Submitted by LUMEY PEREZ ARTILES null (lumeyp@yahoo.es) on 2017-03-06T20:53:35Z No. of bitstreams: 1 Tese_Lumey_Perez_Artiles.pdf: 1138570 bytes, checksum: 01f489085bac51420d9c3526e6ac70a8 (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-03-13T18:59:39Z (GMT) No. of bitstreams: 1 artiles_lp_dr_jabo.pdf: 1138570 bytes, checksum: 01f489085bac51420d9c3526e6ac70a8 (MD5) / Made available in DSpace on 2017-03-13T18:59:39Z (GMT). No. of bitstreams: 1 artiles_lp_dr_jabo.pdf: 1138570 bytes, checksum: 01f489085bac51420d9c3526e6ac70a8 (MD5) Previous issue date: 2017-01-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A doença huanglongbing (HLB) é considerada um dos maiores problemas fitossanitários da cultura dos citros na atualidade e sua disseminação ocorre principalmente por insetos vectores. Desta forma, o presente trabalho teve por objetivos estudar a biologia e os parâmetros populacionais de Diaphorina citri (vector do HLB) em variedades de citros e determinar o controle natural de Tamarixia radiata sobre as populações de D. citri. A biologia de D. citri foi estudada nos hospedeiros, limão Cravo e Citrumelo ‘Swingle’ e as copas de laranjas Pera, Hamilim e Valência em combinações com estes porta-enxertos. Em cada hospedeiro avaliaram-se a duração das fases de ovo, ninfa e ciclo biológico, viabilidade, fecundidade e razão sexual. Os resultados obtidos do estudo da biologia de D. citri mostram que em Citrumelo ‘Swingle’, a praga apresentou o maior tempo de duração da fase de ovo. A duração do ciclo biológico vario de 15,08 dias em Pêra/Cravo a 16,5 dias em Hamilim/Citrumelo ‘Swingle’, diferenciando-se significativamente. As maiores taxas de sobrevivência e viabilidade para ovo e ciclo biológico foram observadas nas copas de laranjas enxertadas em limoeiro Cravo. Na combinação Valência/limoeiro Cravo o inseto apresentou a maior viabilidade dos ovos (82,02%) e a menor (73,66%) em Hamilim/ Citrumelo ‘Swingle’. Os valores de sobrevivência (ovo-adulto) variaram de 71,07 a 56,16 % entre os hospedeiros avaliados, observando-se que a combinação Pêra/Cravo favorece a sobrevivência do inseto. Com base na tabela de fertilidade, o limoeiro Cravo apresentou a maior taxa liquida de reproduço (160,57) e a menor tempo de duração de cada geração (31,54 dias). Estimativas Jackknife da taxa intrínseca de crescimento (rm) variou de 0,152 em Valencia/Limoeiro Cravo para 0,127 em Citrumelo 'Swingle'. Os resultados indicaram que o porta-enxerto limoeiro Cravo favorece os parâmetros reprodutivos da praga, enquanto o Citrumelo ‘Swingle’ foi menos adequado para o desenvolvimento de D. citri, especialmente na copa Hamilim. Para complementar o segundo objetivo os experimentos sobre o controle natural de T. radiata foram desenvolvidos em Artemisa, Cuba, durante os anos 2007, 2008 e 2009, foram realizadas coletas de 125 brotos jovens em Citrus sinensis com uma frequência quinzenal e foi contabilizada a presença de ninfas de 3o a 5o instar e determinado as porcentagens de parasitismo por T. radiata. Foram determinadas as correlações entre as porcentagens de parasitismo e as variáveis climáticas. Foi encontrada uma correlação positiva (0,545 **, p <0,01) entre o número de ninfas parasitadas por T. radiata e a população total de ninfas de D. citri. Das variáveis climáticas estudadas, as precipitações (-0.388, p = 0,0257), dias com chuva (-0.364, p = 0,0374) e umidade relativa (-0.399, p = 0,0219) foram correlacionadas negativamente com o percentual de parasitismo. A percentagem de parasitismo foi estatisticamente significativa diferente entre estações do ano (p <0,01), sendo maior na primavera e outono e menor durante o verão. Tamarixia radiata mostrou-se ser regulador eficientes das populações de D. citri nas condições avaliadas com porcentagens de parasitismos superiores a 50% na primavera. / Huanglongbing disease (HLB) is considered one of the main phytosanitary problems currently in citrus plantations and its dissemination occurs mainly by insect vectors. Therefore, the present work aims to study the biology and population parameters of Diaphorina citri (vector do HLB) in citrus varieties and to evaluate the natural control of Tamarixia radiata on the populations of D. citri. The biology of D. citri was studied in the hosts Rangpur and Citrumelo ‘Swingle’ and the canopy of oranges Pera, Valencia and Hamilim in the combinations with these rootstocks. In each host the duration of the egg, nymph and biological cycle phases, viability, fertility and sexual proportions were evaluated. As for the biology of D. citri the longer time for the egg phase was obtained in Citrumelo Swingle. The biological cycle duration varied from 15.08 days in Pera/Rangpur to 16.5 in Hamilim/Citrumelo Swingle, differing significantly. The highest survival rates and viability of eggs and biological cycle were observed in the varieties of oranges grafted on lemon Rangpur. In the combination Valencia/Rangpur, the insect has presented the highest viability of eggs (82. 02%) and the lowest viability in Hamilim/Citrumelo Swingle (73. 66%). Survival values (egg-adult) ranged from 71.07 to 56.16% in the evaluated hosts, taking into account that the combination of the Pera/Rangpur favors the survival of the insect. Based on the fertility chart, Rangpur lemon had the highest net reproductive rate (160.57) and the shortest duration of each generation time (31.54 days). Jackknife estimates of rm varied from 0.152 on Valencia/Rangpur to 0.127 on Citrumelo Swingle. The results indicate that the rootstock of Rangpur lemon favors the reproductive parameters of the pest, while Citrumelo Swingle was less suitable for the development of D. citri, especially with Hamilim canopy. To complement the second objective the experiments were carried out in Artemisa, Cuba, during the years 2007, 2008 and 2009. Samples of young shoots of Citrus sinensis (125) were taken with a biweekly frequency and counted the number of nymphs in 3rd and 5th instar and the parasitism of T. radiata was determined. The correlation between climatic variables and parasitism was determined. A positive correlation (0.545 ** p <0.01) between the number of nymphs parasitized by T. radiata and the total population of D. citri nymphs found. Of the climatic variables studied rainfall (-0.388, p = 0.0257), the days with rain (-0.364, p = 0.0374) and relative humidity (-0.399, p = 0.0219) were negatively correlated with the parasitism percentage. Parasitism was statistically different between seasons (p <0.01), being higher in spring and autumn and less during summer. T. radiata proved to be effective in regulating D. citri populations under conditions evaluated with percent parasitisms higher than 50% in spring.

The objective of the field of Synthetic Biology is to implement novel functionalities in a biological context or redesign existing biological systems. To achieve this, it employs tried and tested engineering principles, such as standardisation and the design-build-test cycle. A crucial part of this process is the convergence of modelling and experiment. The aim of this thesis is to improve the design principles employed by Synthetic Biology in the context of Gene Regulatory Networks (GRNs). Small Ribonucleic Acids (sRNAs), in particular, are focussed on as a mechanism for post-transcriptional expression regulation, as they present great potential as a tool to be harnessed in GRNs. Modelling sRNA regulation and its interaction with its associated chaperone Host-Factor of Bacteriophage Q&beta; (Hfq) is investigated. Inclusion of Hfq is found to be necessary in stochastic models, but not in deterministic models. Secondly, feedback is core to the thesis, as it presents a means to scale-up designed systems. A linear design framework for GRNs is then presented, focussing on Transcription Factor (TF) interactions. Such frameworks are powerful as they facilitate the design of feedback. The framework supplies a block diagram methodology for visualisation and analysis of the designed circuit. In this context, phase lead and lag controllers, well-known in the context of Control Engineering, are presented as genetic motifs. A design example, employing the genetic phase lag controller, is then presented, demonstrating how the developed framework can be used to design a genetic circuit. The framework is then extended to include sRNA regulation. Four GRNs, demonstrating the simplest forms of genetic feedback, are then modelled and implemented. The feedback occurs at three different levels: autoregulation, through an sRNA and through another TF. The models of these GRNs are inspired by the implemented biological topologies, focussing on steady state behaviour and various setups. Both deterministic and stochastic models are studied. Dynamic responses of the circuits are also briefly compared. Data is presented, showing good qualitative agreement between models and experiment. Both culture level data and cell population data is presented. The latter of these is particularly useful as the moments of the distributions can be calculated and compared to results from stochastic simulation. The fit of a deterministic model to data is attempted, which results in a suggested extension of the model. The conclusion summarises the thesis, stating that modelling and experiment are in good qualitative agreement. The required next step is to be able to predict behaviour quantitatively.

This thesis is concerned with methodologies for the accurate quantitative modelling of molecular biological systems. The first part is devoted to the chemical Langevin equation (CLE), a stochastic differential equation driven by a multidimensional Wiener process. The CLE is an approximation to the standard discrete Markov jump process model of chemical reaction kinetics. It is valid in the regime where molecular populations are abundant enough to assume their concentrations change continuously, but stochastic fluctuations still play a major role. We observe that the CLE is not a single equation, but a family of equations with shared finite-dimensional distributions. On the theoretical side, we prove that as many Wiener processes are sufficient to formulate the CLE as there are independent variables in the equation, which is just the rank of the stoichiometric matrix. On the practical side, we show that in the case where there are m_1 pairs of reversible reactions and m_2 irreversible reactions, there is another, simple formulation of the CLE with only m_1+m_2 Wiener processes, whereas the standard approach uses 2m_1+m_2. Considerable computational savings are achieved with this latter formulation. A flaw of the CLE model is identified: trajectories may leave the nonnegative orthant with positive probability. The second part addresses the challenge when alternative, structurally different ordinary differential equation models of similar complexity fit the available experimental data equally well. We review optimal experiment design methods for choosing the initial state and structural changes on the biological system to maximally discriminate between the outputs of rival models in terms of L_2-distance. We determine the optimal stimulus (input) profile for externally excitable systems. The numerical implementation relies on sum of squares decompositions and is demonstrated on two rival models of signal processing in starving Dictyostelium amoebae. Such experiments accelerate the perfection of our understanding of biochemical mechanisms.