Functional Genomics and Cell Biology of the Dolphin (Tursiops runcatus): Establishment of Novel Molecular Tools to Study Marine Mammals in Changing Environments

The dolphin (Tursiops truncatus) is a mammal that is adapted to life in a totally aquatic environment. Despite the popularity and even iconic status of the dolphin, our knowledge of its physiology, its unique adaptations and the effects on it of environmental stressors are limited. One approach to improve this limited understanding is the implementation of established cellular and molecular methods to provide sensitive and insightful information for dolphin biology. We initiated our studies with the analysis of wild dolphin peripheral blood leukocytes, which have the potential to be informative of the animal’s global immune status. Transcriptomic profiles from almost 200 individual samples were analyzed using a newly developed species-specific microarray to assess its value as a prognostic and diagnostic tool. Functional genomics analyses were informative of stress-induced gene expression profiles and also of geographical location specific transcriptomic signatures, determined by the interaction of genetic, disease and environmental factors. We have developed quantitative metrics to unambiguously characterize the phenotypic properties of dolphin cells in culture. These quantitative metrics can provide identifiable characteristics and baseline data which will enable identification of changes in the cells due to time in culture. We have also developed a novel protocol to isolate primary cultures from cryopreserved tissue of stranded marine mammals, establishing a tissue (and cell) biorepository, a new approach that can provide a solution to the limited availability of samples. The work presented represents the development and application of tools for the study of the biology, health and physiology of the dolphin, and establishes their relevance for future studies of the impact on the dolphin of environmental infection and stress.

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.

Genes involved in germline regulative pathways in metazoa: an integrative approach to investigate a key feature of animal biology

In Metazoa, the germline represents the cell lineage devoted to transmission of genetic heredity across generations. Its functions intuitively evoke the crucial roles that it plays in the development of a new organism and in the evolution of the species. Germline establishment is tightly tied to animal multicellularity itself, in which the complex differentiation of cell lineages is favoured by the confinement of totipotency in specific cell populations. In the present thesis, I addressed the subject of germline characterization in animals through different approaches, in an attempt to cover different sides and scales. First, I investigated the extent and nature of shared differentially transcribed molecular factors in 10 different species germline-related lineages. I observed that newly evolved genes are less likely to be involved in germline-related mechanisms and that the mostly shared transcriptional signal across the species considered was the upregulation of genes associated to proper DNA replication, instead of the expected transcriptional and post-transcriptional regulation, that apparently have a higher level of lineage-specificity. I then focused on the evolutionary history of Tudor domain containing proteins, a gene family that underwent germline-associated expansions in animals. Using data from 24 holozoan phyla, I could confirm the previously proposed evolution of the Tudor domain secondary structure. Also, I associated lineage-specific family reductions and expansions to peculiar genomic dynamics and to the evolution of germline-associated piRNA pathway of retrotransposon silencing. Lastly, I characterized and investigated the expression of the Tudor protein TDRD7 in the clam Ruditapes philippinarum. Through immunolocalization, I could compare its expression profiles in gametogenic specimens to the previously characterized germline marker vasa. Combining results with literature, I proposed that, in this species, TDRD7 is involved in the assembly of germ granules, i.e. cytoplasmic structures associated to germline differentiation in virtually all animals, but whose assemblers can be taxon specific.

A 3D biomimetic in vitro model: a novel strategy to investigate glioma biology

Gliomas are one of the most frequent primary malignant brain tumors. Acquisition of stem-like features likely contributes to the malignant nature of high-grade gliomas and may be responsible for the initiation, growth, and recurrence of these tumors. In this regard, although the traditional 2D cell culture system has been widely used in cancer research, it shows limitations in maintaining the stemness properties of cancer and in mimicking the in vivo microenvironment. In order to overcome these limitations, different three-dimensional (3D) culture systems have been developed to mimic better the tumor microenvironment. Cancer cells cultured in 3D structures may represent a more reliable in vitro model due to increased cell-cell and cell-extracellular matrix (ECM) interaction. Several attempts to recreate brain cancer tissue in vitro are described in literature. However, to date, it is still unclear which main characteristics the ideal model should reproduce. The overall goal of this project was the development of a 3D in vitro model able to reproduce the brain ECM microenvironment and to recapitulate pathological condition for the study of tumor stroma interactions, tumor invasion ability, and molecular phenotype of glioma cells. We performed an in silico bioinformatic analysis using GEPIA2 Software to compare the expression level of seven matrix protein in the LGG tumors with healthy tissues. Then, we carried out a FFPE retrospective study in order to evaluate the percentage of expression of selected proteins. Thus, we developed a 3D scaffold composed by Hyaluronic Acid and Collagen IV in a ratio of 50:50. We used two astrocytoma cell lines, HTB-12 and HTB-13. In conclusion, we developed an in vitro 3D model able to reproduce the composition of brain tumor ECM, demonstrating that it is a feasible platform to investigate the interaction between tumor cells and the matrix.