Statistical methods for the analysis of DNA sequences: application to dinucleotide distribution in the human genome

Questa tesi si inserisce nell'ambito delle analisi statistiche e dei metodi stocastici applicati all'analisi delle sequenze di DNA. Nello specifico il nostro lavoro è incentrato sullo studio del dinucleotide CG (CpG) all'interno del genoma umano, che si trova raggruppato in zone specifiche denominate CpG islands. Queste sono legate alla metilazione del DNA, un processo che riveste un ruolo fondamentale nella regolazione genica. La prima parte dello studio è dedicata a una caratterizzazione globale del contenuto e della distribuzione dei 16 diversi dinucleotidi all'interno del genoma umano: in particolare viene studiata la distribuzione delle distanze tra occorrenze successive dello stesso dinucleotide lungo la sequenza. I risultati vengono confrontati con diversi modelli nulli: sequenze random generate con catene di Markov di ordine zero (basate sulle frequenze relative dei nucleotidi) e uno (basate sulle probabilità di transizione tra diversi nucleotidi) e la distribuzione geometrica per le distanze. Da questa analisi le proprietà caratteristiche del dinucleotide CpG emergono chiaramente, sia dal confronto con gli altri dinucleotidi che con i modelli random. A seguito di questa prima parte abbiamo scelto di concentrare le successive analisi in zone di interesse biologico, studiando l’abbondanza e la distribuzione di CpG al loro interno (CpG islands, promotori e Lamina Associated Domains). Nei primi due casi si osserva un forte arricchimento nel contenuto di CpG, e la distribuzione delle distanze è spostata verso valori inferiori, indicando che questo dinucleotide è clusterizzato. All’interno delle LADs si trovano mediamente meno CpG e questi presentano distanze maggiori. Infine abbiamo adottato una rappresentazione a random walk del DNA, costruita in base al posizionamento dei dinucleotidi: il walk ottenuto presenta caratteristiche drasticamente diverse all’interno e all’esterno di zone annotate come CpG island. Riteniamo pertanto che metodi basati su questo approccio potrebbero essere sfruttati per migliorare l’individuazione di queste aree di interesse nel genoma umano e di altri organismi.

Multicelled Behaviour in E. coli: Design and experimental characterization of an engineered library of hybrid promoters.

Synthetic biology has recently had a great development, many papers have been published and many applications have been presented, spanning from the production of biopharmacheuticals to the synthesis of bioenergetic substrates or industrial catalysts. But, despite these advances, most of the applications are quite simple and don’t fully exploit the potential of this discipline. This limitation in complexity has many causes, like the incomplete characterization of some components, or the intrinsic variability of the biological systems, but one of the most important reasons is the incapability of the cell to sustain the additional metabolic burden introduced by a complex circuit. The objective of the project, of which this work is part, is trying to solve this problem through the engineering of a multicellular behaviour in prokaryotic cells. This system will introduce a cooperative behaviour that will allow to implement complex functionalities, that can’t be obtained with a single cell. In particular the goal is to implement the Leader Election, this procedure has been firstly devised in the field of distributed computing, to identify the process that allow to identify a single process as organizer and coordinator of a series of tasks assigned to the whole population. The election of the Leader greatly simplifies the computation providing a centralized control. Further- more this system may even be useful to evolutionary studies that aims to explain how complex organisms evolved from unicellular systems. The work presented here describes, in particular, the design and the experimental characterization of a component of the circuit that solves the Leader Election problem. This module, composed of an hybrid promoter and a gene, is activated in the non-leader cells after receiving the signal that a leader is present in the colony. The most important element, in this case, is the hybrid promoter, it has been realized in different versions, applying the heuristic rules stated in [22], and their activity has been experimentally tested. The objective of the experimental characterization was to test the response of the genetic circuit to the introduction, in the cellular environment, of particular molecules, inducers, that can be considered inputs of the system. The desired behaviour is similar to the one of a logic AND gate in which the exit, represented by the luminous signal produced by a fluorescent protein, is one only in presence of both inducers. The robustness and the stability of this behaviour have been tested by changing the concentration of the input signals and building dose response curves. From these data it is possible to conclude that the analysed constructs have an AND-like behaviour over a wide range of inducers’ concentrations, even if it is possible to identify many differences in the expression profiles of the different constructs. This variability accounts for the fact that the input and the output signals are continuous, and so their binary representation isn’t able to capture the complexity of the behaviour. The module of the circuit that has been considered in this analysis has a fundamental role in the realization of the intercellular communication system that is necessary for the cooperative behaviour to take place. For this reason, the second phase of the characterization has been focused on the analysis of the signal transmission. In particular, the interaction between this element and the one that is responsible for emitting the chemical signal has been tested. The desired behaviour is still similar to a logic AND, since, even in this case, the exit signal is determined by the hybrid promoter activity. The experimental results have demonstrated that the systems behave correctly, even if there is still a substantial variability between them. The dose response curves highlighted that stricter constrains on the inducers concentrations need to be imposed in order to obtain a clear separation between the two levels of expression. In the conclusive chapter the DNA sequences of the hybrid promoters are analysed, trying to identify the regulatory elements that are most important for the determination of the gene expression. Given the available data it wasn’t possible to draw definitive conclusions. In the end, few considerations on promoter engineering and complex circuits realization are presented. This section aims to briefly recall some of the problems outlined in the introduction and provide a few possible solutions.

Transgenerational effects of temperature on egg and larval development in Cyprinodon variegatus

Transgenerational plasticity (TGP), a type of maternal effect, occurs when the environment experienced by one or both the parents prior to fertilization directly translates, without changing DNA sequences, into changes in offspring reaction norms. Evidence of such effects has been found in several traits throughout many phyla, and, although of great potential importance - especially in a time of rapid climate change - TGP in thermal growth physiology had never been demonstrated for vertebrates until the first experiment on thermal TGP in sheepshead minnows, who, given sufficient time, adaptively program their offspring for maximal egg viability and growth at the temperature experienced before fertilization. This study on sheepshead minnows from South Carolina and Connecticut investigates how population, parent temperature, and offspring temperature affect egg production, size, viability, larval survival and growth rates, whether these effects provide evidence of TGP, and whether and how they vary with length of exposure time (5, 12, 19, 26, 33 and 43 days) of the parents to the new experimental temperatures of either 26°C or 32°C. Several results are consistent with those obtained in the previous TGP study, which outline a sequence of events consisting of an initial adjustment period to the new temperatures, in which egg production decreases and no signs of TGP are present, followed by a shift to TGP (towards 26-33 days of exposure) in which parents start to produce more eggs which are better adapted to the new thermal environment. Other results present new information, such as signs of TGP in the parent temperature effect on egg sizes already around 20 days of exposure. The innovative idea of populations being able to adapt to rapidly shifting environments through non-genetic mechanisms such as TGP opens new possibilities of survival of species and will have important implications on ecology, physiology, and contemporary evolution.

Functional dna nanostructures for in vitro biosensing in live cells

DNA is a fascinating biomolecule that is well known for its genetic role in living systems. The emerging area of DNA nanotechnology provides an alternative view that exploits unparallel self-assembly ability of DNA molecules for material use of DNA. Although many reports exist on the results of DNA self-assembling systems, still few of them focus on the in vitro study about the function of such DNA nanostructures in live cells. Due to this, there are still a limited research about the in vitro functionality of such designs. To address an aspect of this issue, we have designed, synthesized and characterized two multifunctional fluorescencent nanobiosensors by DNA self-assembling. Each structure was designed and implemented to be introduced in live cells in order to give information on their functioning in real-time. Computational tools were used in order to design a graphic model of two new DNA motifs and also to obtain the specific sequences to all the ssDNA molecules. By thermal self-assembly techniques we have successfully synthesized the structure and corroborate their formation by the PAGE technique. In addition, we have established the conditions to characterize their structural conformation change when they perform their sensor response. The sensing behavior was also accomplished by fluorescence spectroscopy techniques; FRET evaluation and fluorescence microscopy imaging. Providing the evidence about their adequate sensing performance outside and inside the cells detected in real-time. In a preliminary evaluation we have tried to show the in vitro functionality of our structures in different cancer cell lines with the ability to perform local sensing responses. Our findings suggest that DNA sensor nanostructures could serve as a platform to exploit further therapeutic achievements in live cells.