da malária demonstrando uma transição abrupta de fase entre estabilidade epidémica e eliminação da doença. No nosso modelo foi demonstrado que na presença de heterogeneidade a transmissão de malária pode sofrer uma amplificação significativa, de aproximadamente três vezes. Foram analisados diferentes aspectos da heterogeneidade tais como a migração humana, a densidade vectorial e a precipitação sazonal. Foi confirmado o potencial benefício de supressão da transmissão da malária na presença de heterogeneidade com a utilização de fármacos gametocidas (primaquina) e ivermectina. O expoente de Hurst tem sido aplicado com sucesso nas áreas da hidrologia e do mercado bolsista. Não houve até agora evidência da sua aplicação à área da infecciologia. No entanto, os dados apresentados sugerem a sua utilidade, a par da entropia de Shannon, na previsão da incidência da malária. Foi demonstrado que o uso combinado de agentes gametocidas (primaquina ou azul de metileno) e ivermectina pode constituir uma abordagem eficaz na prevenção da malári

Role of food material properties on the mechanisms of solid food disintegration during gastric digestion : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand

The stomach is, after the mouth, the major organ for the breakdown of foods by a complex interaction of biochemical and mechanical mechanisms driven by the diffusion of gastric juice and the peristaltic activity of the stomach. The degree of fragmentation of solid food in the stomach and consequent release of nutrients is largely dependent on the food material properties. Despite extensive research directed at the gastric digestion, the establishment of the proper relationship between the initial material properties of foods and their subsequent breakdown during gastric digestion is still far from being fully understood. To bridge the aforementioned knowledge gap, the aim of this thesis was to characterise the relationship between material properties of solid foods (composition and structure) and their disintegration behaviour in the stomach. Sweet potato (steamed and fried) and egg white gels (pH 5 and pH 9 EWGs) were used as starch and protein based-product models, respectively, to develop experimental models to characterise not only the diffusion of gastric juice into the food matrix, but also the mechanisms underlying the biochemical and mechanical degradation of the food matrix during in vitro gastric digestion. Overall results revealed that the porous network created during frying facilitated a faster gastric acid penetration into the sweet potato food matrix than occurred in the less porous steamed sweet potato. Consequently, the fried sweet potato matrix underwent a faster collapsing and quicker softening time during in vitro gastric digestion than the more compact and denser structure of steamed sweet potato. This led to the faster disintegration and subsequent release of β-carotene in the human gastric simulator from the fried sweet potato matrix. A similar effect was demonstrated with the EWG, where the loose protein network of pH 5 EWG exhibited a significantly higher rate of pepsin diffusion, softening, nutrient release and mechanical breakdown compared to the more tightened gel microstructure found in the pH 9 EWG. In conclusion, gastric disintegration and nutrient release within the solid food structures are mainly controlled by the initial food microstructure and composition. Such knowledge will help to identify key factors for the designing of health-promoting food formulations

High-Performance Modelling and Simulation for Big Data Applications

This open access book was prepared as a Final Publication of the COST Action IC1406 “High-Performance Modelling and Simulation for Big Data Applications (cHiPSet)“ project. Long considered important pillars of the scientific method, Modelling and Simulation have evolved from traditional discrete numerical methods to complex data-intensive continuous analytical optimisations. Resolution, scale, and accuracy have become essential to predict and analyse natural and complex systems in science and engineering. When their level of abstraction raises to have a better discernment of the domain at hand, their representation gets increasingly demanding for computational and data resources. On the other hand, High Performance Computing typically entails the effective use of parallel and distributed processing units coupled with efficient storage, communication and visualisation systems to underpin complex data-intensive applications in distinct scientific and technical domains. It is then arguably required to have a seamless interaction of High Performance Computing with Modelling and Simulation in order to store, compute, analyse, and visualise large data sets in science and engineering. Funded by the European Commission, cHiPSet has provided a dynamic trans-European forum for their members and distinguished guests to openly discuss novel perspectives and topics of interests for these two communities. This cHiPSet compendium presents a set of selected case studies related to healthcare, biological data, computational advertising, multimedia, finance, bioinformatics, and telecommunications

Advancing Knowledge on Cyanobacterial Blooms in Freshwaters

Cyanobacterial blooms are a water quality problem that is widely acknowledged to have detrimental ecological and economic effects in drinking and recreational water supplies and fisheries. There is increasing evidence that cyanobacterial blooms have increased globally and are likely to expand in water resources as a result of climate change. Of most concern are cyanotoxins, along with the mechanisms that induce their release and determine their fate in the aquatic environment. These secondary metabolites pose a potential hazard to human health and agricultural and aquaculture products that are intended for animal and human consumption; therefore, strict and reliable control of cyanotoxins is crucial for assessing risk. In this direction, a deeper understanding of the mechanisms that determine cyanobacterial bloom structure and toxin production has become the target of management practices. This Special Issue, entitled “Advancing Knowledge on Cyanobacterial Blooms in Freshwaters”, aims to bring together recent multi- and interdisciplinary research, from the field to the laboratory and back again, driven by working hypotheses based on any aspect of mitigating cyanobacterial blooms, from ecological theory to applied research