Vibration as a method for muscular stimulation

Human reactions to vibration have been extensively investigated in the past. Vibration, as well as whole-body vibration (WBV), has been commonly considered as an occupational hazard for its detrimental effects on human condition and comfort. Although long term exposure to vibrations may produce undesirable side-effects, a great part of the literature is dedicated to the positive effects of WBV when used as method for muscular stimulation and as an exercise intervention. Whole body vibration training (WBVT) aims to mechanically activate muscles by eliciting neuromuscular activity (muscle reflexes) via the use of vibrations delivered to the whole body. The most mentioned mechanism to explain the neuromuscular outcomes of vibration is the elicited neuromuscular activation. Local tendon vibrations induce activity of the muscle spindle Ia fibers, mediated by monosynaptic and polysynaptic pathways: a reflex muscle contraction known as the Tonic Vibration Reflex (TVR) arises in response to such vibratory stimulus. In WBVT mechanical vibrations, in a range from 10 to 80 Hz and peak to peak displacements from 1 to 10 mm, are usually transmitted to the patient body by the use of oscillating platforms. Vibrations are then transferred from the platform to a specific muscle group through the subject body. To customize WBV treatments, surface electromyography (SEMG) signals are often used to reveal the best stimulation frequency for each subject. Use of SEMG concise parameters, such as root mean square values of the recordings, is also a common practice; frequently a preliminary session can take place in order to discover the more appropriate stimulation frequency. Soft tissues act as wobbling masses vibrating in a damped manner in response to mechanical excitation; Muscle Tuning hypothesis suggest that neuromuscular system works to damp the soft tissue oscillation that occurs in response to vibrations; muscles alters their activity to dampen the vibrations, preventing any resonance phenomenon. Muscle response to vibration is however a complex phenomenon as it depends on different parameters, like muscle-tension, muscle or segment-stiffness, amplitude and frequency of the mechanical vibration. Additionally, while in the TVR study the applied vibratory stimulus and the muscle conditions are completely characterised (a known vibration source is applied directly to a stretched/shortened muscle or tendon), in WBV study only the stimulus applied to a distal part of the body is known. Moreover, mechanical response changes in relation to the posture. The transmissibility of vibratory stimulus along the body segment strongly depends on the position held by the subject. The aim of this work was the investigation on the effects that the use of vibrations, in particular the effects of whole body vibrations, may have on muscular activity. A new approach to discover the more appropriate stimulus frequency, by the use of accelerometers, was also explored. Different subjects, not affected by any known neurological or musculoskeletal disorders, were voluntarily involved in the study and gave their informed, written consent to participate. The device used to deliver vibration to the subjects was a vibrating platform. Vibrations impressed by the platform were exclusively vertical; platform displacement was sinusoidal with an intensity (peak-to-peak displacement) set to 1.2 mm and with a frequency ranging from 10 to 80 Hz. All the subjects familiarized with the device and the proper positioning. Two different posture were explored in this study: position 1 - hack squat; position 2 - subject standing on toes with heels raised. SEMG signals from the Rectus Femoris (RF), Vastus Lateralis (VL) and Vastus medialis (VM) were recorded. SEMG signals were amplified using a multi-channel, isolated biomedical signal amplifier The gain was set to 1000 V/V and a band pass filter (-3dB frequency 10 - 500 Hz) was applied; no notch filters were used to suppress line interference. Tiny and lightweight (less than 10 g) three-axial MEMS accelerometers (Freescale semiconductors) were used to measure accelerations of onto patient’s skin, at EMG electrodes level. Accelerations signals provided information related to individuals’ RF, Biceps Femoris (BF) and Gastrocnemius Lateralis (GL) muscle belly oscillation; they were pre-processed in order to exclude influence of gravity. As demonstrated by our results, vibrations generate peculiar, not negligible motion artifact on skin electrodes. Artifact amplitude is generally unpredictable; it appeared in all the quadriceps muscles analysed, but in different amounts. Artifact harmonics extend throughout the EMG spectrum, making classic high-pass filters ineffective; however, their contribution was easy to filter out from the raw EMG signal with a series of sharp notch filters centred at the vibration frequency and its superior harmonics (1.5 Hz wide). However, use of these simple filters prevents the revelation of EMG power potential variation in the mentioned filtered bands. Moreover our experience suggests that the possibility of reducing motion artefact, by using particular electrodes and by accurately preparing the subject’s skin, is not easily viable; even though some small improvements were obtained, it was not possible to substantially decrease the artifact. Anyway, getting rid of those artifacts lead to some true EMG signal loss. Nevertheless, our preliminary results suggest that the use of notch filters at vibration frequency and its harmonics is suitable for motion artifacts filtering. In RF SEMG recordings during vibratory stimulation only a little EMG power increment should be contained in the mentioned filtered bands due to synchronous electromyographic activity of the muscle. Moreover, it is better to remove the artifact that, in our experience, was found to be more than 40% of the total signal power. In summary, many variables have to be taken into account: in addition to amplitude, frequency and duration of vibration treatment, other fundamental variables were found to be subject anatomy, individual physiological condition and subject’s positioning on the platform. Studies on WBV treatments that include surface EMG analysis to asses muscular activity during vibratory stimulation should take into account the presence of motion artifacts. Appropriate filtering of artifacts, to reveal the actual effect on muscle contraction elicited by vibration stimulus, is mandatory. However as a result of our preliminary study, a simple multi-band notch filtering may help to reduce randomness of the results. Muscle tuning hypothesis seemed to be confirmed. Our results suggested that the effects of WBV are linked to the actual muscle motion (displacement). The greater was the muscle belly displacement the higher was found the muscle activity. The maximum muscle activity has been found in correspondence with the local mechanical resonance, suggesting a more effective stimulation at the specific system resonance frequency. Holding the hypothesis that muscle activation is proportional to muscle displacement, treatment optimization could be obtained by simply monitoring local acceleration (resonance). However, our study revealed some short term effects of vibratory stimulus; prolonged studies should be assembled in order to consider the long term effectiveness of these results. Since local stimulus depends on the kinematic chain involved, WBV muscle stimulation has to take into account the transmissibility of the stimulus along the body segment in order to ensure that vibratory stimulation effectively reaches the target muscle. Combination of local resonance and muscle response should also be further investigated to prevent hazards to individuals undergoing WBV treatments.

A clustering method for robust and reliable large scale functional and structural protein sequence annotation

Bioinformatics, in the last few decades, has played a fundamental role to give sense to the huge amount of data produced. Obtained the complete sequence of a genome, the major problem of knowing as much as possible of its coding regions, is crucial. Protein sequence annotation is challenging and, due to the size of the problem, only computational approaches can provide a feasible solution. As it has been recently pointed out by the Critical Assessment of Function Annotations (CAFA), most accurate methods are those based on the transfer-by-homology approach and the most incisive contribution is given by cross-genome comparisons. In the present thesis it is described a non-hierarchical sequence clustering method for protein automatic large-scale annotation, called “The Bologna Annotation Resource Plus” (BAR+). The method is based on an all-against-all alignment of more than 13 millions protein sequences characterized by a very stringent metric. BAR+ can safely transfer functional features (Gene Ontology and Pfam terms) inside clusters by means of a statistical validation, even in the case of multi-domain proteins. Within BAR+ clusters it is also possible to transfer the three dimensional structure (when a template is available). This is possible by the way of cluster-specific HMM profiles that can be used to calculate reliable template-to-target alignments even in the case of distantly related proteins (sequence identity < 30%). Other BAR+ based applications have been developed during my doctorate including the prediction of Magnesium binding sites in human proteins, the ABC transporters superfamily classification and the functional prediction (GO terms) of the CAFA targets. Remarkably, in the CAFA assessment, BAR+ placed among the ten most accurate methods. At present, as a web server for the functional and structural protein sequence annotation, BAR+ is freely available at http://bar.biocomp.unibo.it/bar2.0.

A machine learning based method to detect genomic imbalances exploiting X chromosome exome reads

Whole Exome Sequencing (WES) is rapidly becoming the first-tier test in clinics, both thanks to its declining costs and the development of new platforms that help clinicians in the analysis and interpretation of SNV and InDels. However, we still know very little on how CNV detection could increase WES diagnostic yield. A plethora of exome CNV callers have been published over the years, all showing good performances towards specific CNV classes and sizes, suggesting that the combination of multiple tools is needed to obtain an overall good detection performance. Here we present TrainX, a ML-based method for calling heterozygous CNVs in WES data using EXCAVATOR2 Normalized Read Counts. We select males and females’ non pseudo-autosomal chromosome X alignments to construct our dataset and train our model, make predictions on autosomes target regions and use HMM to call CNVs. We compared TrainX against a set of CNV tools differing for the detection method (GATK4 gCNV, ExomeDepth, DECoN, CNVkit and EXCAVATOR2) and found that our algorithm outperformed them in terms of stability, as we identified both deletions and duplications with good scores (0.87 and 0.82 F1-scores respectively) and for sizes reaching the minimum resolution of 2 target regions. We also evaluated the method robustness using a set of WES and SNP array data (n=251), part of the Italian cohort of Epi25 collaborative, and were able to retrieve all clinical CNVs previously identified by the SNP array. TrainX showed good accuracy in detecting heterozygous CNVs of different sizes, making it a promising tool to use in a diagnostic setting.

RNA interference technology as a potential control method for fruit and horticultural crops pathogens Botrytis cinerea and Plasmopara viticola

Chapter 1, a general introduction on Botrytis cinerea and its threat to crop production is presented. What Botrytis looks like, its life cycle, why it is a threat to agricultural production, its worldwide pest status, and its current state of management is further elaborated on. Chapter 2, a general introduction on Plasmopara viticola, its threat to grape production and management strategies presented. Chapter 3, titled " RNA Interference Strategies for Future Management of Plant Pathogenic Fungi: Prospects and Challenges ", presents the rapid improvement and extensive implementation of RNA interference (RNAi) technology for the management of fungal pathogens. In this chapter, we describe the application of exogenous RNAi involved in plant pathogenic fungi and discuss dsRNA production, formulation, and RNAi delivery methods. Chapter 4, titled " Exogenous dsRNAs against chitin synthase and glucan synthase genes suppress the growth of the pathogenic fungus Botrytis cinerea " addresses two important questions: Is RNAi technology functional for B. cinerea control ? And which target genes can be exploited for RNAi-based B.cinerea disease control ? Upon target genes selections, an exogenous RNAi protocol was set up and we could effectively deliver a known dose of bacterially produced double stranded RNA (dsRNA) to induce RNAi in B. cinerea. Chapter 5, titled " Double-Stranded RNA Targeting Dicer-Like Genes Compromises the Pathogenicity of Plasmopara viticola on Grapevine “, which deals mainly on RNAi induction against Plasmopara viticola. This chapter addresses two main questions: Is RNAi technology functional in contrasting Plasmopara viticola? And which target genes can be exploited for RNAi-based disease control in Plasmopara viticola?. In the last Chapter (Chapter 6) titled “General discussions and perspectives for future research”, the major research findings from this thesis are discussed together with perspectives for future research.