Design and implementation of bioinformatics tools for large scale genome annotation

The continuous increase of genome sequencing projects produced a huge amount of data in the last 10 years: currently more than 600 prokaryotic and 80 eukaryotic genomes are fully sequenced and publically available. However the sole sequencing process of a genome is able to determine just raw nucleotide sequences. This is only the first step of the genome annotation process that will deal with the issue of assigning biological information to each sequence. The annotation process is done at each different level of the biological information processing mechanism, from DNA to protein, and cannot be accomplished only by in vitro analysis procedures resulting extremely expensive and time consuming when applied at a this large scale level. Thus, in silico methods need to be used to accomplish the task. The aim of this work was the implementation of predictive computational methods to allow a fast, reliable, and automated annotation of genomes and proteins starting from aminoacidic sequences. The first part of the work was focused on the implementation of a new machine learning based method for the prediction of the subcellular localization of soluble eukaryotic proteins. The method is called BaCelLo, and was developed in 2006. The main peculiarity of the method is to be independent from biases present in the training dataset, which causes the over‐prediction of the most represented examples in all the other available predictors developed so far. This important result was achieved by a modification, made by myself, to the standard Support Vector Machine (SVM) algorithm with the creation of the so called Balanced SVM. BaCelLo is able to predict the most important subcellular localizations in eukaryotic cells and three, kingdom‐specific, predictors were implemented. In two extensive comparisons, carried out in 2006 and 2008, BaCelLo reported to outperform all the currently available state‐of‐the‐art methods for this prediction task. BaCelLo was subsequently used to completely annotate 5 eukaryotic genomes, by integrating it in a pipeline of predictors developed at the Bologna Biocomputing group by Dr. Pier Luigi Martelli and Dr. Piero Fariselli. An online database, called eSLDB, was developed by integrating, for each aminoacidic sequence extracted from the genome, the predicted subcellular localization merged with experimental and similarity‐based annotations. In the second part of the work a new, machine learning based, method was implemented for the prediction of GPI‐anchored proteins. Basically the method is able to efficiently predict from the raw aminoacidic sequence both the presence of the GPI‐anchor (by means of an SVM), and the position in the sequence of the post‐translational modification event, the so called ω‐site (by means of an Hidden Markov Model (HMM)). The method is called GPIPE and reported to greatly enhance the prediction performances of GPI‐anchored proteins over all the previously developed methods. GPIPE was able to predict up to 88% of the experimentally annotated GPI‐anchored proteins by maintaining a rate of false positive prediction as low as 0.1%. GPIPE was used to completely annotate 81 eukaryotic genomes, and more than 15000 putative GPI‐anchored proteins were predicted, 561 of which are found in H. sapiens. In average 1% of a proteome is predicted as GPI‐anchored. A statistical analysis was performed onto the composition of the regions surrounding the ω‐site that allowed the definition of specific aminoacidic abundances in the different considered regions. Furthermore the hypothesis that compositional biases are present among the four major eukaryotic kingdoms, proposed in literature, was tested and rejected. All the developed predictors and databases are freely available at: BaCelLo http://gpcr.biocomp.unibo.it/bacello eSLDB http://gpcr.biocomp.unibo.it/esldb GPIPE http://gpcr.biocomp.unibo.it/gpipe

Parapoxvirus: basi genomiche della patogenesi

Parapoxvirus (PPV) are member of a genus in the family poxviridae which currently encompasses four species: the prototype orf virus (OV), bovine papular stomatitis virus (BPSV), pseudocowpox virus (PCPV) and parapoxvirus of New Zealand red deer (PVNZ). PPVs cause widespread, but localized diseases of small and large ruminants and they can also be transmitted to man. Knowledge of the molecular biology of PPV is still limited as compared to orthopoxviruses, especially vaccinia virus (VACV). The PPV genome displays a high G+C content and relatively small size for poxvirus. Coventional electron microscopy displays PPV virions with ovoid shape and slightly smaller in size than the brickshaped orthopoxviruses. The most striking feature, which readily enables identification of PPV, is a tubule-like structure that surrounds the particle in a spiral fashion. PPV genome organization and content is very similar to that of other poxviruses, the central region contain 88 genes which are present in all poxviruse, in contrast the terminal regions are variable and contain a set of genes unique to the genus PPV. Genes in the near-terminal regions of the genome are frequently not essential for growth in cultured cells encoding factors with important roles in virushost interactions including modulating host immune responses and determining host range. Recently it was suggested that the open reading frames (ORFs) 109 and 110 of the OV genome have a major role in determining species specificity during natural infection in sheep and goats. This hypothesis is based on the analysis of a few number of sequences of different sheep and goats viral isolates. PPV replicate into the cytoplasm of infected cells and produce three structurally different infectious particles: the intracellular mature virions (IMV), intracellular enveloped virions (IEV) and the extracellular enveloped virions (EEV). The vaccinia A33R and A34R hotologue proteins encoded by the ORFS 109 and 110 are expressed in the envelope of the IEV and EEV. The F1L immunodominant protein of orf virus is the major component of the surface tubule structure of the IMV and can post-translationaly insert into membranes via Cterminal, hydrofobic anchor sequence like its orthologue VACV H3L protein. Moreover the F1L protein binds to glycosaminoglycans on the cell surface and has an important role in IMV adsorption to mammalian cells. In this study we investigated the morphogenesis of the PPV through the construction of a mutant virus deleted of the F1L protein. A study of the deleted virus life cycle was conducted in different type of cells and its morphology was observed with electron microscopy. It was demonstared that F1L protein have important role in morphogenesis and infectivity. Moreover it is essential to determine the spiral fashion of the tubule like structure of the virion surface. Some pathogenetic aspects of the PPV infection were studied, in particular the protein implicated in the host range were analysed in detail. An experimental infection with OV and PCPV was conducted in goats and sheep. After infection, the severity of the lesions were comparable in both the animal species. The OV did not result in severe disease neither in sheep nor in goats, suggesting that host factors, rather than virus strain characteristics, may play an important role in the pathogenesis of the Parapoxvirus infections. The PCPV failed to produce any lesion in both sheep and goats, ruling out the possibility of any recombination between PCPV and OV during natural infection in these animal species. The phylogenetic analysis of the ORFs 109 and 110 from several goats and sheep viral isolates showed a clustering based on the antigenic content of the protein that was independent from species and geographic origin.

Visual scanning in sports actions: comparison between soccer goalkeepers and judo fighters

Visual search and oculomotor behaviour are believed to be very relevant for athlete performance, especially for sports requiring refined visuo-motor coordination skills. Modern coaches believe that a correct visuo-motor strategy may be part of advanced training programs. In this thesis two experiments are reported in which gaze behaviour of expert and novice athletes were investigated while they were doing a real sport specific task. The experiments concern two different sports: judo and soccer. In each experiment, number of fixations, fixation locations and mean fixation duration (ms) were considered. An observational analysis was done at the end of the paper to see perceptual differences between near and far space. Purpose: The aim of the judo study was to delineate differences in gaze behaviour characteristics between a population of athletes and one of non athletes. Aspects specifically investigated were: search rate, search order and viewing time across different conditions in a real-world task. The second study was aimed at identifying gaze behaviour in varsity soccer goalkeepers while facing a penalty kick executed with instep and inside foot. Then an attempt has been done to compare the gaze strategies of expert judoka and soccer goalkeepers in order to delineate possible differences related to the different conditions of reacting to events occurring in near (peripersonal) or far (extrapersonal) space. Judo Methods: A sample of 9 judoka (black belt) and 11 near judoka (white belt) were studied. Eye movements were recorded at 500Hz using a video based eye tracker (EyeLink II). Each subject participated in 40 sessions for about 40 minutes. Gaze behaviour was considered as average number of locations fixated per trial, the average number of fixations per trial, and mean fixation duration. Soccer Methods: Seven (n = 7) intermediate level male volunteered for the experiment. The kickers and goalkeepers, had at least varsity level soccer experience. The vision-in-action (VIA) system (Vickers 1996; Vickers 2007) was used to collect the coupled gaze and motor behaviours of the goalkeepers. This system integrated input from a mobile eye tracking system (Applied Sciences Laboratories) with an external video of the goalkeeper’s saving actions. The goalkeepers took 30 penalty kicks on a synthetic pitch in accordance with FIFA (2008) laws. Judo Results: Results indicate that experts group differed significantly from near expert for fixations duration, and number of fixations per trial. The expert judokas used a less exhaustive search strategy involving fewer fixations of longer duration than their novice counterparts and focused on central regions of the body. The results showed that in defence and attack situation expert group did a greater number of transitions with respect to their novice counterpart. Soccer Results: We found significant main effect for the number of locations fixated across outcome (goal/save) but not for foot contact (instep/inside). Participants spent more time fixating the areas in instep than inside kick and in goal than in save situation. Mean and standard error in search strategy as a result of foot contact and outcome indicate that the most gaze behaviour start and finish on ball interest areas. Conclusions: Expert goalkeepers tend to spend more time in inside-save than instep-save penalty, differences that was opposite in scored penalty kick. Judo results show that differences in visual behaviour related to the level of expertise appear mainly when the test presentation is continuous, last for a relatively long period of time and present a high level of uncertainty with regard to the chronology and the nature of events. Expert judoist performers “anchor” the fovea on central regions of the scene (lapel and face) while using peripheral vision to monitor opponents’ limb movements. The differences between judo and soccer gaze strategies are discussed on the light of physiological and neuropsychological differences between near and far space perception.

Epidemiological and functional characterization of Streptococcus pneumoniae pili

Streptococcus pneumoniae is an important life threatening human pathogen causing agent of invasive diseases such as otitis media, pneumonia, sepsis and meningitis, but is also a common inhabitant of the respiratory tract of children and healthy adults. Likewise most streptococci, S. pneumoniae decorates its surface with adhesive pili, composed of covalently linked subunits and involved in the attachment to epithelial cells and virulence. The pneumococcal pili are encoded by two genomic regions, pilus islet 1 (PI-1), and pilus islet-2 (PI-2), which are present in about 30% and 16% of the pneumococcal strains, respectively. PI-1 exists in three clonally related variants, whereas PI-2 is highly conserved. The presence of the islets does not correlate with the serotype of the strains, but with the genotype (as determined by Multi Locus Sequence Typing). The prevalence of PI-1 and PI-2 positive strains is similar in isolates from invasive disease and carriage. To better dissect a possible association between PIs presence and disease we evaluated the distribution of the two PIs in a panel of 113 acute otitis media (AOM) clinical isolates from Israel. PI-1 was present in 30.1% (N=34) of the isolates tested, and PI-2 in 7% (N=8). We found that 50% of the PI-1 positive isolates belonged to the international clones Spain9V-3 (ST156) and Taiwan19F-14 (ST236), and that PI-2 was not present in the absence of Pl-1. In conclusion, there was no correlation between PIs presence and AOM, and, in general, the observed differences in PIs prevalence are strictly dependent upon regional differences in the distribution of the clones. Finally, in the AOM collection the prevalence of PI-1 was higher among antibiotic resistant isolates, confirming previous indications obtained by the in silico analysis of the MLST database collection. Since the pilus-1 subunits were shown to confer protection in mouse models of infection both in active and passive immunization studies, and were regarded as potential candidates for a new generation of protein-based vaccines, the functional characterization was mainly focused on S. pneumoniae pilus -1 components. The pneumococcal pilus-1 is composed of three subunits, RrgA, RrgB and RrgC, each stabilized by intra-molecular isopeptide bonds and covalently polymerized by means of inter-molecular isopeptide bonds to form an extended fibre. The pilus shaft is a multimeric structure mainly composed by the RrgB backbone subunit. The minor ancillary proteins are located at the tip and at the base of the pilus, where they have been proposed to act as the major adhesin (RrgA) and as the pilus anchor (RrgC), respectively. RrgA is protective in in vivo mouse models, and exists in two variants (clades I and II). Mapping of the sequence variability onto the RrgA structure predicted from X-ray data showed that the diversity was restricted to the “head” of the protein, which contains the putative binding domains, whereas the elongated “stalk” was mostly conserved. To investigate whether this variability could influence the adhesive capacity of RrgA and to map the regions important for binding, two full-length protein variants and three recombinant RrgA portions were tested for adhesion to lung epithelial cells and to purified extracellular matrix (ECM) components. The two RrgA variants displayed similar binding abilities, whereas none of the recombinant fragments adhered at levels comparable to those of the full-length protein, suggesting that proper folding and structural arrangement are crucial to retain protein functionality. Furthermore, the two RrgA variants were shown to be cross-reactive in vitro and cross-protective in vivo in a murine model of passive immunization. Taken together, these data indicate that the region implicated in adhesion and the functional epitopes responsible for the protective ability of RrgA may be conserved and that the considerable level of variation found within the “head” domain of RrgA may have been generated by immunologic pressure without impairing the functional integrity of the pilus.

Gold and the Stock Market: 3 Essays on Gold Investments

This thesis gives an overview of the history of gold per se, of gold as an investment good and offers some institutional details about gold and other precious metal markets. The goal of this study is to investigate the role of gold as a store of value and hedge against negative market movements in turbulent times. I investigate gold’s ability to act as a safe haven during periods of financial stress by employing instrumental variable techniques that allow for time varying conditional covariance. I find broad evidence supporting the view that gold acts as an anchor of stability during market downturns. During periods of high uncertainty and low stock market returns, gold tends to have higher than average excess returns. The effectiveness of gold as a safe haven is enhanced during periods of extreme crises: the largest peaks are observed during the global financial crises of 2007-2009 and, in particular, during the Lehman default (October 2008). A further goal of this thesis is to investigate whether gold provides protection from tail risk. I address the issue of asymmetric precious metal behavior conditioned to stock market performance and provide empirical evidence about the contribution of gold to a portfolio’s systematic skewness and kurtosis. I find that gold has positive coskewness with the market portfolio when the market is skewed to the left. Moreover, gold shows low cokurtosis with the market returns during volatile periods. I therefore show that gold is a desirable investment good to risk averse investors, since it tends to decrease the probability of experiencing extreme bad outcomes, and the magnitude of losses in case such events occur. Gold thus bears very important and under-researched characteristics as an asset class per se, which this thesis contributed to address and unveil.

Attenuation of Eph Receptor Kinase Activation in Cancer Cells by Coexpressed Ephrin Ligands

The Eph receptor tyrosine kinases mediate juxtacrine signals by interacting “in trans” with ligands anchored to the surface of neighboring cells via a GPI-anchor (ephrin-As) or a transmembrane segment (ephrin-Bs), which leads to receptor clustering and increased kinase activity. Additionally, soluble forms of the ephrin-A ligands released from the cell surface by matrix metalloproteases can also activate EphA receptor signaling. Besides these trans interactions, recent studies have revealed that Eph receptors and ephrins coexpressed in neurons can also engage in lateral “cis” associations that attenuate receptor activation by ephrins in trans with critical functional consequences. Despite the importance of the Eph/ephrin system in tumorigenesis, Eph receptor-ephrin cis interactions have not been previously investigated in cancer cells. Here we show that in cancer cells, coexpressed ephrin-A3 can inhibit the ability of EphA2 and EphA3 to bind ephrins in trans and become activated, while ephrin-B2 can inhibit not only EphB4 but also EphA3. The cis-inhibition of EphA3 by ephrin-B2 implies that in some cases ephrins that cannot activate a particular Eph receptor in trans can nevertheless inhibit its signaling ability through cis association. We also found that an EphA3 mutation identified in lung cancer enhances cis interaction with ephrin-A3. These results suggest a novel mechanism that may contribute to cancer pathogenesis by attenuating the tumor suppressing effects of Eph receptor signaling pathways activated by ephrins in trans (Falivelli et al. 2013).

The Effect of Metronome Pacing on Gait in Healthy Subjects

The complex process of gait is rendered partially automatic by central pattern generators (CPGs). To further our understanding of their role in gait control in healthy subjects, we applied a paradigm of anti-phase, or syncopated, movement to gait. To provide a context for our results, we reviewed the literature on in-phase, or synchronized, gait. The review results are as follows. Auditory cueing increased step/stride rate for older subjects, but not younger. Stride rate variability decreased for younger subjects, perhaps because the metronome’s cue acted as a temporal ‘anchor point’ for each step. Step width increased in half of the treadmill studies, but none of the overground ones, suggesting a cumulative effect of the attentional demands of synchronizing gait while on a treadmill. Time series analysis revealed that the α exponent was the most sensitive parameter reported, decreasing toward anti-persistence in almost all cued-gait studies. This project compares in-phase (IN) and anti-phase gait (ANTI) in young and old healthy subjects. We expected gait to be less disrupted during ANTI trials at preferred speed, when the facilitating effect of CPGs would be strongest. The measures step time variability, jerk index, and harmonic ratio quantified gait perturbation: none indicated that ANTI was easiest at preferred walking speed. Surprisingly, the gait of older subjects was no more perturbed than that of younger subjects. When they successfully matched the pace of the beat, they unwittingly synchronized to it. The temporal relationship of their steps to the beat was the same in the IN and ANTI conditions. Younger subjects, visibly struggling during ANTI trials, were able to walk in syncopation. This result suggests that cognitive resources available only to the younger group are required to resist synchronizing to the beat.

Self-Assembled Monolayers (SAMs) in Organic Field-Effect Transistors

Organic printed electronics is attracting an ever-growing interest in the last decades because of its impressive breakthroughs concerning the chemical design of π-conjugated materials and their processing. This has an impact on novel applications, such as flexible-large-area displays, low- cost printable circuits, plastic solar cells and lab-on-a-chip devices. The organic field-effect transistor (OFET) relies on a thin film of organic semiconductor that bridges source and drain electrodes. Since its first discovery in the 80s, intensive research activities were deployed in order to control the chemico-physical properties of these electronic devices and consequently their charge. Self-assembled monolayers (SAMs) are a versatile tool for tuning the properties of metallic, semi-conducting, and insulating surfaces. Within this context, OFETs represent reliable instruments for measuring the electrical properties of the SAMs in a Metal/SAM/OS junction. Our experimental approach, named Charge Injection Organic-Gauge (CIOG), uses OTFT in a charge-injection controlled regime. The CIOG sensitivity has been extensively demonstrated on different homologous self-assembling molecules that differ in either chain length or in anchor/terminal group. One of the latest applications of organic electronics is the so-called “bio-electronics” that makes use of electronic devices to encompass interests of the medical science, such as biosensors, biotransducers etc… As a result, thee second part of this thesis deals with the realization of an electronic transducer based on an Organic Field-Effect Transistor operating in aqueous media. Here, the conventional bottom gate/bottom contact configuration is replaced by top gate architecture with the electrolyte that ensures electrical contact between the top gold electrode and the semiconductor layer. This configuration is named Electrolyte-Gated Field-Effect Transistor (EGOFET). The functionalization of the top electrode is the sensing core of the device allowing the detection of dopamine as well as of protein biomarkers with ultra-low sensitivity.

Application of Submerged Grouted Anchors in Sheet Pile Quay Walls

Sheet pile walls are one of the oldest earth retention systems utilized in civil engineering projects. They are used for various purposes; such as excavation support system, cofferdams, cut-off walls under dams, slope stabilization, waterfront structures, and flood walls. Sheet pile walls are one of the most common types of quay walls used in port construction. The worldwide increases in utilization of large ships for transportation have created an urgent need of deepening the seabed within port areas and consequently the rehabilitation of its wharfs. Several methods can be used to increase the load-carrying capacity of sheet-piling walls. The use of additional anchored tie rods grouted into the backfill soil and arranged along the exposed wall height is one of the most practical and appropriate solutions adopted for stabilization and rehabilitation of the existing quay wall. The Ravenna Port Authority initiated a project to deepen the harbor bottom at selected wharves. An extensive parametric study through the finite element program, PLAXIS 2D, version 2012 was carried out to investigate the enhancement of using submerged grouted anchors technique on the load response of sheet-piling quay wall. The influence of grout-ties area, length of grouted body, anchor inclination and anchor location were considered and evaluated due to the effect of different system parameters. Also a comparative study was conducted by Plaxis 2D and 3D program to investigate the behavior of these sheet pile quay walls in terms of horizontal displacements induced along the sheet pile wall and ground surface settlements as well as the anchor force and calculated factor of safety. Finally, a comprehensive study was carried out by using different constitutive models to simulate the mechanical behavior of the soil to investigate the effect of these two models (Mohr-Coulomb and Hardening Soil) on the behavior of these sheet pile quay walls.