Transposable elements: structure and dynamic of the autonomous retrotransposon R2 in Arthropoda with non-canonical reproduction

Eukaryotic ribosomal DNA constitutes a multi gene family organized in a cluster called nucleolar organizer region (NOR); this region is composed usually by hundreds to thousands of tandemly repeated units. Ribosomal genes, being repeated sequences, evolve following the typical pattern of concerted evolution. The autonomous retroelement R2 inserts in the ribosomal gene 28S, leading to defective 28S rDNA genes. R2 element, being a retrotransposon, performs its activity in the genome multiplying its copy number through a “copy and paste” mechanism called target primed reverse transcription. It consists in the retrotranscription of the element’s mRNA into DNA, then the DNA is integrated in the target site. Since the retrotranscription can be interrupted, but the integration will be carried out anyway, truncated copies of the element will also be present in the genome. The study of these truncated variants is a tool to examine the activity of the element. R2 phylogeny appears, in general, not consistent with that of its hosts, except some cases (e.g. Drosophila spp. and Reticulitermes spp.); moreover R2 is absent in some species (Fugu rubripes, human, mouse, etc.), while other species have more R2 lineages in their genome (the turtle Mauremys reevesii, the Japanese beetle Popilia japonica, etc). R2 elements here presented are isolated in 4 species of notostracan branchiopods and in two species of stick insects, whose reproductive strategies range from strict gonochorism to unisexuality. From sequencing data emerges that in Triops cancriformis (Spanish gonochoric population), in Lepidurus arcticus (two putatively unisexual populations from Iceland) and in Bacillus rossius (gonochoric population from Capalbio) the R2 elements are complete and encode functional proteins, reflecting the general features of this family of transposable elements. On the other hand, R2 from Italian and Austrian populations of T. cancriformis (respectively unisexual and hermaphroditic), Lepidurus lubbocki (two elements within the same Italian population, gonochoric but with unfunctional males) and Bacillus grandii grandii (gonochoric population from Ponte Manghisi) have sequences that encode incomplete or non-functional proteins in which it is possible to recognize only part of the characteristic domains. In Lepidurus couesii (Italian gonochoric populations) different elements were found as in L. lubbocki, and the sequencing is still in progress. Two hypothesis are given to explain the inconsistency of R2/host phylogeny: vertical inheritance of the element followed by extinction/diversification or horizontal transmission. My data support previous study that state the vertical transmission as the most likely explanation; nevertheless horizontal transfer events can’t be excluded. I also studied the element’s activity in Spanish populations of T. cancriformis, in L. lubbocki, in L. arcticus and in gonochoric and parthenogenetic populations of B. rossius. In gonochoric populations of T. cancriformis and B. rossius I found that each individual has its own private set of truncated variants. The situation is the opposite for the remaining hermaphroditic/parthenogenetic species and populations, all individuals sharing – in the so far analyzed samples - the majority of variants. This situation is very interesting, because it isn’t concordant with the Muller’s ratchet theory that hypothesizes the parthenogenetic populations being either devoided of transposable elements or TEs overloaded. My data suggest a possible epigenetic mechanism that can block the retrotransposon activity, and in this way deleterious mutations don’t accumulate.

Novel tools for conservation genetics in marine fish: population structure and evolution of the European hake (Merluccius merluccius) inferred by SNP variation and applications to traceability

The research presented in my PhD thesis is part of a wider European project, FishPopTrace, focused on traceability of fish populations and products. My work was aimed at developing and analyzing novel genetic tools for a widely distributed marine fish species, the European hake (Merluccius merluccius), in order to investigate population genetic structure and explore potential applications to traceability scenarios. A total of 395 SNPs (Single Nucleotide Polymorphisms) were discovered from a massive collection of Expressed Sequence Tags, obtained by high-throughput sequencing, and validated on 19 geographic samples from Atlantic and Mediterranean. Genome-scan approaches were applied to identify polymorphisms on genes potentially under divergent selection (outlier SNPs), showing higher genetic differentiation among populations respect to the average observed across loci. Comparative analysis on population structure were carried out on putative neutral and outlier loci at wide (Atlantic and Mediterranean samples) and regional (samples within each basin) spatial scales, to disentangle the effects of demographic and adaptive evolutionary forces on European hake populations genetic structure. Results demonstrated the potential of outlier loci to unveil fine scale genetic structure, possibly identifying locally adapted populations, despite the weak signal showed from putative neutral SNPs. The application of outlier SNPs within the framework of fishery resources management was also explored. A minimum panel of SNP markers showing maximum discriminatory power was selected and applied to a traceability scenario aiming at identifying the basin (and hence the stock) of origin, Atlantic or Mediterranean, of individual fish. This case study illustrates how molecular analytical technologies have operational potential in real-world contexts, and more specifically, potential to support fisheries control and enforcement and fish and fish product traceability.

New computational biology tools for the systematic analysis of the structure and expression of human genes

From the late 1980s, the automation of sequencing techniques and the computer spread gave rise to a flourishing number of new molecular structures and sequences and to proliferation of new databases in which to store them. Here are presented three computational approaches able to analyse the massive amount of publicly avalilable data in order to answer to important biological questions. The first strategy studies the incorrect assignment of the first AUG codon in a messenger RNA (mRNA), due to the incomplete determination of its 5' end sequence. An extension of the mRNA 5' coding region was identified in 477 in human loci, out of all human known mRNAs analysed, using an automated expressed sequence tag (EST)-based approach. Proof-of-concept confirmation was obtained by in vitro cloning and sequencing for GNB2L1, QARS and TDP2 and the consequences for the functional studies are discussed. The second approach analyses the codon bias, the phenomenon in which distinct synonymous codons are used with different frequencies, and, following integration with a gene expression profile, estimates the total number of codons present across all the expressed mRNAs (named here "codonome value") in a given biological condition. Systematic analyses across different pathological and normal human tissues and multiple species shows a surprisingly tight correlation between the codon bias and the codonome bias. The third approach is useful to studies the expression of human autism spectrum disorder (ASD) implicated genes. ASD implicated genes sharing microRNA response elements (MREs) for the same microRNA are co-expressed in brain samples from healthy and ASD affected individuals. The different expression of a recently identified long non coding RNA which have four MREs for the same microRNA could disrupt the equilibrium in this network, but further analyses and experiments are needed.

Spectroscopic investigation on photoreactivity, structure and polymorphism of organic molecular crystals

The study of the spectroscopic phenomena in organic solids, in combination with other techniques, is an effective tool for the understanding of the structural properties of materials based on these compounds. This Ph.D. work was dedicated to the spectroscopic investigation of some relevant processes occurring in organic molecular crystals, with the goal of expanding the knowledge on the relationship between structure, dynamics and photoreactivity of these systems. Vibrational spectroscopy has been the technique of choice, always in combination with X-ray diffraction structural studies and often the support of computational methods. The vibrational study of the molecular solid state reaches its full potential when it includes the low-wavenumber region of the lattice-phonon modes, which probe the weak intermolecular interactions and are the fingerprints of the lattice itself. Microscopy is an invaluable addition in the investigation of processes that take place in the micro-meter scale of the crystal micro-domains. In chemical and phase transitions, as well as in polymorph screening and identification, the combination of Raman microscopy and lattice-phonon detection has provided useful information. Research on the fascinating class of single-crystal-to-single-crystal photoreactions, has shown how the homogeneous mechanism of these transformations can be identified by lattice-phonon microscopy, in agreement with the continuous evolution of their XRD patterns. On describing the behavior of the photodimerization mechanism of vitamin K3, the focus was instead on the influence of its polymorphism in governing the product isomerism. Polymorphism is the additional degree of freedom of molecular functional materials, and by advancing in its control and properties, functionalities can be promoted for useful applications. Its investigation focused on thin-film phases, widely employed in organic electronics. The ambiguities in phase identification often emerging by other experimental methods were successfully solved by vibrational measurements.

Reconstruction and analysis of the NF-kB pathway interactome: a systems biology approach

Background. One of the phenomena observed in human aging is the progressive increase of a systemic inflammatory state, a condition referred to as “inflammaging”, negatively correlated with longevity. A prominent mediator of inflammation is the transcription factor NF-kB, that acts as key transcriptional regulator of many genes coding for pro-inflammatory cytokines. Many different signaling pathways activated by very diverse stimuli converge on NF-kB, resulting in a regulatory network characterized by high complexity. NF-kB signaling has been proposed to be responsible of inflammaging. Scope of this analysis is to provide a wider, systemic picture of such intricate signaling and interaction network: the NF-kB pathway interactome. Methods. The study has been carried out following a workflow for gathering information from literature as well as from several pathway and protein interactions databases, and for integrating and analyzing existing data and the relative reconstructed representations by using the available computational tools. Strong manual intervention has been necessarily used to integrate data from multiple sources into mathematically analyzable networks. The reconstruction of the NF-kB interactome pursued with this approach provides a starting point for a general view of the architecture and for a deeper analysis and understanding of this complex regulatory system. Results. A “core” and a “wider” NF-kB pathway interactome, consisting of 140 and 3146 proteins respectively, were reconstructed and analyzed through a mathematical, graph-theoretical approach. Among other interesting features, the topological characterization of the interactomes shows that a relevant number of interacting proteins are in turn products of genes that are controlled and regulated in their expression exactly by NF-kB transcription factors. These “feedback loops”, not always well-known, deserve deeper investigation since they may have a role in tuning the response and the output consequent to NF-kB pathway initiation, in regulating the intensity of the response, or its homeostasis and balance in order to make the functioning of such critical system more robust and reliable. This integrated view allows to shed light on the functional structure and on some of the crucial nodes of thet NF-kB transcription factors interactome. Conclusion. Framing structure and dynamics of the NF-kB interactome into a wider, systemic picture would be a significant step toward a better understanding of how NF-kB globally regulates diverse gene programs and phenotypes. This study represents a step towards a more complete and integrated view of the NF-kB signaling system.

Practical Management of the Endangered African Wild Dog (Lycaon Pictus): Implementation of Research Methodologies, Management Tools and Validation of Non-Invasive Endocrinology Applications

The porpoise of this study was to implement research methodologies and assess the effectiveness and impact of management tools to promote best practices for the long term conservation of the endangered African wild dog (Lycaon pictus). Different methods were included in the project framework to investigate and expand the applicability of these methodologies to free-ranging African wild dogs in the southern African region: ethology, behavioural endocrinology and ecology field methodologies were tested and implemented. Additionally, research was performed to test the effectiveness and implication of a contraceptive implant (Suprenolin) as a management tool for the species of a subpopulation hosted in fenced areas. Attention was especially given to social structure and survival of treated packs. This research provides useful tools and advances the applicability of these methods for field studies, standardizing and improving research instruments in the field of conservation biology and behavioural endocrinology. Results reported here provide effective methodologies to expand the applicability of non-invasive endocrine assessment to previously prohibited fields, and validation of sampling methods for faecal hormone analysis. The final aim was to fill a knowledge gap on behaviours of the species and provide a common ground for future researchers to apply non-invasive methods to this species research and to test the effectiveness of the contraception on a managed metapopulation.

NMR, Metabonomics and Molecular Profiles: Applications to the Quality Assessment of Foodstuffs

Nuclear Magnetic Resonance (NMR) is a branch of spectroscopy that is based on the fact that many atomic nuclei may be oriented by a strong magnetic field and will absorb radiofrequency radiation at characteristic frequencies. The parameters that can be measured on the resulting spectral lines (line positions, intensities, line widths, multiplicities and transients in time-dependent experi-ments) can be interpreted in terms of molecular structure, conformation, molecular motion and other rate processes. In this way, high resolution (HR) NMR allows performing qualitative and quantitative analysis of samples in solution, in order to determine the structure of molecules in solution and not only. In the past, high-field NMR spectroscopy has mainly concerned with the elucidation of chemical structure in solution, but today is emerging as a powerful exploratory tool for probing biochemical and physical processes. It represents a versatile tool for the analysis of foods. In literature many NMR studies have been reported on different type of food such as wine, olive oil, coffee, fruit juices, milk, meat, egg, starch granules, flour, etc using different NMR techniques. Traditionally, univariate analytical methods have been used to ex-plore spectroscopic data. This method is useful to measure or to se-lect a single descriptive variable from the whole spectrum and , at the end, only this variable is analyzed. This univariate methods ap-proach, applied to HR-NMR data, lead to different problems due especially to the complexity of an NMR spectrum. In fact, the lat-ter is composed of different signals belonging to different mole-cules, but it is also true that the same molecules can be represented by different signals, generally strongly correlated. The univariate methods, in this case, takes in account only one or a few variables, causing a loss of information. Thus, when dealing with complex samples like foodstuff, univariate analysis of spectra data results not enough powerful. Spectra need to be considered in their wholeness and, for analysing them, it must be taken in consideration the whole data matrix: chemometric methods are designed to treat such multivariate data. Multivariate data analysis is used for a number of distinct, differ-ent purposes and the aims can be divided into three main groups: • data description (explorative data structure modelling of any ge-neric n-dimensional data matrix, PCA for example); • regression and prediction (PLS); • classification and prediction of class belongings for new samples (LDA and PLS-DA and ECVA). The aim of this PhD thesis was to verify the possibility of identify-ing and classifying plants or foodstuffs, in different classes, based on the concerted variation in metabolite levels, detected by NMR spectra and using the multivariate data analysis as a tool to inter-pret NMR information. It is important to underline that the results obtained are useful to point out the metabolic consequences of a specific modification on foodstuffs, avoiding the use of a targeted analysis for the different metabolites. The data analysis is performed by applying chemomet-ric multivariate techniques to the NMR dataset of spectra acquired. The research work presented in this thesis is the result of a three years PhD study. This thesis reports the main results obtained from these two main activities: A1) Evaluation of a data pre-processing system in order to mini-mize unwanted sources of variations, due to different instrumental set up, manual spectra processing and to sample preparations arte-facts; A2) Application of multivariate chemiometric models in data analy-sis.