Modeling and simulation of a synthetic genetic circuit that implements a multicelled behavior in a growing microcolony of E. coli

Synthetic Biology is a relatively new discipline, born at the beginning of the New Millennium, that brings the typical engineering approach (abstraction, modularity and standardization) to biotechnology. These principles aim to tame the extreme complexity of the various components and aid the construction of artificial biological systems with specific functions, usually by means of synthetic genetic circuits implemented in bacteria or simple eukaryotes like yeast. The cell becomes a programmable machine and its low-level programming language is made of strings of DNA. This work was performed in collaboration with researchers of the Department of Electrical Engineering of the University of Washington in Seattle and also with a student of the Corso di Laurea Magistrale in Ingegneria Biomedica at the University of Bologna: Marilisa Cortesi. During the collaboration I contributed to a Synthetic Biology project already started in the Klavins Laboratory. In particular, I modeled and subsequently simulated a synthetic genetic circuit that was ideated for the implementation of a multicelled behavior in a growing bacterial microcolony. In the first chapter the foundations of molecular biology are introduced: structure of the nucleic acids, transcription, translation and methods to regulate gene expression. An introduction to Synthetic Biology completes the section. In the second chapter is described the synthetic genetic circuit that was conceived to make spontaneously emerge, from an isogenic microcolony of bacteria, two different groups of cells, termed leaders and followers. The circuit exploits the intrinsic stochasticity of gene expression and intercellular communication via small molecules to break the symmetry in the phenotype of the microcolony. The four modules of the circuit (coin flipper, sender, receiver and follower) and their interactions are then illustrated. In the third chapter is derived the mathematical representation of the various components of the circuit and the several simplifying assumptions are made explicit. Transcription and translation are modeled as a single step and gene expression is function of the intracellular concentration of the various transcription factors that act on the different promoters of the circuit. A list of the various parameters and a justification for their value closes the chapter. In the fourth chapter are described the main characteristics of the gro simulation environment, developed by the Self Organizing Systems Laboratory of the University of Washington. Then, a sensitivity analysis performed to pinpoint the desirable characteristics of the various genetic components is detailed. The sensitivity analysis makes use of a cost function that is based on the fraction of cells in each one of the different possible states at the end of the simulation and the wanted outcome. Thanks to a particular kind of scatter plot, the parameters are ranked. Starting from an initial condition in which all the parameters assume their nominal value, the ranking suggest which parameter to tune in order to reach the goal. Obtaining a microcolony in which almost all the cells are in the follower state and only a few in the leader state seems to be the most difficult task. A small number of leader cells struggle to produce enough signal to turn the rest of the microcolony in the follower state. It is possible to obtain a microcolony in which the majority of cells are followers by increasing as much as possible the production of signal. Reaching the goal of a microcolony that is split in half between leaders and followers is comparatively easy. The best strategy seems to be increasing slightly the production of the enzyme. To end up with a majority of leaders, instead, it is advisable to increase the basal expression of the coin flipper module. At the end of the chapter, a possible future application of the leader election circuit, the spontaneous formation of spatial patterns in a microcolony, is modeled with the finite state machine formalism. The gro simulations provide insights into the genetic components that are needed to implement the behavior. In particular, since both the examples of pattern formation rely on a local version of Leader Election, a short-range communication system is essential. Moreover, new synthetic components that allow to reliably downregulate the growth rate in specific cells without side effects need to be developed. In the appendix are listed the gro code utilized to simulate the model of the circuit, a script in the Python programming language that was used to split the simulations on a Linux cluster and the Matlab code developed to analyze the data.

Fusarium species responsible for mycotoxin production in wheat crop: involvement in food safety

Fusarium Head Blight (FHB) is a worldwide cereal disease responsible of significant yield reduction, inferior grain quality, and mycotoxin accumulation. Fusarium graminearum and F. culmorum are the prevalent causal agents. FHB has been endemic in Italy since 1995, while there are no records about its presence in Syria. Forty-eight and forty-six wheat kernel samples were collected from different localities and analyzed for fungal presence and mycotoxin contamination. Fusarium strains were identified morphologically but the molecular confirmation was performed only for some species. Further differentiation of the chemotypes for trichothecene synthesis by F. graminearum and F. culmorum strains was conducted by PCR assays. Fusarium spp. were present in 62.5% of Syrian samples. 3Acetyl-Deoxynivalenol and nivalenol chemotypes were found in F. culmorum whilst all F. graminearum strains belonged to NIV chemotype. Italian samples were infected with Fusarium spp for 67.4%. 15Ac-DON was the prevalent chemotype in F. graminearum, while 3Ac-DON chemotype was detected in F. culmorum. The 60 Syrian Fusarium strains tested for mycotoxin production by HPLC-MS/MS have shown the prevalence of zearalenone while the emerging mycotoxins were almost absent. The analysis of the different Syrian and Italian samples of wheat kernels for their mycotoxin content showed that Syrian kernels were mainly contaminated with storage mycotoxins, aflatoxins and ochratoxin whilst Italian grains with mainly Fusarium mycotoxins. The aggressiveness of several Syrian F. culmorum isolates was estimated using three different assays: floret inoculation in growth chamber, ear inoculation in the field and a validated new Petri-dish test. The study of the behaviour of different Syrian wheat cultivars, grown under different conditions, has revealed that Jory is a FHB Syrian tolerant cultivar. This is the first study in Syria on Fusarium spp. associated to FHB, Fusarium mycotoxin producers and grain quality.

Association mapping of stem rust resistance in durum wheat at the seedling and adult plant stages

In wheat, stem rust is known to rapidly evolve new virulence to resistance genes. While more than 50 stem rust resistance (Sr) loci have been identified in wheat, only a few remain effective, particularly against the highly virulent race Ug99 (TTKSK race) and a mixture of durum-specific races. An association mapping (AM) study based on 183 durum wheat accessions was utilized to identify resistance loci for stem rust response in Ethiopia over four seasons and artificial inoculation with Ug99 (TTKSK race) and a mixture of durum-specific races under field conditions as well as in greenhouse test at seedling stage under controlled conditions for resistance to four highly virulent stem rust races: TRTTF, TTTTF, (TTKSK (Ug99) and JRCQC. The panel was profiled with 1,253 SSR and DArT markers. Twelve QTL-tagging markers were significant (P < 0.05) across three to four seasons. The role of Sr13, Sr9, Sr14, Sr17, and Sr28 was confirmed. Thirteen significant markers were in regions with no Sr genes/QTLs. The results under controlled conditions showed that 15, 20, 19 and 19 chromosome regions harbored markers that showed significant effects for races TRTTF, TTTTF, TTKSK and JRCQC, respectively. These genomic regions showed marker R2 values ranging from 1.13 to 8.34, 1.92 to 17.64, 1.75 to 23.12 and 1.51 to 15.33% for races TRTTF, TTTTF, TTKSK and JRCQC, respectively. The study demonstrates that stem rust resistance in durum wheat is governed in part by shared loci and in part by race-specific ones. The QTLs identified in this study through AM will be useful in the marker-assisted development of durum wheat cultivars with durable stem rust resistance.

Studio di caratteri correlati a stress idrico in una collezione di cultivar di frumento duro (Triticum durum Desf.)

L’agricoltura si trova ad affrontare una diminuzione della disponibilità d’acqua ed una crescente domanda della produzione di cereali per scopi alimentari. Sono perciò necessarie strategie di coltivazione innovative per migliorare la produttività e nuovi genotipi migliorati nell'efficienza dell’uso delle risorse in condizioni di siccità. Questi rappresentano gli obietti principali del progetto “DROPS” (Drought tolerant yielding Plants) all’interno del quale ha avuto luogo il mio progetto di Dottorato. La mia attività di ricerca è stata svolta come segue: 1. Caratterizzazione molecolare di un panel di188 accessioni di frumento duro con marcatori SSR e DaRT; 2. Esperimenti in serra su 100 accessioni del panel per valutare la Water-Use Efficiency (WUE) in sei repliche secondo un Alpha Lattice design; 3. Prove sul campo, effettuate secondo un Alpha Lattice design, in due stagioni di crescita: a. 2010/11, valutazione di 100 accessioni presso l’Azienda sperimentale dell'Università di Cadriano (BO); b. 2011/12, valutazione del panel completo in 3 ambienti, con due diversi regimi irrigui In entrambi gli anni, abbiamo valutato caratteri agronomici correlati con il ciclo di sviluppo, la resa di granella e sue componenti, nonché diversi fattori ambientali e del suolo. Per quanto riguarda WUE, abbiamo trovato differenze altamente significative tra accessioni; inoltre, cinque accessioni hanno mostrato elevati valori di WUE e cinque accessioni valori molto bassi di WUE in tutte e sei le repliche. Gli esperimenti di campo nelle stagioni 2011 e 2012 hanno evidenziato differenze altamente significative tra le accessioni del panel per la maggior parte dei caratteri analizzati, confermando inoltre che il panel di fiorisce entro una settimana. L'esperimento del secondo anno ci ha permesso osservare un significativa interazione Genotipo X Ambiente. Questi risultati saranno integrati con ulteriori analisi QTL, per identificare regioni cromosomiche coinvolte nel controllo genetico dei caratteri di interesse e verificare la stabilità dei QTL in diversi ambienti.

A new global wheat marketmodel (GLOWMM) for the analysis of wheat export prices

Food commodity prices fluctuations have important impacts on poverty and food insecurity across the world. Conventional models have not provided a complete picture of recent price spikes in agricultural commodity markets, while there is an urgent need for appropriate policy responses. Perhaps new approaches are needed in order to better understand international spill-overs, the feedback between the real and the financial sectors and also the link between food and energy prices. In this paper, we present results from a new worldwide dynamic model that provides short and long-run impulse responses of wheat international prices to various real shocks.

Fine tuning of on-growing diets for farmed marine fish species as a key tool to sustain the aquaculture development

The aim of this thesis was to investigate some important key factors able to promote the prospected growth of the aquaculture sector. The limited availability of fishmeal and fish oil led the attention of the aquafeed industry to reduce the dependency on marine raw materials in favor of vegetable ingredients. In Chapter 2, we reported the effects of fishmeal replacement by a mixture of plant proteins in turbot (Psetta maxima L.) juveniles. At the end of the trial, it was found that over the 15% plant protein inclusion can cause stress and exert negative effects on growth performance and welfare. Climate change aroused the attention of the aquafeed industry toward the production of specific diets capable to counteract high temperatures. In Chapter 3, we investigated the most suitable dietary lipid level for gilthead seabream (Sparus aurata L.) reared at Mediterranean summer temperature. In this trial, it was highlighted that 18% dietary lipid allows a protein sparing effect, thus making the farming of this species economically and environmentally more sustainable. The introduction of new farmed fish species makes necessary the development of new species-specific diets. In Chapter 4, we assessed growth response and feed utilization of common sole (Solea solea L.) juveniles fed graded dietary lipid levels. At the end of the trial, it was found that increasing dietary lipids over 8% led to a substantial decline in growth performance and feed utilization indices. In Chapter 5, we investigated the suitability of mussel meal as alternative ingredient in diets for common sole juveniles. Mussel meal proved to be a very effective alternative ingredient for enhancing growth performance, feed palatability and feed utilization in sole irrespectively to the tested inclusion levels. This thesis highlighted the importance of formulating more specific diets in order to support the aquaculture growth in a sustainable way.

A multiparental cross population for mapping QTL for relevant agronomic traits in durum wheat (Triticum durum Desf.)

Multiparental cross designs for mapping quantitative trait loci (QTL) in crops are efficient alternatives to conventional biparental experimental populations because they exploit a broader genetic basis and higher mapping resolution. We describe the development and deployment of a multiparental recombinant inbred line (RIL) population in durum wheat (Triticum durum Desf.) obtained by crossing four elite cultivars characterized by different traits of agronomic value. A linkage map spanning 2,663 cM and including 7,594 single nucleotide polymorphisms (SNPs) was produced by genotyping 338 RILs with a wheat-dedicated 90k SNP chip. A cluster file was developed for correct allele calling in the framework of the tetraploid durum wheat genome. Based on phenotypic data collected over four field experiments, a multi-trait quantitative trait loci (QTL) analysis was carried out for 18 traits of agronomic relevance (including yield, yield-components, morpho-physiological and seed quality traits). Across environments, a total of 63 QTL were identified and characterized in terms of the four founder haplotypes. We mapped two QTL for grain yield across environments and 23 QTL for grain yield components. A novel major QTL for number of grain per spikelet/ear was mapped on chr 2A and shown to control up to 39% of phenotypic variance in this cross. Functionally different QTL alleles, in terms of direction and size of genetic effect, were distributed among the four parents. Based on the occurrence of QTL-clusters, we characterized the breeding values (in terms of effects on yield) of most of QTL for heading and maturity as well as yield component and quality QTL. This multiparental RIL population provides the wheat community with a highly informative QTL mapping resource enabling the dissection of the genetic architecture of multiple agronomic relevant traits in durum wheat.

Genetic basis of variation for root traits and response to heat stress in durum wheat

Durum wheat is the second most important wheat species worldwide and the most important crop in several Mediterranean countries including Italy. Durum wheat is primarily grown under rainfed conditions where episodes of drought and heat stress are major factors limiting grain yield. The research presented in this thesis aimed at the identification of traits and genes that underlie root system architecture (RSA) and tolerance to heat stress in durum wheat, in order to eventually contribute to the genetic improvement of this species. In the first two experiments we aimed at the identification of QTLs for root trait architecture at the seedling level by studying a bi-parental population of 176 recombinant inbred lines (from the cross Meridiano x Claudio) and a collection of 183 durum elite accessions. Forty-eight novel QTLs for RSA traits were identified in each of the two experiments, by means of linkage- and association mapping-based QTL analysis, respectively. Important QTLs controlling the angle of root growth in the seedling were identified. In a third experiment, we investigated the phenotypic variation of root anatomical traits by means of microscope-based analysis of root cross sections in 10 elite durum cultivars. The results showed the presence of sizeable genetic variation in aerenchyma-related traits, prompting for additional studies aimed at mapping the QTLs governing such variation and to test the role of aerenchyma in the adaptive response to abiotic stresses. In the fourth experiment, an association mapping experiment for cell membrane stability at the seedling stage (as a proxy trait for heat tolerance) was carried out by means of association mapping. A total of 34 QTLs (including five major ones), were detected. Our study provides information on QTLs for root architecture and heat tolerance which could potentially be considered in durum wheat breeding programs.

Escape mechanisms after antiangiogenic treatment, or why are the tumors growing again?

Inhibitors of angiogenesis and radiation induce compensatory changes in the tumor vasculature both during and after cessation of treatment. In numerous preclinical studies, angiogenesis inhibitors were shown to be efficient in the treatment of many pathological conditions, including solid cancers. In most clinical trials, however, this approach turned out to have no significant effect, especially if applied as monotherapy. Recovery of tumors after therapy is a major problem in the management of cancer patients. The mechanisms underlying tumor recovery (or therapy resistance) have not yet been explicitly elucidated. This review deals with the transient switch from sprouting to intussusceptive angiogenesis, which may be an adaptive response of tumor vasculature to cancer therapy that allows the vasculature to maintain its functional properties. Potential candidates for molecular targeting of this angioadaptive mechanism are yet to be elucidated in order to improve the currently poor efficacy of contemporary antiangiogenic therapies.