Review of Immunoinformatic approaches to in-silico B-cell epitope prediction

In this paper, the current state of in-silico, B-cell epitope prediction is discussed. Recommendations for improving some of the approaches encountered are outlined, along with the presentation of an entirely novel technique, which uses molecular mechanics for epitope classification, evaluation and prediction

Knowledge Extraction from Textual Resources through Semantic Web Tools and Advanced Machine Learning Algorithms for Applications in Various Domains

Nowadays there is a tremendous amount of unstructured data, often represented by texts, which is created and stored in variety of forms in many domains such as patients' health records, social networks comments, scientific publications, and so on. This volume of data represents an invaluable source of knowledge, but unfortunately it is challenging its mining for machines. At the same time, novel tools as well as advanced methodologies have been introduced in several domains, improving the efficacy and the efficiency of data-based services. Following this trend, this thesis shows how to parse data from text with Semantic Web based tools, feed data into Machine Learning methodologies, and produce services or resources to facilitate the execution of some tasks. More precisely, the use of Semantic Web technologies powered by Machine Learning algorithms has been investigated in the Healthcare and E-Learning domains through not yet experimented methodologies. Furthermore, this thesis investigates the use of some state-of-the-art tools to move data from texts to graphs for representing the knowledge contained in scientific literature. Finally, the use of a Semantic Web ontology and novel heuristics to detect insights from biological data in form of graph are presented. The thesis contributes to the scientific literature in terms of results and resources. Most of the material presented in this thesis derives from research papers published in international journals or conference proceedings

Genetic and phenotypic variation of the equine infectious anemia virus surface unit envelope glycoprotein during disease progression

Lentiviruses are single-stranded RNA viruses generally associated with chronic diseases of the immune and central nervous systems. In contrast to the insidious, progressive nature of most lentiviral diseases, equine infectious anemia virus (EIAV) infection results in rapid onset of a variable disease course in equids. Acute disease is accompanied with high-titered viremia, thrombocytopenia, fever, depression, and inappetance. The chronic stage is usually characterized by recurrent episodes of disease. Equids that survive recurrent disease episodes progress to the inapparent stage of disease where no clinical signs are evident; however, there is persistent, ongoing virus replication. Lentiviruses exist within the host as a population of closely related genotypes, termed a quasispecies. Variation in the virus surface unit envelope glycoprotein (SU) has been demonstrated to contribute to immune evasion of host responses during chronic disease. However, little is known about the SU genotypes and phenotypes associated with disease progression to the inapparent stage of disease. The goal of this research is a genotypic and phenotypic characterization of the SU quasispecies during clinical and inapparent stages of disease. To accomplish this goal, I undertook a longitudinal study of SU variation in a pony experimentally inoculated with the virulent, wild-type, EIAVWyo. There was a marked increase in quasispecies diversity and divergence that coincided with maturation of the immune response and progression to the inapparent stage of disease. Variation was characterized by point mutations in each SU variable region as well as deletion/insertions within the principal neutralizing domain (PND). Genotypes representative of predominant PND variants were used to construct chimeric proviral clones for virus neutralization assays. A type-specific virus neutralizing antibody response was associated with resolution of acute disease. Variants predominant at later stages of disease showed increasing resistance to both type- and group-specific neutralizing antibody. Variants most resistant to group-specific antibody showed reduced replication fitness in vitro. These studies provide evidence that neutralizing antibody selects for resistant SU variants and thereby plays an important role in immune control of virus replication during the inapparent stage of disease

Detecting LINE-1 mediated structural variants from sequencing data: computational characterization of genomic rearrangements occurring in human post-mortem brains in the pathologic context of Alzheimer’s disease and in mouse olfactory epithelium at physiological conditions

One of the most intriguing discoveries in the recent decades is that “the genome is a work in progress”, constantly gaining and loosing chunks of sequence, in order to provide new potentially favorable combinations for adaptation. The old genetic concept that the genome is static has prevailed until the 1950s, when it was first suggested that there is a lot more to DNA than just genes. Indeed, genetic material is dynamic and the greatest part of most organisms’ genome is occupied by non-coding DNA, especially DNA fragments deriving from elements capable of moving to new locations: Transposable Elements (TEs). TEs are mobile DNA fragments, whose remnants occupy nearly half of mammalian genome and up to 90% of the genome of some plants (SanMiguel et al., 1996). Since 1951, when Barbara McClintock discovered them in maize (McClintock, 1951), extensive efforts have been devoted to understand the function of these interspersed repeats. Unfortunately, due to their hidden activity, TEs have been largely underappreciated and dismissed as ‘junk DNA’. When researchers identified long interspersed element-1 (LINE-1 or L1) insertions to be responsible for haemophilia A, in 1988 (Kazazian et al., 1988), TEs gained new attention. LINE-1 elements are the only active, autonomous TE present in the mammalian genome. These molecules, able to create polymorphisms among individuals and genomic mosaicism among populations of cells, are major sources of Structural Variations (SVs) in humans and are responsible for 124 genetic diseases (Hancks and Kazazian, 2016). In particular, the discovery of LINE- 1 mobilization in neurogenesis (Muotri et al., 2005, Coufal et al., 2009) urged the scientific community to investigate the potential involvement of mobile elements in neuropsychiatric disorders (Bundo et al., 2014 , Guffanti et al., 2016, Shpyleva et al., 2017 ) and neurodegenerative diseases (Li et al., 2012). Nowadays, LINE-1 activity has been proven in vitro (Moran et al., 1996) and in vivo (Ostertag et al., 2002) while the real rate of retrotransposition remains an open question. One of the main reasons for this lack of knowledge is the absence of reliable methods to detect elements present in a small minority of cells, or unique to a single cell. This is exacerbated by the technical complexity of deconstructing non-reference, chimeric regions of the genomes through experimental or computational means. Until very recently, assays using ligation-mediated PCR techniques have been considered the gold standard for proving and quantifying current retrotransposon activity. vi Unfortunately, both positive and negative changes in the number of repeats detected with these techniques can occur by a multitude of mechanisms not directly related to retrotransposition. Among the most common retrotransposition-independent rearrangements there are non-homologous recombination-mediated deletions and duplications. In this thesis, I focus on the effects of LINE-1 elements on genome stability. To this purpose, I describe three different bioinformatics methods for the study of the hallmarks of LINE-1-mediated genome instability: direct insertion, post-insertional rearrangements and Double Strand Breaks (DSBs). The increasing availability of large amounts of sequencing data produced by Next- generation sequencing (NGS) calls for the development of new genomics technologies and bioinformatics pipelines targeted to study retrotransposons, to fully exploit the available resources. Therefore a scalable approach, such as the Splinkerette Analysis of Mobile Elements (SPAM) method proposed here, is of substantial interest to assist the current and future developments in the study of TEs. Importantly, SPAM allowed us to target exclusively Full-Length LINE-1 elements (FL-L1) present in Frontal Cortex (FC) and Kidney (K) of Alzheimer’s Disease (AD) and controls (CTRL) post-mortem tissues and to test whether LINE-1 polymorphisms can be a relevant source of SVs associated to AD risks. This is accomplished combining a PCR-based enrichment of FL-L1 elements with an ad hoc bioinformatic pipeline. The performance of our integrative method is achieved for its ability to detect LINE-1 insertion sites with great precision and for its scalability. Embedded in the methodology is the flexibility to perform the same technique in different organisms and for different classes of TEs. Using SPAM, we observed for the first time an unexpectedly high levels of retrotransposition in the K. In association with the SPAM approach, we performed TaqMan based Copy Number Variation (CNV) analysis to evaluate the content of potentially active L1s in the different tissues of AD and CTRL individuals. Overall, we show that the content of FL-L1 sequences in AD is significantly lower than in CTRL, that de-novo integrations are not associated to the disease but that FL-L1 polymorphisms can be a relevant source of SVs. Then, we investigated which mechanism underlies the regulation of Olfactory Receptor (OR) choice in the mouse Olfactory Epithelium (OE), characterizing Olfr2 locus-specific SVs. To perform this task, we combined whole genome amplification from small number vii of cells with PacBio single molecule sequencing and a complementary high-fidelity paired-end Illumina sequencing. This approach allowed us an accurate identification of breakpoints in a locus where a very high repeat concentration, especially LINE elements, provides more chances for recombination events to occur between retrotransposon fragments. Surprisingly, the analysis revealed hundreds of heterozygous structural variants in the vicinity of the locus, among which deletions are the most abundant. The presence and characteristics of particular genomic features associated with the observed deletions, suggest us that Micro-homology Mediated End Joining (MMEJ) of Double Strand Breaks (DSB) seems to be the main mechanism operating in the formation of deletions. Further experiments will tell us if the observed SVs are involved in the regulation of the expression of ORs. Intrigued by the idea that OR genes can present somatic SVs, we profiled endogenous DSB distribution in the mouse OE at p6 and 1m and in the liver at p6. To this purpose, we performed a Chromatin ImmunoPrecipitation and Sequencing (ChIP-Seq) analysis of γ-H2AX (an early response marker for DNA-DSBs). Little is known about the differential distribution of γ-H2AX throughout the genome at physiological conditions. In the light of our results, γ-H2AX signal is stronger in gene-rich, transcribed regions where it co- localizes with regulatory sites. These results suggest a potential involvement of DBSs in resolving topological stress and promoting interactions between regulatory regions. The research described in this thesis is aimed at enhancing our understanding of the role of LINE-1-mediated SVs in health and disease