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

Representation of distribution networks of ships using graph-theory

CETENA S.p.A., SISSA (International School for Advanced Studies) and Lloyd\u2019s Register (Class Society) have recently been involved in a challenge aimed at developing smart algorithms capable to evaluate the effect of different failure modes \u2014 caused by a fire or a flooding\u2014on the systems of passenger ships in order to improve the design of new passenger ships [1]. Considering that a failure may cause serious accidents both to the vessel and human lives, the goal of this project is to evaluate the best reconfiguration of current ship plants after each casualty scenario so as to guarantee the minimal functioning requirements. This implies a continuous cross check activity (design against installation) that follows the whole ship construction process. The urgency of this work is motivated by the necessity to meet the International Maritime Organizations (IMO) Safety Of Life At Sea (Solas) design prescriptions defined in the Safe Return to Port (SRtP) regulations [2]. According to these criteria, a vessel should be able to safely return to port under its own propulsion after an adverse event not exceeding any of the defined casualty thresholds and criteria imposed by the regulations. Thus, the identification of all the possible failure modes and their propagation through the on-board systems has become a task of paramount importance for the proper design of the ship\u2019s systems against failure events. Currently, in accordance with IMO MSC.1/circ.1369 [3], CETENA produces the Operating Manuals that allow the crew to reconfigure the essential systems after a SRtP casualty so as to be able to bring the ship to a port with adequate comfort and safety standards. However, the ship can be operated in a different way from what is planned in the design stage. In these scenarios, the present static Operational Manuals can be a limitation. In order to be effective during emergency operation, Operational Manuals must be dynamic so as to provide interactive information and guidance to crew members about the reconfiguration of the ship and the recovery of her functions based on the systems configuration at the moment of the casualty. The focus of this work is the study of domino effects triggered by fire or flooding casualties in passenger ships in order to provide crew with a tool which speeds up and facilitates the decision-making process when choices have to be made to optimize the ship residual capability after a casualty. The framework of this study may be extended to other types of domino escalation

Mental sleep activity and disturbing dreams in the lifespan

Sleep significantly changes across the lifespan, and several studies underline its crucial role in cognitive functioning. Similarly, mental activity during sleep tends to covary with age. This review aims to analyze the characteristics of dreaming and disturbing dreams at dierent age brackets. On the one hand, dreams may be considered an expression of brain maturation and cognitive development, showing relations with memory and visuo-spatial abilities. Some investigations reveal that specific electrophysiological patterns, such as frontal theta oscillations, underlie dreams during sleep, as well as episodic memories in the waking state, both in young and older adults. On the other hand, considering the role of dreaming in emotional processing and regulation, the available literature suggests that mental sleep activity could have a beneficial role when stressful events occur at dierent age ranges. We highlight that nightmares and bad dreams might represent an attempt to cope the adverse events, and the degrees of cognitive-brain maturation could impact on these mechanisms across the lifespan. Future investigations are necessary to clarify these relations. Clinical protocols could be designed to improve cognitive functioning and emotional regulation by modifying the dream contents or the ability to recall/non-recall them

EEG oscillations during sleep and dream recall. State- or trait-like individual differences?

Dreaming represents a peculiar form of cognitive activity during sleep. On the basis of the well-known relationship between sleep and memory, there has been a growing interest in the predictive role of human brain activity during sleep on dream recall. Neuroimaging studies indicate that rapid eye movement (REM) sleep is characterized by limbic activation and prefrontal cortex deactivation. This pattern could explain the presence of emotional contents in dream reports. Furthermore, the morphoanatomical measures of amygdala and hippocampus predict some features of dream contents (bizarreness, vividness, and emotional load). More relevant for a general view of dreaming mechanisms, empirical data from neuropsychological and electroencephalographic (EEG) studies support the hypothesis that there is a sort of continuity between the neurophysiological mechanisms of encoding and retrieval of episodic memories across sleep and wakefulness. A notable overlap between the electrophysiological mechanisms underlying emotional memory formation and some peculiar EEG features of REM sleep has been suggested. In particular, theta (5–8 Hz) EEG oscillations on frontal regions in the pre-awakening sleep are predictive of dream recall, which parallels the predictive relation during wakefulness between theta activity and successful retrieval of episodic memory. Although some observations support an interpretation more in terms of an intraindividual than interindividual mechanism, the existing empirical evidence still precludes from definitely disentangling if this relation is explained by state- or trait-like differences

The functional role of dreaming in emotional processes

Dream experience (DE) represents a fascinating condition linked to emotional processes and the human inner world. Although the overlap between REM sleep and dreaming has been overcome, several studies point out that emotional and perceptually vivid contents are more frequent when reported upon awakenings from this sleep stage. Actually, it is well-known that REM sleep plays a pivotal role in the processing of salient and emotional waking-life experiences, strongly contributing to the emotional memory consolidation. In this vein, we highlighted that, to some extent, neuroimaging studies showed that the processes that regulate dreaming and emotional salience in sleep mentation share similar neural substrates of those controlling emotions during wakefulness. Furthermore, the research on EEG correlates of the presence/absence of DE and the results on EEG pattern related to the incorporated memories converged to assign a crucial role of REM theta oscillations in emotional re-processing. In particular, the theta activity is involved in memory processes during REM sleep as well as during the waking state, in line with the continuity hypothesis. Also, the gamma activity seems to be related to emotional processes and dream recall as well as to lucid dreams. Interestingly, similar EEG correlates of DE have been found in clinical samples when nightmares or dreams occur. Research on clinical samples revealed that promoting the rehearsal of frightening contents aimed to change them is a promising method to treat nightmares, and that lucid dreams are associated with an attenuation of nightmares. In this view, DE can defuse emotional traumatic memories when the emotional regulation and the fear extinction mechanisms are compromised by traumatic and frightening events. Finally, dreams could represent a sort of simulation of reality, providing the possibility to create a new scenario with emotional mastery elements to cope with dysphoric items included in nightmares. In addition, it could be hypothesized that the insertion of bizarre items besides traumatic memories might be functional to “impoverish” the negative charge of the experiences

State- or trait-like individual differences in dream recall. Preliminary findings from a within-subjects study of multiple nap REM sleep awakenings

We examined the question whether the role of EEG oscillations in predicting presence/absence of dream recall (DR) is explained by "state-" or "trait-like" factors. Six healthy subjects were awakened from REM sleep in a within-subjects design with multiple naps, until a recall and a non-recall condition were obtained. Naps were scheduled in the early afternoon and were separated by 1 week. Topographical EEG data of the 5-min of REM sleep preceding each awakening were analyzed by power spectral analysis [Fast Fourier Transform (FFT)] and by a method to detect oscillatory activity [Better OSCillations (BOSC)]. Both analyses show that REC is associated to higher frontal theta activity (5-7 Hz) and theta oscillations (6.06 Hz) compared to NREC condition, but only the second comparison reached significance. Our pilot study provides support to the notion that sleep and wakefulness share similar EEG correlates of encoding in episodic memories, and supports the "state-like hypothesis": DR may depend on the physiological state related to the sleep stage from which the subject is awakened rather than on a stable individual EEG pattern

Oscillatory EEG activity during REM sleep in elderly people predicts subsequent dream recall after awakenings

Several findings underlined that the electrophysiological (EEG) background of the last segment of sleep before awakenings may predict the presence/absence of dream recall (DR) in young subjects. However, little is known about the EEG correlates of DR in elderly people. Only an investigation found differences between recall and non-recall conditions during NREM sleep EEG in older adults, while—surprisingly—no EEG predictor of DR was found for what concerns REMsleep. Considering REMsleep as a privileged scenario to produce mental sleep activity related to cognitive processes, our study aimed to investigate whether specific EEG topography and frequency changes during REM sleep in elderly people may predict a subsequent recall of mental sleep activity. Twenty-one healthy older volunteers (mean age 69.2 ± 6.07 SD) and 20 young adults (mean age 23.4 ± 2.76 SD) were recorded for one night from19 scalp derivations. Dreams were collected upon morning awakenings from REM sleep. EEG signals of the last 5min were analyzed by the Better OSCillation algorithm to detect the peaks of oscillatory activity in both groups. Statistical comparisons revealed that older as well as young individuals recall their dream experience when the last segment of REM sleep is characterized by frontal theta oscillations. No Recall (Recall vs. Non-Recall) × Age (Young vs. Older) interaction was found. This result replicated the previous evidence in healthy young subjects, as shown in within- and between-subjects design. The findings are completely original for older individuals, demonstrating that theta oscillations are crucial for the retrieval of dreaming also in this population. Furthermore, our results did not confirm a greater presence of the theta activity in healthy aging. Conversely, we found a greater amount of rhythmic theta and alpha activity in young than older participants. It is worth noting that the theta oscillations detected are related to cognitive functioning. We emphasize the notion that the oscillatory theta activity should be distinguished from the non-rhythmic theta activity identified in relation to other phenomena such as (a) sleepiness and hypoarousal conditions during the waking state and (b) cortical slowing, considered as an EEG alteration in clinical samples

Chemical Constituents and Biological Properties of Genus Doronicum (Asteraceae)

The genus Doronicum, belonging to tribe Senecioneae (Fam. Asteraceae), is found mainly in the Asia, Europe and North Africa. This genus of plant has always been used in traditional medicinal treatments due to the many biological properties shown such as killing parasitic worms and for relieving constipation, as well as to improve heart health, to alleviate pain and inflammation, to treat insect bites, etc. According to the World Flora the genus Doronicum contains 39 subordinate taxa.[1-3] The purpose of this article, which covers data published from 1970 to 2021 with more than 110 articles, aims to carry out a complete and critical review of the Doronicum genus, examining traditional uses and reporting the antioxidant, antimicrobial, anti-inflammatory and antitumor activity shown from crude extracts or essential oils, and from single isolated compounds. Furthermore, critical considerations of the published data have been highlighted by comparing them with the results obtained from species of other genus belonging to the Asteraceae family

Bilateral 5 Hz transcranial alternating current stimulation on fronto-temporal areas modulates resting-state EEG

Rhythmic non-invasive brain stimulations are promising tools to modulate brain activity by entraining neural oscillations in specific cortical networks. The aim of the study was to assess the possibility to influence the neural circuits of the wake-sleep transition in awake subjects via a bilateral transcranial alternating current stimulation at 5 Hz (theta-tACS) on fronto-temporal areas. 25 healthy volunteers participated in two within-subject sessions (theta-tACS and sham), one week apart and in counterbalanced order. We assessed the stimulation effects on cortical EEG activity (28 derivations) and self-reported sleepiness (Karolinska Sleepiness Scale). theta-tACS induced significant increases of the theta activity in temporo-parieto-occipital areas and centro-frontal increases in the alpha activity compared to sham but failed to induce any online effect on sleepiness. Since the total energy delivered in the sham condition was much less than in the active theta-tACS, the current data are unable to isolate the specific effect of entrained theta oscillatory activity per se on sleepiness scores. On this basis, we concluded that theta-tACS modulated theta and alpha EEG activity with a topography consistent with high sleep pressure conditions. However, no causal relation can be traced on the basis of the current results between these rhythms and changes on sleepines

The spatiotemporal pattern of the human electroencephalogram at sleep onset after a period of prolonged wakefulness

During the sleep onset (SO) process, the human electroencephalogram (EEG) is characterized by an orchestrated pattern of spatiotemporal changes. Sleep deprivation (SD) strongly affects both wake and sleep EEG, but a description of the topographical EEG power spectra and oscillatory activity during the wake-sleep transition after a period of prolonged wakefulness is still missing. The increased homeostatic sleep pressure should induce an earlier onset of sleep-related EEG oscillations. The aim of the present study was to assess the spatiotemporal EEG pattern at SO following SD. A dataset of a previous study was analyzed. We assessed the spatiotemporal EEG changes (19 cortical derivations) during the SO (5 min before vs. 5 min after the first epoch of Stage 2) of a recovery night after 40 h of SD in 39 healthy subjects, analyzing the EEG power spectra (fast Fourier transform) and the oscillatory activity [better oscillation (BOSC) detection method]. The spatiotemporal pattern of the EEG power spectra mostly confirmed the changes previously observed during the wake-sleep transition at baseline. The comparison between baseline and recovery showed a wide increase of the post- vs. pre-SO ratio during the recovery night in the frequency bins 10 Hz. We found a predominant alpha oscillatory rhythm in the pre-SO period, while after SO the theta oscillatory activity was prevalent. The oscillatory peaks showed a generalized increase in all frequency bands from delta to sigma with different predominance, while beta activity increased only in the fronto-central midline derivations. Overall, the analysis of the EEG power replicated the topographical pattern observed during a baseline night of sleep but with a stronger intensity of the SO-induced changes in the frequencies 10 Hz, and the detection of the rhythmic activity showed the rise of several oscillations at SO after SD that was not observed during the wake-sleep transition at baseline (e.g., alpha and frontal theta in correspondence of their frequency peaks). Beyond confirming the local nature of the EEG pattern at SO, our results show that SD has an impact on the spatiotemporal modulation of cortical activity during the falling-asleep process, inducing the earlier emergence of sleep-related EEG oscillations