Understanding the Organization and functional Control of Polysomes by integrative Approaches

Background and rationale Translation is a fundamental biological process occurring in cells, carried out by ribosomes simultaneously bound to an mRNA molecule (polyribosomes). It has been exhaustively demonstrated that dysregulation of translation is implicated in a wide collection of pathologies including tumours and neurological disorders. Latest findings reveal the existence of translational regulatory mechanisms acting in cis or trans with respect to the mRNAs and governing the movement and the position of ribosomes along transcripts or directly impacting on the ribosome catalogue of its constituent proteins. For this reason, translational controls also account for widespread uncoupling between transcript and protein abundances in cells. To explain the poor correlation between transcripts and protein levels, many computational models of translation have been developed. Usually, these approaches aim at predicting protein abundances in cells starting from the mRNA abundance. Despite the efforts of these modelling studies, a consensus model remains elusive, drawing to contradictory conclusions concerning the role of mRNA regulatory elements such as the usage of codons (codon usage bias) and slowdown mechanism at the beginning of the coding sequence (ramp). More recently, following the rapid and widespread diffusion of ribosome footprinting assays (RiboSeq), which enables the dissection of translation at single nucleotide resolution, a number of computational pipelines dedicated to the analysis of RiboSeq data have been proposed. These tools are typically designed for extracting gene expression alterations at the translational level, while the positional information describing fluxes and positions of ribosomes along the transcript is still underutilized. Therefore, the polysome organization, in term of number and position of ribosomes along the transcript and the translational controls directed in shaping cellular phenotypes is still open to breakthrough discoveries. Broad objectives The aim of my thesis is the development of mathematical and computational tools integrated with experimental data for a comprehensive understanding of translation regulation and polysome organization rules governing the number of ribosomes per polysome and the ribosome position along transcripts. Project design and methods With this purpose, I developed riboWaves, an integrated bioinformatics suite divided in two branches. riboWaves includes in the first branch two modeling modules: riboAbacus, predicting the number of ribosomes per transcript, and riboSim, predicting ribosome localization along mRNAs. In the second branch, riboWaves provides two pipelines, riboWaltz and riboScan, for detailed analyses of ribosome profiling data aimed at providing meaningful and yet unexplored ribosome positional information. The models and the pipelines are implemented in C and R, respectively. riboAbacus and riboWaltz are available on GitHub. Results To predict the number of ribosomes per transcript and the position of ribosomes on mRNAs, I applied riboAbacus and riboSim, respectively, to transcriptomes of different organisms (yeast, mouse, human) for understanding the role of translational regulatory elements in tuning polysome in different organisms. First, I trained and validated performances of riboAbacus taking advantage of Atomic Force Microscopy images of polysomes, while performances of riboSim were assessed employing ribosome profiling data. Predictions provided by riboAbacus and riboSim were evaluated in parallel. I showed that the average number of ribosomes translating a molecule of mRNA can be well explained by the deterministic model, riboAbacus, that includes as features the mRNA levels, the mRNA sequences, the codon usage bias and a slowdown mechanism at the beginning of the CDS (ramp hypothesis). The predictions of ribosome localization by riboSim that used as features the mRNA sequence, the codon usage and the ramp, were run for yeast, mouse and human. I observed a good similarity between the predicted and experimental positions of ribosomes along transcripts in yeast, while poor similarity was obtained between predicted and experimental ribosome positions in the two mammals, suggesting the presence of more elaborate controls that tune ribosomes movement in higher eukaryotes than in simple species. After having developed two tools for the analyses of RiboSeq data and extraction of positional information on ribosome localization along transcripts, I applied both riboWaltz and riboScan in a case study. The aim was to dissect possible defects in ribosome localization in tissues of a mouse model of Spinal Muscular Atrophy (SMA). SMA is a neurodegenerative disorder caused by low levels of the Survival of Motor Neuron protein (SMN) in which translational impairments are recently emerging as possible cause of the disease. I analysed ribosome profiling data obtained from three different types of RiboSeq variants in healthy and SMA-affected mouse brains at the early-symptomatic stage of the disease. I observed i) a significant drop-off of translating ribosomes along the coding sequence in the SMA condition (using riboWaltz); ii) in SMA-affected mice, the possible accumulation of ribosomes along the 3' UTR in neuro-related mRNAs (using riboScan); iii) the involvement of SMN-specialized ribosomes in playing a very intimate role with the elongation stage of translation of the first codons of transcripts (riboWaltz), iv) the loss of ribosomes at the 3rd codon in SMA in transcripts bound by SMN-specialized ribosomes and v) a remarkable connection between SMN and the down-regulation of genes in SMA-affected mice. Overall, these findings confirmed previous observation about possible SMN-related dysregulations of local protein synthesis in neurons. More importantly, they unravel a completely new role of SMN in tuning translation at multiple levels (initiation, elongation and the recycling of terminating ribosomes), opening new hypotheses and scenarios for explaining the most devastating genetic disease, leading cause worldwide of infant mortality. Conclusions The present work provides a new comprehensive and integrated scenario for better understanding translation and demonstrates that this approach is a very powerful strategy to pave the way for new understanding of fine alteration in polysome organization and functional control in both physiological and pathological conditions

Monte Carlo evaluation of the impact of subsequent strokes on backflashover rate

The paper deals with the impact of subsequent strokes on the backflashover rate (BFR) of HV overhead transmission lines (OHLs), assessed by means of an ATP-EMTP Monte Carlo procedure. The application to a typical 150 kV Italian OHL is discussed, simulating several tower grounding system arrangements. Subsequent strokes parameters are added to the statistical simulation variables: Peak current, front time, time-to-half value, lightning polarity, line insulation withstand, lightning location and phase angle of the power frequency voltage. The input data are fed to an ATP-EMTP complete circuit model of the OHL, including line insulation, lightning representation and tower grounding system, the latter simulated by a pi-circuit model able to simulate the effects due to propagation and soil ionization, at modest computational costs. Numerical results evidence a non-negligible BFR increase (in relative terms) due to subsequent strokes: for spatially concentrated grounding systems the BFR increase approximatively vary in inverse proportion with the low frequency grounding resistance, whereas for spatially extended grounding systems the BFR increase depends on the grounding system behavior at high frequencies

An equivalent circuit for the evaluation of cross-country fault currents in medium voltage (MV) distribution networks

A Cross-Country Fault (CCF) is the simultaneous occurrence of a couple of Line-to-Ground Faults (LGFs), affecting different phases of same feeder or of two distinct ones, at different fault locations. CCFs are not uncommon in medium voltage (MV) public distribution networks operated with ungrounded or high-impedance neutral: despite the relatively small value of LGF current that is typical of such networks, CCF currents can be comparable to those that are found in Phase-To-Phase Faults, if the affected feeder(s) consists of cables. This occurs because the faulted cables' sheaths/screens provide a continuous, relatively low-impedance metallic return path to the fault currents. An accurate evaluation is in order, since the resulting current magnitudes can overheat sheaths/screens, endangering cable joints and other plastic sheaths. Such evaluation, however, requires the modeling of the whole MV network in the phase domain, simulating cable screens and their connections to the primary and secondary substation earth electrodes by suitable computer programs, such as ATP (which is the acronym for alternative transient program) or EMTP (the acronym for electromagnetic transient program), with substantial input data being involved. This paper presents a simplified yet accurate circuit model of the faulted MV network, taking into account the CCF currents' return path (cable sheaths/screens, ground conductors, and earthing resistances of secondary substations). The proposed CCF model can be implemented in a general-purpose simulation program, and it yields accurate fault currents estimates: for a 20 kV network case study, the comparison with accurate ATP simulations evidences mismatches mostly smaller than 2%, and never exceeding 5%

Tower grounding improvement versus line surge arresters: comparison of remedial measures for high-BFOR subtransmission lines

This paper presents a technical/economic comparison between remedial measures aimed at improving the lightning performance of an existing Italian three-phase 150-kV overhead line. The line is characterized by a very high back-flashover rate (BFOR), due to large grounding resistance values. Two countermeasures are proposed: grounding system improvement with additional vertical rods and line metal oxide surge arrester (MOSA) installation on one or all phases. A Monte Carlo ATP-EMTP procedure developed by the authors, which takes into account both the tower grounding nonlinear transient response due to soil ionization and MOSA nonlinear response, has been applied to evaluate and compare the effectiveness of the proposed countermeasures. The installation of MOSA on all phases is technically the best option, but it is relatively expensive. Tower grounding improvement and MOSA installation on the lower phase yield very similar BFORs: the economic comparison strongly depends on tower's accessibility and soil nature

Estimating Wind Farm Transformers Rating through Lifetime Characterization Based on Stochastic Modeling of Wind Power

This paper deals with the problem of the optimal rating of mineral-oil-immersed transformers in large wind farms. The optimal rating is derived based on the probabilistic analyses of wind power generation through the Ornstein–Uhlenbeck stochastic process and on thermal model of the transformer through the integration of stochastic differential equations. These analyses allow the stochastic characterization of lifetime reduction of the transformer and then its optimal rating through a simple closed form. The numerical application highlights the effectiveness and easy applicability of the proposed methodology. The proposed methodology allows deriving the rating of transformers which better fits the specific peculiarities of wind power generation. Compared to the conventional approaches, the proposed method can better adapt the transformer size to the intermittence and variability of the power generated by wind farms, thus overcoming the often-recognized reduced lifetime

Genetic variants of Y chromosome are associated with a protective lipid profile in black men

Objective— Gender and ethnicity modulate the phenotypic expression of cardiovascular risk factors. In particular, men are at higher risk of developing cardiovascular diseases compared to women, whereas black populations of African origin display reduced mortality from coronary heart disease (CHD) as compared to both whites and South Asians. Because the male-specific region (MSY) of the human Y chromosome is an obvious candidate for gender-related differences in the development of cardiovascular diseases, we aimed to identify genetic variants of MSY influencing cardiovascular risk profile in different ethnic groups. Methods and Results— We genotyped 4 polymorphisms of MSY (HindIII±, rs768983 of TBL1Y, rs3212292 of USP9Y, and rs9341273 of UTY genes) in 579 men of different ethnic groups (blacks, South Asians, and whites) from UK and in 301 whites in Italy. We found that the TBL1YA USP9YA haplotype, present only in blacks in whom it represents the most frequent allelic combinations (AA: n=125; all other combinations: n=45), was associated with lower levels of triglycerides (P=0.025) and higher levels of HDL-cholesterol (P=0.005) as compared to the other haplotypes. Conclusion— The TBL1YA USP9YA haplotype of the Y chromosome, present only in black people of African origin, attributes a favorable lipoprotein pattern, likely to contribute to their reduced susceptibility to coronary heart disease. The study evaluated the association of genetic variants of the male-specific region of the Y chromosome with cardiovascular risk factors in different ethnic groups. The most frequently observed haplotype in black people was associated with a favorable lipoprotein pattern, thus contributing to the lower rate of cardiovascular diseases in blacks

Analytical Description of Overhead Transmission Lines Loadability

The loadability characteristics of overhead transmission lines (OHLs) is certainly not a new topic. However, driven by sustainability issues, the increasing need to exploit existing electrical infrastructures as much as possible, has given OHL loadability a renowned central role and, recently, new investigations on this subject have been carried out. OHL loadability is generally investigated by means of numerical methods. Even though this approach allows deducing useful information in both planning and operation stage, it does not permit to capture all the insights obtainable by an analytical approach. The goal of this paper is to tailor a general analytical formulation for the loadability of OHLs. The first part of the paper is devoted to the base-case of uncompensated OHLs. Later, aiming to demonstrate the inherent feasibility and flexibility of the novel approach proposed, the less frequent case of shunt compensated radial OHLs is investigated as well. The analytical formulation is combined with the use of circular diagrams. Such diagrams allow a geometrical interpretation of the analytical relationships and are very useful to catch the physical insights of the problem. Finally, in order to show the applicability of the new analytical approach, a practical example is provided. The example concerns calculation of the loadability characteristics of typical 400 kV single-circuit OHLs

riboWaltz: optimization of ribosome P-site positioning in ribosome profiling data

<div><p>Ribosome profiling is a powerful technique used to study translation at the genome-wide level, generating unique information concerning ribosome positions along RNAs. Optimal localization of ribosomes requires the proper identification of the ribosome P-site in each ribosome protected fragment, a crucial step to determine the trinucleotide periodicity of translating ribosomes, and draw correct conclusions concerning where ribosomes are located. To determine the P-site within ribosome footprints at nucleotide resolution, the precise estimation of its offset with respect to the protected fragment is necessary. Here we present riboWaltz, an R package for calculation of optimal P-site offsets, diagnostic analysis and visual inspection of ribosome profiling data. Compared to existing tools, riboWaltz shows improved accuracies for P-site estimation and neat ribosome positioning in multiple case studies. riboWaltz was implemented in R and is available as an R package at <a href="https://github.com/LabTranslationalArchitectomics/RiboWaltz" target="_blank">https://github.com/LabTranslationalArchitectomics/RiboWaltz</a>.</p></div

METHYLOMIC SIGNATURE AND MOLECULAR MODELLING TO BETTER UNDERSTAND AUTOPHAGY INDUCED BY PHYTOCHEMICAL IN CACO-2 CELLS

The binomial “autophagy-cancer” is intricate and methylomic studies can help to understand it by changing point of view from a gene level to an -omic one. Recently, autophagy-modulating properties of several phytochemicals have attracted attention in anticancer research. We evaluated whether Indicaxanthin (IND), the peculiar known beneficial phytochemical of prickly pear, seasonally available in the southern Italy, could induce autophagy in Caco2 cells, and whether it results from an epigenomic modification and/or a direct molecular interaction. IND increased autophagy in Caco-2 cells; the methylomic signature, obtained by Reduced Representation Bisulfite Sequencing (15 million of clusters) reported that 14 main genes of autophagy, showed a different methylation consistent with the induction of this phenomenon. Among these: MTOR, ATG13, BECN1, TFEB, ATG3, WIPI2, TECPR1, SNAP29, VPS11, VPS16. By traditional approaches we confirmed the demethylation of BECN1 gene and the increase of Beclin1 levels. By in-silico molecular modelling, we displayed a possible interference of IND, by competitive mechanisms, in the Beclin1-Bcl2 interaction. Methylomic signature and molecular modelling has been helpful to understand autophagy IND-induced in intestinal epithelial tumour cells. Our results suggest that the pro-autophagic action promoted by this phytochemical involves both epigenomic modulation and post-translation mechanisms by direct interaction with key targets of autophagy pathway